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Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy
Abstract
The light-driven proton pump bacteriorhodopsin occurs naturally as two-dimensional crystals. A three-dimensional density map of the structure, at near-atomic resolution, has been obtained by studying the crystals using electron cryo-microscopy to obtain electron diffraction patterns and high-resolution micrographs. New methods were developed for analysing micrographs from tilted specimens, incorporating methods previously developed for untilted specimens that enable large areas to be analysed and corrected for distortions. Data from 72 images, from both tilted and untilted specimens, were analysed to produce the phases of 2700 independent Fourier components of the structure. The amplitudes of these components were accurately measured from 150 diffraction patterns. Together, these data represent about half of the full three-dimensional transform to 3.5 A. The map of the structure has a resolution of 3.5 A in a direction parallel to the membrane plane but lower than this in the perpendicular direction. It shows many features in the density that are resolved from the main density of the seven alpha-helices. We interpret these features as the bulky aromatic side-chains of phenylalanine, tyrosine and tryptophan residues. There is also a very dense feature, which is the beta-ionone ring of the retinal chromophore. Using these bulky side-chains as guide points and taking account of bulges in the helices that indicate smaller side-chains such as leucine, a complete atomic model for bacteriorhodopsin between amino acid residues 8 and 225 has been built. There are 21 amino acid residues, contributed by all seven helices, surrounding the retinal and 26 residues, contributed by five helices, forming the proton pathway or channel. Ten of the amino acid residues in the middle of the proton channel are also part of the retinal binding site. The model also provides a useful basis for consideration of the mechanism of proton pumping and allows a consistent interpretation of a great deal of other experimental data. In particular, the structure suggests that pK changes in the Schiff base must act as the means by which light energy is converted into proton pumping pressure in the channel. Asp96 is on the pathway from the cytoplasm to the Schiff base and Asp85 is on the pathway from the Schiff base to the extracellular surface.
... The capabilities of electron microscopy have expanded so much that when combined with various methodological approaches to the preparation of objects, it has become possible to solve issues related to detailing the structural features of individual elements included in the object [1,2]. Currently, electron microscopic equipment is being rapidly improved, and special analytical electron microscopes are being created to determine the composition of micro-objects using electronic and x-ray spectra. ...
... A theoretical description of the density distribution inside a macromolecular coil has been proposed, representing it as a spherically symmetric object [10]. ρ(r) = n 3 2π(2R) 2 3 2 exp − 3r 2 2R 2 (1) where n is the number of segments of the macromolecule, R is the radius of gyration of the macromolecule, and r is the current radius. The found segment density distribution functions are in good agreement with experiment. ...
... However, X-ray studies provide results averaged over an ensemble of macromolecules. In this case, the radial density distribution function is well described using Equation (1). In order to analyze each individual macromolecule without averaging over the ensemble and to identify intra-ball density fluctuations, transmission electron microscopy should be used. ...
More than five hundred images of individual macromolecules of random styrene-butadiene copolymers and styrene-isoprene block copolymers dissolved in a polystyrene matrix were analyzed. The presence of density fluctuations inside the macromolecular coil has been established. Within the framework of the model of harmonic oscillations, the radial distribution of such density fluctuations is estimated.
... Less than a decade ago, before the "resolution revolution," cryo-electron microscopy (cryo-EM) was indulgently called "blobology" [1][2][3][4]. Whereas seminal work of cryo-EM experts resulted in high-resolution 3D maps and atomic models of ordered assemblies such as 2D crystals, helical arrays, and icosahedral viruses [5][6][7][8][9][10][11], commonly obtained 3D maps of less regular or asymmetric objects could be interpreted only in terms of global 3D architecture, domain organization, and-at most-secondary structure elements. Atomic model building was the privilege and expertise of crystallographers, requiring careful consideration of structural details such as bond geometry, steric clashes, and hydrogen bonds. ...
With the rapid improvement of cryo-electron microscopy (cryo-EM) resolution, new computational tools are needed to assist and improve upon atomic model building and refinement options. This communication demonstrates that microscopists can now collaborate with the players of the computer game Foldit to generate high-quality de novo structural models. This development could greatly speed the generation of excellent cryo-EM structures when used in addition to current methods.
... In H. salinarum cell membranes, bacteriorhodopsin forms two-dimensional crystals called purple membranes (PMs). They have a hexagonal lattice with a parameter a ≈62 Å, with each unit cell corresponding to a single HsBR trimer [6] (Fig. 1). The mass ratio of HsBR to lipids in PMs reaches 3 : 1 [7]. ...
... A typical approach to determining the 3D structure of protein 2D lattices at high resolution is through crystallographic methods, such as X-ray and cryogenic electron crystallography [13][14][15][16] . In this technique, the lattice, embedded in vitri ed ice to maintain its near-native state, is reconstructed into a 3D map through the combined analysis of electron microscopic images and diffraction patterns acquired at different tilt angles. ...
Programmable and self-assembled two-dimensional (2D) protein lattices hold significant potential in synthetic biology, nanoscale catalysis, and biological devices. However, achieving high-order 2D lattices from three-dimensional (3D) nanoscale objects remains challenging due to structural heterogeneity caused by the flexibility and distortions of building blocks and their connectivity in a unit cell, leading to the formation of lattices with imperfections. This flexibility largely limits the analysis of key structural parameters at unit-cell resolutions due to the need to average 3D reconstructions in current methods. Here, we utilized advances in individual-particle cryo-electron tomography (IPET) to analyze the 3D structure of a designed 2D lattice formed by DNA-origami octahedral cages (unit-cell particles) encapsulating ferritin by determining the non-averaged 3D structure of each unit-cell particle. These protein-carrying DNA cages were analyzed at ferritin loading percentages of 100%, 70%, and 0%. Correlation analysis revealed that neither the ferritin loading percentage nor off-centralized placement in cages significantly affected lattice parameters, flexibility, or long-range order. Instead, the soft nature of DNA cages and interparticle linkages were the primary reasons for lattice imperfections. Structural improvements for enhancing lattice orders were evaluated through a series of molecular dynamics simulations. The developed cryo-EM 3D imaging reveals the molecular origin of heterogeneity of DNA-origami 2D lattices and highlights a path toward improved lattice designs.
... HeAR was successfully expressed in E. coli BL21 (DE3) and spontaneously assembled into trimers (SEC analysis: 129 kDa, Figure 2), with circular dichroism (CD) spectral characteristics highly consistent with those of bacterial bacteriorhodopsin (BR) (Figure 3). This similarity suggests that HeAR and BR share comparable transmembrane helical topologies [52]. However, HeAR exhibits a 10 nm red-shift in maximum absorption wavelength compared to BR (535 nm vs. 525 nm), and its protonated Schiff base counterion Asp-95 has a significantly higher pKa value (3.5) than BR's Asp-85 (2.6) ( Figure 6). ...
This study elucidates the structural determinants and optogenetic potential of Archaerhodopsin HeAR, a proton pump from Halorubrum sp. Ejinoor isolated from Inner Mongolian salt lakes. Through heterologous expression in E. coli BL21 (DE3) and integrative biophysical analyses, we demonstrate that HeAR adopts a stable trimeric architecture (129 kDa) with detergent-binding characteristics mirroring bacteriorhodopsin (BR); however, it exhibits a 10 nm bathochromic spectral shift (λmax = 550 nm) and elevated proton affinity (Asp-95 pKa = 3.5 vs. BR Asp-85 pKa = 2.6), indicative of evolutionary optimization in its retinal-binding electrostatic microenvironment. Kinetic profiling reveals HeAR’s prolonged photocycle (100 ms vs. BR’s 11 ms), marked by rapid M-state decay (3.3 ms) and extended dark-adaptation half-life (160 min), a bistable behavior attributed to enhanced hydrogen bond persistence (80%) and reduced conformational entropy (RMSD = 2.0 Å). Functional assays confirm light-driven proton extrusion (0.1 ng H⁺/mg·s) with DCCD-amplified flux (0.3 ng H⁺/mg·s) and ATP synthesis (0.3 nmol/mg·s), underscoring its synergy with H⁺-ATPase. Phylogenetic and structural analyses reveal 95% homology with Halorubrum AR4 and conservation of 11 proton-wire residues, despite divergent Trp/Tyr/Ser networks that redefine chromophore stabilization. AlphaFold-predicted models (TM-score > 0.92) and molecular docking identify superior retinoid-binding affinity (ΔG = −12.27 kcal/mol), while spectral specificity (550–560 nm) and acid-stable photoresponse highlight its adaptability for low-irradiance neuromodulation. These findings position HeAR as a precision optogenetic tool, circumventing spectral overlap with excitatory opsins and enabling sustained hyperpolarization with minimized phototoxicity. By bridging microbial energetics and optobioengineering, this work expands the archaeal rhodopsin toolkit and provides a blueprint for designing wavelength-optimized photoregulatory systems.
... Cryo-electron microscopy, a state-of-the-art technique, enables highresolution visualization of protein complexes in near-native states without requiring protein crystallization. [20][21][22] However, in mixture samples, scattered light from minerals interferes with the production of high-resolution images, making this technique unsuitable for analyzing biomineral proteins involved in organic-inorganic interactions. ...
Biominerals form via organic–inorganic interactions, but their molecular mechanisms remain unclear. Solution NMR with dispersive mineral particles enables conformational analysis, providing insights for biomimetic mineral synthesis.
... The chromophore of bR monomer is a retinal moiety attached to a highly conserved lysine residue (Lys216) at the middle of the seventh helix (G) via a protonated Schiff-base linkage. The lipids connect three bR monomers to form trimers assembled in two-dimensional hexagonal crystalline structure in the form of a circular purple patches called purple membrane (PM) [11][12][13] . The contributing role of lipid-protein beside to the protein-protein interactions in connecting the bR trimer should be regarded not only in the structure of bR, but also in its function 11,12 . ...
The bacteriorhodopsin of purple membrane is the first discovered light-sensing protein among ion transporting microbial rhodopsins, some of which (e.g. Archaerhodopsin 3) could be broadly used as tools in optogenetics having wide potential of medical applications. Since its discovery as early as in 1971, bacteriorhodopsin has attracted wide interests in nano-biotechnology, particularly in optoelectronics devices. Therefore, the present work has been motivated due to two topics; firstly, anisotropy demand became indispensible in bioelectronics; secondly, the stationary level of electric response in bacteriorhodopsin within the pH range of proton pumping (pH 3 – pH 10) implies, in turn, raising here a question about whether the electric anisotropy is implicated for reducing (or switching off) such level beyond such pH range. Noteworthy is that the purple membrane converts to blue form upon acidification, while to reddish purple form upon alkalization. In the present study, the acidic and alkaline forms of bacteriorhodopsin have exhibited most probable state of reversal for the dielectric anisotropy around pH 2.5 and pH 10.5, respectively. This is underscored by proposing a correlation seemingly found between disassembly of the crystalline structure of bacteriorhodopsin and the reversal of dielectric anisotropy, at such acidic and alkaline reversal pH’s, in terms of the essence of the crystalline lattice. Most importantly, the results have substantiated dual frequency characteristics and logic gate-based dielectric anisotropy reversal to bacteriorhodopsin, which may implicate it for potential applications in bioelectronics.
Supplementary Information
The online version contains supplementary material available at 10.1038/s41598-024-80512-0.
... Life arose in aqueous environments and removing water from the biological samples normally destroys the sample's structural integrity. Early landmark successes in cryogenic EM were achieved by low-dose imaging of 2D crystals like purple membrane and porins that are sometimes found naturally in cell membranes of and which samples could be embedded in sugars for structural preservation [Unwin 1975;Sass 1989;Henderson 1990]. It was especially the remarkable discovery of the "vitreous-ice" state of water which enabled the direct visualization of individual biological complexes in their hydrated state, within the EM [Fernandez Moran 1960;Taylor 1976;Chanzy 1976;Brüggeller 1980;Dubochet 1981;Jeng 1984;Chiu 1986], and of the "freeze-plunging" specimen-preparation technique [Adrian1984; Dubochet 1988] that gave rise to today's Cryo-EM routine. ...
Electron microscopy has been an important method for visualising biological structures and processes since the 1940s. The discovery of a practical vitreous-ice specimen-preparation technique in the mid-1980s [Adrian 1984] led to modern-day Cryogenic Electron Microscopy (Cryo-EM) which in recent years has become a major technique for studying the architecture of biological macromolecules. Many further instrumental and data-analysis improvements were established in the decades after the introduction of the “vitreous-ice” state of water. Especially the advent of direct electron detectors boosted the quality of the recorded data, allowing atomic-resolution information of biological complexes to be harvested in the early 2010s, developments that truly revolutionized the use of Cryo-EM in structural biology. Single-Particle Analysis (SPA) of isolated molecules, prepared in a thin layer of vitreous water, has proven a most successful approach in structural biology and now often supplants the use of classical techniques like X-ray crystallography, especially for large biological complexes. The ever-increasing number of researchers using Cryo-EM is reflected by the growing number of depositions in the Electron Microscopy Data Bank (EMDB). Explaining this methodology to a new generation of researchers has now become a priority. In writing this review we were reminded of some persistent confusions that emerged in the early days of Cryo-EM but that continue to muddle the field. A new problem with the prolific use of Graphic User Interfaces (GUIs), is that the underlying methodology is often no longer transparent to the users of these “black boxes”. Complicated procedures, well hidden behind a GUI window, may contain methodological flaws that the user must be aware of. The conquering of markets in this booming Cryo-EM field – crucial for developing new pharmaceuticals – must not prevail over scientific integrity. We here describe and critically review the principles of single-particle Cryo-EM. We warn for procedures that have gone astray and could generate serious problems especially in the quality-control of Single-Particle Cryogenic Electron Microscopy.
... Life arose in aqueous environments and removing water from the biological samples normally destroys the sample's structural integrity. Early landmark successes in cryogenic EM were achieved by low-dose imaging of 2D crystals like purple membrane and porins that are sometimes found naturally in cell membranes of and which samples could be embedded in sugars for structural preservation [Unwin 1975;Sass 1989;Henderson 1990]. It was especially the remarkable discovery of the "vitreous-ice" state of water which enabled the direct visualization of individual biological complexes in their hydrated state, within the EM [Fernandez Moran 1960;Taylor 1976;Chanzy 1976;Brüggeller 1980;Dubochet 1981;Jeng 1984;Chiu 1986], and of the "freeze-plunging" specimen-preparation technique [Adrian1984; Dubochet 1988] that gave rise to today's Cryo-EM routine. ...
Electron microscopy has been an important method for visualising biological structures and processes since the 1940s. The discovery of a practical vitreous-ice specimen-preparation technique in the mid-1980s [Adrian 1984] led to modern-day Cryogenic Electron Microscopy (Cryo-EM) which in recent years has become a major technique for studying the architecture of biological macromolecules. Many further instrumental and data-analysis improvements were established in the decades after the introduction of the “vitreous-ice” state of water. Especially the advent of direct electron detectors boosted the quality of the recorded data, allowing atomic-resolution information of biological complexes to be harvested in the early 2010s, developments that truly revolutionized the use of Cryo-EM in structural biology. Single-Particle Analysis (SPA) of isolated molecules, prepared in a thin layer of vitreous water, has proven a most successful approach in structural biology and now often supplants the use of classical techniques like X-ray crystallography, especially for large biological complexes. The ever-increasing number of researchers using Cryo-EM is reflected by the growing number of depositions in the Electron Microscopy Data Bank (EMDB). Explaining this methodology to a new generation of researchers has now become a priority. In writing this review we were reminded of some persistent confusions that emerged in the early days of Cryo-EM but that continue to muddle the field. A new problem with the prolific use of Graphic User Interfaces (GUIs), is that the underlying methodology is often no longer transparent to the users of these “black boxes”. Complicated procedures, well hidden behind a GUI window, may contain methodological flaws that the user must be aware of. The conquering of markets in this booming Cryo-EM field – crucial for developing new pharmaceuticals – must not prevail over scientific integrity. We here describe and critically review the principles of single-particle Cryo-EM. We warn for procedures that have gone astray and could generate serious problems especially in the quality-control of Single-Particle Cryogenic Electron Microscopy.
... The first and by far the most extensively used type of proton pump in this process is bacteriorhodopsin (bR), originating from the archaea Halobacterium salinarium. [14,15] In 1974, Racker and Stoeckenius were the first to demonstrate ATP formation under light illumination by reconstituting a vesicle with a protein fraction containing mitochondrial ATP synthase and the bR-containing purple membrane. [16] Following this study, numerous researchers have used the combination of bR and ATP synthase, mainly to explore ways to increase ATP production efficiency or to utilize produced ATP in various cellular functions ( Table 1). ...
Autonomous generation of energy, specifically adenosine triphosphate (ATP), is critical for sustaining the engineered functionalities of synthetic cells constructed from the bottom‐up. In this mini‐review, we categorize studies on ATP‐producing synthetic cells into three different approaches: photosynthetic mechanisms, mitochondrial respiration mimicry, and utilization of non‐conventional approaches such as exploiting synthetic metabolic pathways. Within this framework, we evaluate the strengths and limitations of each approach and provide directions for future research endeavors. We also introduce a concept of building ATP‐generating synthetic organelle that will enable us to mimic cellular respiration in a simpler way than current strategies. This review aims to highlight the importance of energy self‐production in synthetic cells, providing suggestions and ideas that may help overcome some longstanding challenges in this field.
... Previously, electron diffraction was primarily used for 2D crystals, which can be described as a single crystal layer embedded in a lipid bilayer or proteins forming sheets or tubular structures. 9 Early examples of electron diffraction usage include structures of bacteriorhodopsin 10,11 and aquaporin-0. 12 While these methods can result in high-resolution structures, the number of suitable samples forming such crystals remains extremely limited. ...
Microcrystal electron diffraction (MicroED) is an emerging structural technique in which submicron crystals are used to generate diffraction data for structural studies. Structures allow for the study of molecular-level architecture and drive hypotheses about modes of action, mechanisms, dynamics, and interactions with other molecules. Combining cryoelectron microscopy (cryo-EM) instrumentation with crystallographic techniques, MicroED has led to three-dimensional structural models of small molecules, peptides, and proteins and has generated tremendous interest due to its ability to use vanishingly small crystals. In this perspective, we describe the current state of the field for MicroED methodologies, including making and detecting crystals of the appropriate size for the technique, as well as ways to best handle and characterize these crystals. Our perspective provides insight into ways to unlock the full range of potential for MicroED to access previously intractable samples and describes areas of future development.
... Since the 1980s, nuclear magnetic resonance (NMR) spectroscopy techniques have also contributed thousands of structures, albeit largely limited to relatively small (and soluble) molecules. Electron diffraction was already used in the 1970s to investigate the structure of an integral membrane protein, bacteriorhodopsin, although an atomic model was not described until 1990 (Henderson et al., 1990). During the 1980s, pioneers in the cryogenic-specimen electron microscopy (cryoEM) field developed experimental and computational methods (notably, embedding specimens in vitreous ice, low-dose electron imaging and detection, and particle reconstruction from projection images) which enabled increasingly high-resolution studies of a variety of biological specimens, and eventually, in what has been termed the 'resolution revolution' (Kü hlbrandt, 2014), atomistic modelling. ...
In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for the deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop’s motivation and history, the topics discussed, and the resulting consensus recommendations. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.
... [138][139][140][141] Flash-freezing protocols for cryogenic electron microscopy (EM) that vitrify water and preserve the structure of biomolecules in cells and viruses enabled investigations of biological samples at atomic resolution. [142][143][144][145][146][147] Nowadays, AI-based structure prediction increasingly complements high-resolution EM imaging of cell structures, besides viruses, and enhances the reconstruction of previously intractable macromolecular complexes, such as the nuclear pore complex. 148,149 EM image analysis problems can in principle be tackled using the same approaches as in fluorescence microscopy. ...
Despite advances in virological sciences and antiviral research, viruses continue to emerge, circulate, and threaten public health. We still lack a comprehensive understanding of how cells and individuals remain susceptible to infectious agents. This deficiency is in part due to the complexity of viruses, including the cell states controlling virus-host interactions. Microscopy samples distinct cellular infection stages in a multi-parametric, time-resolved manner at molecular resolution and is increasingly enhanced by machine learning and deep learning. Here we discuss how state-of-the-art artificial intelligence (AI) augments light and electron microscopy and advances virological research of cells. We describe current procedures for image denoising, object segmentation, tracking, classification, and super-resolution and showcase examples of how AI has improved the acquisition and analyses of microscopy data. The power of AI-enhanced microscopy will continue to help unravel virus infection mechanisms, develop antiviral agents, and improve viral vectors.
... These programs continue to be useful for calculating starting models and helical parameters from structures to be calculated by single-particle processing. The 2D crystal processing programs have also been maintained in their original form (Henderson et al., 1990), but many of them have been incorporated into or improved in the 2dx package (Gipson et al., 2007). Ximdisp (Smith, 1999) has been extensively modified and now includes editing a stack of images (Bö ttcher et al., 1997) displayed as a gallery (Fig. 1), boxing of helical filaments (Short et al., 2016) (Fig. 2), improvements to spline fitting a helical filament, new colour tables for display, interactive Fourier transform display from any section from a stack using the scrolling section option and numerous other options. ...
The great success of single-particle electron cryo-microscopy (cryoEM) during the last decade has involved the development of powerful new computer programs and packages that guide the user along a recommended processing workflow, in which the wisdom and choices made by the developers help everyone, especially new users, to obtain excellent results. The ability to carry out novel, non-standard or unusual combinations of image-processing steps is sometimes compromised by the convenience of a standard procedure. Some of the older programs were written with great flexibility and are still very valuable. Among these, the original MRC image-processing programs for structure determination by 2D crystal and helical processing alongside general-purpose utility programs such as Ximdisp, label, imedit and twofile are still available. This work describes an updated version of the MRC software package (MRC2020) that is freely available from CCP-EM. It includes new features and improvements such as extensions to the MRC format that retain the versatility of the package and make it particularly useful for testing novel computational procedures in cryoEM.
Using a nanoelectrospray ion source as a vapour deposition device, it has been possible to synthesise an environment in which large, transportable lipid bilayer membranes with integrated membrane proteins can assemble. Homogeneous membranes 3 mm in diameter were produced by integrating the monomolecular pore protein outer membrane porin G (OmpG) and the non-covalent trimer bacteriorhodopsin. Under these conditions, the large non-covalent 30 mer protein pore complex from listeriolysin O was able to form and insert itself into a membrane. Self-assembly occurs upon detergent extraction in a thin layer of glycerol formed on the liquid surface of a buffer solution in a small vessel. A layered environment was created by spraying different materials in several successive steps, with pauses inserted where self-assembly was required. First, a sufficient amount of phosphatidylcholine is sprayed onto the liquid surface of a buffer to generate a lipid bilayer. After several hours of assembly time, a thin glycerol layer is added to create a hydrophilic base. Then, enough phosphatidylcholine is added to enable the formation of a lipid monolayer. The next layer incorporates detergent-solubilised protein and additional lipids to enable the formation of a lipid bilayer with integrated proteins upon detergent extraction. Finally, another glycerol layer is added to complete the hydrophilic environment. The layered structure is then incubated for several days to allow the detergent to be extracted from the surface using SM-2 Biobeads. Using this technique, it may be possible to incorporate the biochemical properties of any membrane protein or protein complex in the form of membranes into electronic devices as has been achieved in MinION DNA sequencers. Biological membranes consist of many different molecules, primarily lipids and membrane proteins. In a biological context, the function of membrane proteins often depends on both the protein component and its lipid environment. This manuscript focuses primarily on the protein content of membranes, intentionally neglecting the lipid component of the physiological function of membrane proteins. This aspect is to be addressed in future research.
Photoorientation in motile fungal zoospores is mediated by rhodopsin guanylyl cyclases (RGCs). In certain chytrids, these photoreceptors form heterodimers consisting of a visible-light-absorbing RGC paired with neorhodopsin (NeoR), a rhodopsin distinguished by its unique spectral properties: far-red absorption and high fluorescence. Leveraging the native fluorescence of NeoR, we detected RGCs in living zoospores of the fungus Rhizoclosmatium globosum . The reversible photoswitching of bistable NeoR enabled super-resolution microscopy, facilitating single-molecule detection and quantification of NeoR proteins within individual zoospores. This approach also revealed the precise localization of RGCs within the rumposome, a chytrid-specific organelle hypothesized to mediate photoreception. Fluorescence tracking across different stages of the chytrid life cycle and the analysis of transcriptomic data confirmed that RGCs are predominantly present during the zoospore stage. Functional assays of recombinantly expressed RGC heterodimers with modified substrate specificity revealed that only one of the two pseudo-symmetric nucleotide-binding sites is catalytically active. Strikingly, disrupting nucleotide binding in the non-catalytic site enhanced light-triggered cyclase activity by up to ninefold, indicating an allosteric regulatory mechanism in heterodimeric RGCs.
Studies of membrane protein folding have progressed from simple systems such as bacteriorhodopsin to complex structures such as ATP-binding cassette transporters and voltage-gated ion channels. Advances in techniques such as single-molecule force spectroscopy and in vivo force profiling now allow for the detailed examination of membrane protein folding pathways at amino acid resolutions. These proteins navigate rugged energy landscapes partly shaped by the absence of hydrophobic collapse and the viscous nature of the lipid bilayer, imposing biophysical limitations on folding speeds. Furthermore, many transmembrane (TM) helices display reduced hydrophobicity to support functional requirements, simultaneously increasing the energy barriers for membrane insertion, a manifestation of the evolutionary trade-off between functionality and foldability. These less hydrophobic TM helices typically insert and fold as helical hairpins, following the protein synthesis direction from the N terminus to the C terminus, with assistance from endoplasmic reticulum (ER) chaperones like the Sec61 translocon and the ER membrane protein complex. The folding pathways of multidomain membrane proteins are defined by allosteric networks that extend across various domains, where mutations and folding correctors affect seemingly distant domains. A common evolutionary strategy is likely to be domain specialization, where N-terminal domains enhance foldability and C-terminal domains enhance functionality. Thus, despite inherent biophysical constraints, evolution has finely tuned membrane protein sequences to optimize foldability, stability, and functionality.
Ice lithography holds the potential to bridge cryogenic electron microscopy and electron-beam lithography and achieve direct high-precision functionalization of fragile biomaterials. Here we demonstrate that 5 keV electron irradiation of ethanol ice creates a material, patterned with <100 nm resolution, that is stable in the solid phase under ambient conditions. Employing the purple membrane from Halobacterium salinarum as a test target, we additionally show that the fabrication process results in minimal biomaterial mass loss. Ketene, an unstable intermediate, was identified in the irradiated ice via Fourier transform infrared spectroscopy and is likely an important factor triggering formation of the ethanol-based material. Surface-enhanced Raman spectroscopy and additional characterization methodologies revealed that the material contains disordered graphite similar to carbon fiber and is mechanically stiff and electrically insulating. This work demonstrates a novel material for additive manufacturing in general and for the precise functionalization of biological membranes in particular.
Electron cryomicroscopy (cryo‐EM) has evolved as a widely used approach to understand a range of structure‐function questions, particularly of membrane proteins. Studies by both electron crystallography and single particle analysis have provided a wealth of information on membrane transport proteins. Cryo‐EM methods with an emphasis on electron crystallography, which has yielded the most membrane transport protein structural information of any of the cryo‐EM techniques, are described here. Two‐dimensional crystallization approaches are outlined, as well as advances in cryo‐EM specimen preparation, data collection, and image processing. Examples of membrane transport protein structure described serve to illustrate some of the advances in both structural understanding and methods. Further examples outline impressive results that were obtained by a combination of electron crystallography and X‐ray crystallography as well as additional complementary methods. © 2012 American Physiological Society. Compr Physiol 2:283‐293, 2012.
Fourier transform infrared difference spectroscopy was used to study the effect of millimeter-range electromagnetic radiation on the structure of bacteriorhodopsin under lighting conditions. A detailed analysis of the Fourier IR spectra revealed pronounced structural changes in the region of amide I, amide II and rearrangement of the hydrogen bond network. The well-resolved peaks of the amide bands made it possible to recognize two different components (α-I and α-II) of the α-helical conformation of the opsin. Irreversible conformational changes of bacteriorhodopsin in purple membranes detected by Fourier IR difference spectroscopy suggested that microwaves induced structural rearrangements of proteins unrelated to temperature.
Structure-based drug design attempts to design small molecule ligands by docking them directly into the binding pocket of a target protein. This requires a 3D structure of the reference protein. The goal is to optimally fill the binding pocket with a ligand by satisfying non-bonded interactions with the functional groups of the binding site residues. The structure-based design process starts with a detailed analysis of the binding pocket to elucidate the hot spots for the putative interactions with the protein. Either experimental methods or computational tools can be used to perform a mapping of the active site using molecular probes or small solvent-like molecules. In an iterative process of structure determination, modeling of modified ligands, docking and screening, synthesis, and biological testing, the properties of small molecule ligands are refined to optimize their binding to the target protein. Databases have been developed to retrieve and compare structural information from the exponentially growing body of structural data on protein-ligand complexes. They allow comparison of binding poses, active site interaction geometries, protein-ligand binding motifs, and the original solvation structures in the binding pocket of the protein. Proteins can be compared in terms of their exposed binding pockets. The shape and exposure of groups with special physicochemical properties in binding pockets are compared and help to design small molecule ligands with the desired selectivity. Ideas for isosteric replacements on the ligand scaffold can also be generated in this way. If an experimentally determined structure of the target protein is not available, a homology model can be constructed using a related protein of known architecture as a template. The accuracy and success of such a homology model is strongly dependent on the homology of the sequence to the structure of the template. Tools for secondary structure prediction and amino acid substitution propensities have been developed to improve the reliability of the sequence assignment of proteins to the 3D structure of the reference template. Recently, AI-programs have been developed based on neural networks. They were trained to learn structure predictions based on experimentally determined protein structures. De novo design approaches start with a small molecule seed or fragment and grow them into putative ligands in the binding pocket. Two non-overlapping fragments can also be linked together to form a larger ligand with improved binding properties. In all structure-based design strategies, the geometry of a constructed protein-ligand complex must be evaluated in terms of expected binding affinity. A variety of scoring functions are used to predict binding affinity based on the geometry of the formed complex.
https://sn.pub/0alfsy
CAPRI challenges offer a range of blind tests for biomolecule interaction prediction. This study evaluates the performance of our prediction protocols for the human group Zou and the server group MDockPP in CAPRI rounds 47–55, highlighting the impact of AlphaFold2 (AF2) and the effectiveness of massive sampling approaches. Prior to AlphaFold2's release, our methods relied on homology modeling and docking‐based protocols, achieving limited accuracy due to constraints in structural templates and inherent docking limitations. After AlphaFold2's public release, which demonstrated breakthrough accuracy in protein structure prediction, we integrated its multimer models and massive sampling techniques into our protocols. This integration significantly improved prediction accuracy, with human predictions increasing from 1 correct interface of 19 pre‐AlphaFold2 to 4 of 8 post‐AlphaFold2. The massive sampling approach further enhanced performance, particularly for targets T231 and T233, yielding medium‐quality models that default parameters could not achieve.
Microcrystal electron diffraction (MicroED) has advanced structural methods across a range of sample types, from small molecules to proteins. This cryogenic electron microscopy (cryo-EM) technique involves the continuous rotation of small 3D crystals in the electron beam, while a high-speed camera captures diffraction data in the form of a movie. The crystal structure is subsequently determined by using established X-ray crystallographic software. MicroED is a technique still under development, and hands-on expertise in sample preparation, data acquisition and processing is not always readily accessible. This comprehensive guide on MicroED sample preparation addresses commonly used methods for various sample categories, including room temperature solid-state small molecules and soluble and membrane protein crystals. Beyond detailing the steps of sample preparation for new users, and because every crystal requires unique growth and sample-preparation conditions, this resource provides instructions and optimization strategies for MicroED sample preparation. The protocol is suitable for users with expertise in biochemistry, crystallography, general cryo-EM and crystallography data processing. MicroED experiments, from sample vitrification to final structure, can take anywhere from one workday to multiple weeks, especially when cryogenic focused ion beam milling is involved.
Fourier transform infrared (FTIR) difference spectroscopy was used to study the effects of microwaves radiation on the structure of bacteriorhodopsin under light condition. The detailed FTIR spectral analysis revealed the pronounced structural changes in amide I and amide II regions as well as the rearrangements of the hydrogen-bonding network. Well-resolved peaks of amide bands allow accurate determination of two different components (α-I and α-II) of an α-helical conformation of opsin. Irreversible conformational changes of bacteriorhodopsin in purple membranes, detected by FTIR difference spectroscopy, suggest that regardless of temperature, microwaves induce protein structural rearrangements.
Bacteriorhodopsin (bR), a pivotal light-driven proton pump intricately associated with the purple membrane (PM) and lipids, demonstrates considerable promise for diverse applications in nanotechnology and technical fields, owing to its distinctive crystalline structure. The removal of lipids from bR is often imperative, contingent upon the specific research objectives, with established methodologies typically relying on chemical detergents like CHAPS, Sodium Cholate, and Triton X-100. In this innovative investigation, we introduce chitosan as a novel delipidation agent, capitalizing on its sustainable, eco-friendly, and cost-effective characteristics, notably its capacity for cationic lipid binding. Derived from chitin, a component of crustaceans, chitosan emerges as a versatile biomaterial with extensive utility. Through a meticulous review of the literature, we present the inaugural utilization of chitosan for bR delipidation. The purification of bR was accomplished utilizing an aqueous two-phase system (ATPS), with the efficacy of lipid removal verified through meticulous analyses employing SDS-Page and UV–Vis Spectrum techniques. The successful application of chitosan in bR delipidation not only underscores its potential as a sustainable alternative to traditional chemical detergents but also prompts further exploration into biomaterial-based nanotechnological pursuits, thus broadening the horizons of scientific inquiry and innovation.
Multiple proton transfers in cyclic homogenous and mixed H2O‐H2S clusters from trimers to pentamers have been investigated at an affordable and accurate level of theory. Reaction force and Wiberg bond index analyses showed the process to be nearly synchronous in all the homogenous clusters, while asynchronous in the mixed clusters; always initiated by movement of the SH protons to the H‐bonded O atoms. The barriers for proton transfers exhibited a complex pattern, decreasing initially from the homogeneous H2O cluster upon replacement of one H2O by an H2S moiety and subsequently rising monotonically with each subsequent replacement(s) to becoming highest in pure H2S clusters. Finally, the impact of the energetic and mechanistic modulations on the proton transfer kinetics has been studied.
Extremophile and extremozyme capabilities to uphold catalytic actions under extreme situations open up a varied array of biotechnological applications. Extremophiles are a rich supply of biocatalysts used for innumerable purposes. Bioactive molecules and enzymes isolated from organisms inhabiting risky environments being used in biological innovation pipelines and pharmaceutical have positive claims. The species biodiversity has favourable reservoir of the unexploited amalgams with biotechnological significance. Prospective solicitations of extremozymes, chiefly as catalysis of multistep progressions, quorum sensing, bioremediation, biofuel, biodiversity and prospecting, biomining, and genetic technology are explored. To boost the biotechnological uses of extremozymes, research and development efforts are needed to address hurdles such as extremophile culture, gene expression in host cells, and extremozyme bioprocessing. Extremophiles can be a resource for innovative biotechnological comprising industrial biotechnology, agriculture, medical, food, and environmental biotechnology.
Structure-based drug design (SBDD) has gained popularity owing to its ability to develop more potent drugs compared to conventional drug-discovery methods. The success of SBDD relies heavily on obtaining the three-dimensional structures of drug targets. X-ray crystallography is the primary method used for solving structures and aiding the SBDD workflow; however, it is not suitable for all targets. With the resolution revolution, enabling routine high-resolution reconstruction of structures, cryogenic electron microscopy (cryo-EM) has emerged as a promising alternative and has attracted increasing attention in SBDD. Cryo-EM offers various advantages over X-ray crystallography and can potentially replace X-ray crystallography in SBDD. To fully utilize cryo-EM in drug discovery, understanding the strengths and weaknesses of this technique and noting the key advancements in the field are crucial. This review provides an overview of the general workflow of cryo-EM in SBDD and highlights technical innovations that enable its application in drug design. Furthermore, the most recent achievements in the cryo-EM methodology for drug discovery are discussed, demonstrating the potential of this technique for advancing drug development. By understanding the capabilities and advancements of cryo-EM, researchers can leverage the benefits of designing more effective drugs. This review concludes with a discussion of the future perspectives of cryo-EM-based SBDD, emphasizing the role of this technique in driving innovations in drug discovery and development. The integration of cryo-EM into the drug design process holds great promise for accelerating the discovery of new and improved therapeutic agents to combat various diseases.
A handful of biological proton-selective ion channels exist. Some open at positive or negative membrane potentials, others open at low or high pH, and some are light activated. This review focuses on common features that result from the unique properties of protons. Proton conduction through water or proteins differs qualitatively from that of all other ions. Extraordinary proton selectivity is needed to ensure that protons permeate and other ions do not. Proton selectivity arises from a proton pathway comprising a hydrogen-bonded chain that typically includes at least one titratable amino acid side chain. The enormously diverse functions of proton channels in disparate regions of the phylogenetic tree can be summarized by considering the chemical and electrical consequences of proton flux across membranes. This review discusses examples of cells in which proton efflux serves to increase pH i , decrease pH o , control the membrane potential, generate action potentials, or compensate transmembrane movement of electrical charge.
Expected final online publication date for the Annual Review of Physiology, Volume 86 is February 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Science does not take place in a vacuum: The physical and social workplace has a profound influence on scientific discoveries. Everyone at a research institute can contribute to its scientific output and productivity, from faculty research groups to facilities and platforms staff to administration and corporate services. Although the researchers addressing exciting scientific questions are key, their efforts can be fostered and directed by the overarching strategy of the institute, interconnection with facilities and platforms, and strong and directed support of the administration and corporate services. Everybody counts and everybody should be empowered to contribute. But what are the characteristics that make scientific organizations and their people flourish? This Essay looks at the structure and culture of successful research institutes, laying out different operational strategies and highlighting points that need be taken into consideration during their implementation.
To study their role in the structure and function of bacteriorhodopsin, three prolines, presumed to be in the membrane-embedded α-helices, have been individually replaced as follows: Pro-50 and Pro-91 each by Gly and Ala and Pro-186 by Ala, Gly, and Val. The mutants of Pro-50 and Pro-91 all showed normal chromophore and proton pumping. However, the rates of regeneration of the chromophore in Pro-50→Ala, Pro-91→Ala and →Gly with all-trans-retinal were about 30-fold slower than that in the wild-type, whereas the chromophore regeneration rate in Pro-50→Gly was 10-fold faster than in the wild-type. While, Pro-186→Ala regenerated the wild-type chromophore, the mutants Pro-186→Val and Pro-186→Gly showed large blue shifts (about 80 nm) in the chromophore regenerated with all-trans-retinal and showed no apparent dark-light adaptation. Pro-186→Gly first regenerated the wild-type chromophore with 13-cis-retinal which was thermally unstable and rapidly converted to the blue-shifted chromophore obtained with all-trans-retinal. High salt concentration restored the wild-type purple chromophore in the Pro-186→Gly mutant. Thus, in this mutant, the protein interconverts between two conformational states. Pro-186→Ala and Pro-186→Gly showed about 65%, whereas Pro-186→Val showed 10–20% of the normal proton pumping.
We have individually replaced all 7 of the arginine residues in bacteriorhodopsin by glutamine. The mutants with substitutions at positions 7, 164, 175, and 225 showed essentially the wild-type phenotype in regard to chromophore regeneration, chromophore λmax, and proton pumping, although the mutant Arg-175→Gln showed decreased rate of chromophore regeneration. Glutamine substitutions of Arg-82, −134, and −227 affected proton pumping ability, and caused specific alterations in the bacteriorhodopsin photocycle. Finally, electrostatic interactions are proposed between Arg-82 and −227, and specific carboxylic acid residues in helices C and G, which regulate the purple to blue transition and proton transfers during the photocycle.
Bacteriorhodopsin contains 8 tryptophan residues distributed across the membrane-embedded helices. To study their possible functions, we have replaced them one at a time by phenylalanine; in addition, Trp-137 and −138 have been replaced by cysteine. The mutants were prepared by cassette mutagenesis of the synthetic bacterio-opsin gene, expression and purification of the mutant apoproteins, renaturation, and chromophore regeneration. The replacement of Trp-10, Trp-12 (helix A), Trp-80 (helix C), and Trp-138 (helix E) by phenylalanine and of Trp-137 and Trp-138 by cysteine did not significantly alter the absorption spectra or affect their proton pumping. However, substitution of the remaining tryptophans by phenylalanine had the following effects. 1) Substitution of Trp-86 (helix C) and Trp-137 gave chromophores blue-shifted by 20 nm and resulted in reduced proton pumping to about 30%. 2) As also reported previously (Hackett, N. R., Stern, L. J., Chao, B. H., Kronis, K. A., and Khorana, H. G. (1987) J. Biol. Chem. 262, 9277–9284), substitution of Trp-182 and Trp-189 (helix F) caused large blue shifts (70 and 40 nm, respectively) in the chromophore and affected proton pumping. 3) The substitution of Trp-86 and Trp-182 by phenylalanine conferred acid instability on these mutants. The spectral shifts indicate that Trp-86, Trp-182, Trp-189, and possibly Trp-137 interact with retinal.
It is proposed that these tryptophans, probably along with Tyr-57 (helix B) and Tyr-185 (helix F), form a retinal binding pocket. We discuss the role of tryptophan residues that are conserved in bacteriorhodopsin, halorhodopsin, and the related family of opsin proteins.
Halobacterium sp. GRB (Ebert, K., Goebel, W., and Pfeifer, F. (1984) Mol. & Gen. Genet. 194, 91–97) was used to isolate bacteriorhodopsin (BR) mutants. A procedure is described which allows the enrichment of any type of mutant unable to grow under the selection conditions applied. Its use for the isolation of phototrophically negative, retinal-positive mutants of Halobacterium sp. GRB is demonstrated. Single cell clones of this phenotype were further characterized. The expression of bacterioopsin was tested with a monoclonal antibody directed against the C terminus of the protein. The expressed bacteriorhodopsins were characterized by their specific activity for proton pumping, their spectral properties, and photocycle kinetics. About 15 independent mutants carrying bacteriorhodopsins of three distinct phenotypic classes could be isolated, including BR with a different absorption maximum, BR of lower specific activity, and BR characterized by a slower photocycle and a lack of proton pumping activity.
The bacterioopsin genes of Halobacterium sp. GRB (Ebert, K., Goebel, W., and Pfeifer, F. (1984) Mol. & Gen. Genet. 194, 91–97) wild type and 10 independent mutants of different phenotypes have been cloned and sequenced. The wild type gene has two conservative changes compared to the gene of Halobacterium halobium, so that the proteins of the two species are identical. Six different mutations at five different codons have been found, leading to the following amino acid changes compared to the wild type: Trp¹⁰ → Cys (three cases), Tyr⁵⁷ → Asn, Asp⁸⁵ → Glu, Asp⁰⁶ → Asn (three cases), Asp⁹⁶ → Gly, Trp¹³⁸ → Arg. A first characterization of the mutant proteins is given, and their implications for models of bacteriorhodopsin structure and function are discussed.
Bacteriorhodopsins (BRs) containing retinal, 13-ethylretinal, 13-methoxyretinal, and 13-demethylretinal were investigated flash spectroscopically and photoelectrically. In all preparations, bathochromic absorption bands occurred within less than 5 ns although the analogue BRs, due to the chemical structure of the retinal compounds, were in an invariant 13-cis or all-trans form. The lifetimes of these K states varied over 6 orders of magnitude. Photoelectric measurements demonstrated that a rapid charge separation process occurred within less than 100 ps for all preparations. The magnitude and the direction of the charge separation process were nearly the same for all BRs, except of that of 13-demethylretinal-containing BR, which under identical conditions exhibited at 3 times larger amplitude. Thus, an early charge separation connected with a red shift of the absorption maximum is the common consequence of light absorption. This process takes place, regardless of whether the chromophore is originally bound to the protein in the all-trans configuration with the anti-geometry for the Schiff base group or in the 13-cis configuration with the syn geometry for the Schiff base group. The K intermediates form as the result of the disruption of salt bridges between the protonated Schiff bases and their unprotonated counterions for the cis or trans states, respectively, and the subsequent association of the Schiff base groups to another protonated group. On the basis of the generally accepted mechanism, a possible explanation for the exclusively trans-coupled proton translocation and the malfunction of the cis cycle is discussed.
The contrast in high resolution electron micrographs of three different thin crystals has been compared quantitatively with that predicted theoretically from separate measurements of thier electron diffraction patterns. The crystals were vermiculite, a mineral which is not greatly affected by the electron beam, and two organic specimens, n-paraffin and purple membrane, which are both destroyed by doses of about 1 electron/Å2. The results, all at 4.0 to 4.5 Å resolution, show that the absolute contrast in images of vermiculite is roughly 1/5th of that expected for a theoretically perfect microscope, whereas images of paraffin and purple membrane seldom reach more than 1/25th of theoretical contrast. Much of this loss of contrast can be explained on the basis of known microscope parameters in the case of the non-beam-sensitive specimens. However, for the images of paraffin and purple membrane, it is necessary to postulate that beam-induced specimen movement results in further substantial blurring of the image. The tendency for such movement to occur may be unavoidable since the molecular structure is being destroyed during the exposure. The magnitude of this movement must be reduced before the image contrast will be able to approach the theoretical limit.
The orientation of bacteriorhodopsin in the purple membrane of Halobacterium halobium has been studied by proteolytic degradation of purple membrane sheets, reconstituted vesicles, and whole cells, with the following results: (i) Bacteriorhodopsin in purple membrane sheets is cleaved at a single site by Pronase or trypsin; a polypeptide segment of about 15 amino acids is lost from the carboxyl end. Carboxypeptidase A sequentially releases amino acids from the carboxyl end; the tetrapeptide sequence -Ala-Ala-Thr-Ser(COOH) was tentatively deduced for this terminus. (ii) The apomembrane, which lacks retinal, undergoes a second cleavage with trypsin releasing a fragment of approximately 6300 molecular weight from the amino terminus. (iii) Vesicles reconstituted from the purple membrane sheets and synthetic lecithins, in which the direction of proton pumping is opposite to that in the whole cells, have the carboxyl terminus of bacteriorhodopsin accessible to proteolysis. (iv) In envelope vesicles, which largely pump protons in the same direction as the whole cells, the carboxyl terminus is largely protected against proteolysis. (v) Treatment of whole cells with proteinase K hydrolyzes the cell wall proteins but has no effect on acteriorhodopsin. However, the same treatment after lysis of the cells results in degradation of the hydrophilic region at the carboxyl terminus. The results show that the carboxyl terminus as well as the additional cleavage site near the amino terminus observed in apomembrane are on the cytoplasmic side of the purple membrane.
Likely mechanisms for proton transport through biomembranes are explored. The fundamental structural element is assumed to be continuous chains of hydrogen bonds formed from the protein side groups, and a molecular example is presented. From studies in ice, such chains are predicted to have low impedance and can function as proton wires. In addition, conformational changes in the protein may be linked to the proton conduction. If this possibility is allowed, a simple proton pump can be described that can be reversed into a molecular motor driven by an electrochemical potential across the membrane.
The complete primary structure of the purple membrane protein bacteriorhodopsin, which contains 248 amino acid residues, has been determined. Methods used for separation of the hydrophobic fragments included gel permeation and reverse-phase high-pressure liquid chromatography in organic solvents. The amino acid sequence was determined by a combination of automatic Edman degradation and mass spectrometric methods. The total sequence was derived by ordering of the CNBr fragments on the basis of methionine-containing peptides identified by gas chromatographic mass spectrometry and by analysis of N-bromosuccinimide fragments containing overlaps between CNBr fragments. The present sequence differs from that recently reported by Ovchinnikov and coworkers with respect to an additional tryptophan (position 138) and several amino acid assignments.
Bacteriorhodopsin, a membrane protein from Halobacteria, forms two-dimensional crystals which often have diameters of 20 microns. Several crystal forms have been obtained with cell dimensions of about 60 Å and diffraction to beyond 3 Å resolution. The structure of one of these crystal forms has recently been determined in projection at 3.5 Å resolution using images recorded at low temperature, together with computer image analysis methods that enable the averaging of information from areas of up to 1 micron in diameter. Electron diffraction intensities have also been recorded from tilted and untilted specimens providing three-dimensional amplitude measurements to 3 Å resolution. We are now analysing images from tilted specimens that should provide phases for the Fourier components of the structure to high resolution in three dimensions.
Correlation and averaging methods have made it possible to extend electron microscopic structure research to distorted crystals and nonperiodic objects. With the recent introduction of multivariate data analysis methods, molecule projections imaged with the electron microscope can be objectively divided into classes, according to significant structural differences. The projections within each class can be separately averaged and characterized. This new technique has the potential to become an analytical tool in molecular biology that discriminates on the basis of structural differences at the quaternary level.
High resolution images of thin paraffin crystals and of purple membrane (i.e., crystalline bacteriorhodopsin) have been recorded with illumination spots that are confined to a size of 1000 to 2000 Å. Images recorded in this way show three to five times greater contrast than do images which are recorded with more conventional flood-beam (ca. 3 μm diameter) illumination. In addition, optical diffraction patterns of small areas of such images show much better preservation of contrast in all directions than is normally the case. Both effects substantiate the idea that beam-induced movement of radiation-sensitive organic specimens has been a major factor which has caused low dose images to be greatly inferior in their quality compared to electron diffraction patterns of the same type of specimens. In spite of the marked improvement that is obtained, we often observe an unexplained degree of contrast variation within the small illuminated area, demonstrating that even further improvement in the quality of low dose images should be possible.
A high resolution, liquid nitrogen-cooled specimen stage has been designed and constructed for use on the JEOL 100B and 100C electron microscopes. This stage will be useful for imaging biological macromolecular arrays in the frozen-hydrated or glucose-embedded states at low temperature. Images thus obtained should have an increased signal-to-noise ratio due to the radiation damage protection offered by low temperature.
High-resolution, solid-state {sup 15}N NMR has been used to study the chemical shift anisotropies of the Schiff bases in bacteriorhodopsin (bR) and in an extensive series of model compounds. Using slow-spinning techniques, the authors are able to obtain sufficient rotational sideband intensity to determine the full {sup 15}N chemical shift anisotropy for the Schiff base nitrogen in bR{sub 548} and bR{sub 568}. Comparisons are made between all-trans-bR{sub 568} and N-all-trans-retinylidene butylimine salts with halide, phenolate, and carboxylate counterions. It is argues that for the model compounds the variation in {sup 15}N chemical shift reflects the variation in (hydrogen) bond strength with the various counterions. The results suggest that carboxylates and tyrosinates may form hydrogen bonds of comparable strength in a hydrophobic environment. Thus, the hydrogen bonding strength of a counterion depends on factors that are not completely reflected in the solution pK{sub a} of its conujugate acid. For the model compounds, the two most downfield principal values of the {sup 15}N chemical shift tensor, {sigma}{sub 22} and {sigma}{sub 33}, vary dramatically with different counterions, whereas {sigma}{sub 11} remains essentially unaffected. In addition, there exists a linear correlation between {sigma}{sub 22} and {sigma}{sub 33}, which suggests that a single mechanism ismore » responsible for the variation in chemical shifts present in all three classes of model compounds. The data for bR{sub 568} follow this trend, but the isotropic shift is 11 ppm further upfield than any of the model compounds. This extreme value suggests an unusually weak hydrogen bond in the protein.« less
A survey of various types of superconducting lenses is given. Cryogenic problems, particularly material problems, are discussed which must be solved in order to construct lens systems applicable for electron microscopy. The results obtained up to now with the few superconducting lens systems in operation prove a better resolution than is achievable with conventional lenses. The specimen has a temperature of approximately 4 K since it is completely surrounded by walls directly cooled by liquid helium, and the stability of the superconducting lens system permits long exposure times of the micrographs which make it possible to minimize the heating of the object by the beam. For this reason due to cryoprotection one can take advantage of a drastic reduction of radiation damage by the beam.
Alignment by means of current commutating and defocusing of the objective does not yield the desired rotational symmetry of the imaging pencils. This was found while aligning a transmission electron microscope with a single field condenser objective. A series of optical diffractograms of micrographs taken under the same tilted illumination yet under various azimuths have been arranged in a tableau, wherein strong asymmetry is exhibited. Quantitative evaluation yields the most important asymmetric aberration to be the axial coma, which becomes critical when a resolution better than 5 Å is obtained. The tableau also allows an assessment of the three-fold astigmatism.
A procedure has been developed which aligns the microscope onto the coma-free and dispersion-free pencil axis and does not rely on current commutation. The procedure demand equal appearance of astigmatic carbon film images produced under the same tilt yet diametrical azimuth.
A model building and refinement system is described for use with a Vector General 3400 display. The system allows the user to build models using guide atoms and angles to arrive at the final conformation. It has been used to assist in difference Fourier map interpretation at medium and high resolution, and to build a protein molecule into a multiple isomorphous replacement phased electron density map.
Orientations of IR transition moments of the retinal chromophore of bacteriorhodopsin (BR) and of the apo-protein are investigated by FTIR linear dichroism and photoselection measurements. Low temperature difference spectra for the photoinduced transitions of BR to its photocycle intermediates K and L are evaluated using improved methods. Quantum chemical calculations of directions of IR and electronic transition moments of model chromophores are employed to analyze corresponding observations. The chromophore of light-adapted BR568 is shown to exhibit small (15–30°) twists around the CC single bonds of retinals polyene chain but no large overall helicity (⩽15°). The average retinal plane is demonstrated to form an angle of 90±20° with the plane of the purple membrane. The C9C10 double bond of retinal is found approximately parallel to the plane of the membrane. Upon photoisomerization the orientation of the chromophore moiety from C1 to C13 is estimated to be largely conserved. The single bond twists of the chromophore in L are shown to be larger than those in BR568. This result is in agreement with the previous prediction of increased single bond twists in L, which can cause a pK decrease of the chromophore and, thereby, enforce its deprotonation in the L→M transition [Schulten and Tavan, Nature, 272 (1978) 85].
Retention of known geometry, with regard to mean atomic positions, has proved useful in the refinement of macromolecules. In structures with a paucity of diffraction data and large displacements of the atoms from their mean positions, it is also of value to restrain the thermal factors to be consistent with known stereochemistry. This paper presents a technique for accomplishing this by restraining the variances of the interatomic distributions (which are functions of the mean atomic positions and the thermal parameters) to suitably small values. This procedure allows meaningful anisotropic refinement of macromolecules to be carried out with low-resolution diffraction data. Anisotropic thermal parameters obtained in this way should prove useful in understanding the dynamics of the biological functions of macromolecules.
Distortions of different origin in the images of two-dimensional crystal result in reconstruction procedures on large crystals not being likely to give better results than on relatively small crystals. Only if the specimen is exceptionally stable [1] can the processing of large arrays be successful.
A simple specimen holder is described for a Siemens electron microscope which will allow the specimen grid to be set at inclinations up to 75° to the electron beam in any azimuthal direction. This device is suitable for measuring tilted images for the three-dimensional reconstruction of crystalline specimens. A method is also described for calculating the tilt angles for such crystalline specimens by comparing the unit cell dimensions in tilted and untilted images.
The imaging of tilted objects in a CTEM is treated from the point of view of a posteriori correction (e.g. as preliminary step for 3D reconstruction). Strictly periodic as well as thin objects of a more general type are discussed. For weak thin objects a new a posteriori correction of tilt is proposed.
This chapter summarizes the information that can be used to identify the helical regions and attempt to improve the assignments based on several approaches. An important source of information is provided by the action of proteolytic enzymes on the bacteriorhodopsin structure. The underlying assumption in such studies is that the soluble enzymes will act to cleave only polypeptide links that are exposed to the aqueous environment. Consequently, one expects that such cleavage sites will be in connecting loops between helices and can, therefore, be used to identify the spacer regions between helices. In addition to proteolysis, information from other kinds of modification may be useful. One important result comes from the application of lactoperoxidase-catalyzed iodination. Researchers have used an immobilized lactoperoxidase molecule to modify purple membrane fragments. Subsequent analysis showed 75% of the radioactive iodine to be located in the cyanogen bromide fragment from amino acid 118 to 145. In this region only two tyrosines are present, tyrosine 131 and 133. Consequently, the expectation is that one or both of these tyrosines must be exposed at the aqueous surface of the bactcriorhodopsin molecule. Another modification of interest is the derivitization of the membrane by a biotinyl reagent that reacts with lysine amino groups. In this study it is thought that a single lysine was modified by the reagent in such a way as to permit binding of an avidin-fcrritin complex. The use of electron microscopy resulted in a determination that only one side of the membrane, that part oriented toward the outside of the plasma membrane of the Halobacterium, is labeled.
Resonance Raman spectra of the O640 photointermediate of bacteriorhodopsin have been obtained with a dual-beam, time-resolved technique. A flowing purple membrane suspension is first illuminated with a 514-nm laser beam to initiate photocycling. Raman scattering from O640 is excited 3-6 ms "downstream" with a probe beam at 752 nm. Raman spectra of the O640 intermediate have been obtained from native bacteriorhodopsin in H2O and D2O, as well as from bacteriorhodopsin regenerated with 15-deuterio- and 12,14-dideuterioretinals. O640 has a Schiff base line at 1628 cm-1, which shifts to 1589 cm-1 in D2O, demonstrating that the Schiff base is protonated. The pattern of vibrations in the 1100-1400-cm-1 fingerprint region of the O640 Raman spectrum is very similar to that observed in BR568, which contains an all-trans protonated Schiff base chromophore. Further-more, the frequency and intensity changes observed in the Raman spectra of 15-deuterio- and 12,14-dideuterio-O640 correspond closely with the deuteration-induced changes displayed by BR568. However, the changes observed upon deuteration of the chromophore in BR548, the 13-cis component of dark-adapted bacteriorhodopsin, are very different from those observed in O640. These comparisons demonstrate that the retinal chromophore in O640 has an all-trans configuration. Therefore, the M412 to O640 conversion involves 13-cis to all-trans chromophore isomerization and protonation of the retinal-lysine Schiff base. In addition, the O640 spectrum exhibits strong hydrogen out-of-plane wagging vibrations that are not seen in BR568. This indicates that the 13-trans chromophore in O640 is conformationally distorted.
The spatial organization and the antigenic structure of the bacteriorhodopsin molecule in the purple membrane were studied by immunochemical techniques. Five monoclonal antibodies directed against exposed parts of the protein molecule in the membrane were prepared and characterized. Antigenic determinants were localized in the bacteriorhodopsin polypeptide chain by analysis of the interaction between monoclonal antibodies and protein fragments. The structure of antigenic determinants was revealed by the interaction of monoclonal antibodies with (i) isolated bacteriorhodopsin fragments further modified by sequential Edman degradation and (ii) derivatives of bacteriorhodopsin obtained biosynthetically or by selective chemical modification. Five antigenic determinants were localized in the following parts of bacteriorhodopsin: < Glu'-Met20 involving one of the 3 amino acid residues of the N-terminal part; Gly33-Met56 involving Asp36 and/or Asp38 and Phe42; Phe156-Met163 involving Phe156; Glu194-Leu207 involving Glu194; Pro200-Leu207.
The three-dimensional structure of the deoxycholate-treated form of purple membrane has been determined to a resolution of about 6 . Using low temperature electron diffraction data, room temperature electron microscope images and improved methods of data analysis, higher resolution has been reached than was obtained using native membranes of the same size. Statistical analysis of the data shows that the new map is considerably better than earlier maps. The map indicates the probable sites for the lipid molecules that remain in the deoxycholate-treated membranes; some of these sites differ from those suggested by the projection map of Glaeser et al. (1985). Comparison of the bacteriorhodopsin structures now determined independently from three crystal forms shows that the monomer structure is independent of the detailed contacts with lipid molecules. The average of the three structures gives a picture with very little noise showing seven similar rod-like features which are clearly best interpreted as -helices; there is no indication that part of the structure is -sheet as suggested by Jap et al. (1983). Phases from the averaged structure at 6 resolution will enable better refinement of the parameters that will be required in the analysis of higher resolution images from tilted specimens needed to extend the projection map at 3.5 resolution (Henderson et al. 1986) to produce a three-dimensional atomic resolution map.
Electron micrographs of the purple membrane have been recorded using liquid nitrogen and liquid helium cooling on three cryoelectron microscopes. The best micrographs show optical diffraction spots, arising from the two-dimensional crystal, out to resolutions of around 6 Å. Large areas of several of these micrographs have been analysed using a procedure which determines the strength of the very weak high resolution Fourier components of the image of the crystal. The procedure consists of reciprocal space filtering followed by real space correlation analysis to characterise image distortions, removal of the distortions by interpolation, and finally extraction of the amplitudes and phases of the Fourier components from the distortion-corrected image of the crystal. These raw image amplitudes and phases are then used, together with previously measured amplitude and phase information, to refine the beam tilt and crystal tilt, phase origin and amount of defocus and astigmatism of the image. The phases can then be corrected for the effects of the contrast transfer function, beam tilt and phase origin. The amplitudes of all the spots which are expected to be strong from their known electron diffraction intensity are observed to be significantly above the background noise level, and the independent phases from different images, and from symmetry-related directions in the same image, show excellent agreement out to a resolution of 3.5 Å. Although only images from untilted or slightly tilted ( < 5°) crystals have been analysed using the procedure described in this paper, a simple additional step enables analysis of images at any tilt angle, providing a complete practical method for high resolution analysis of images of two-dimensional crystalline arrays.
Electron diffraction patterns from two different crystal forms of purple membrane have been obtained. In both cases, the diffraction spots extend to beyond 3.0 Å resolution. Using specimens tilted at angles up to 60°, the three-dimensional intensities were recorded as a large number of two-dimensional patterns. We describe here the procedures used to record and digitise the patterns, and subsequently to measure and combine the individual spot intensities to produce a complete merged, refined and evaluated three-dimensional diffraction pattern. Approximately 100 patterns were required for adequate sampling in each crystal form.
Two-step excitation of retinal in bacteriorhodopsin by visible light is followed by an energy transfer to amino acids that is seen as fluorescent emission around 350 nm. The fluorescence spectrum obtained after two-step excitation (2 × 527 nm) differs from the fluorescence spectrum obtained after one-step ultraviolet excitation (263.5 nm) by a strongly quenched emission with a fluorescence lifetime of 10 ± 5 ps and a smaller spectral width. The two-step absorption process presumably selects tryptophan residues which strongly couple to the retinal chromophore.
Electron micrographs of monolayer crystals of paraffin have been recorded with a spot-scan mode of imaging which uses a small 50 nm diameter moving beam. In comparison with normal stationary beam images using 5 μm illuminating beams, the spot-scan micrographs show a higher and more consistent contrast from the 3.8–4.2 Å hydrocarbon chain spacings. On average the improvement in contrast is twofold, but this still leaves scope for further improvement: the best spot-scan images still do not quite reach the level of contrast calculated theoretically from electron diffraction. We believe that the cause of the low contrast in paraffin images must be specimen motion caused by radiation damage rather than a charging effect, for two reasons. First, it does not occur in control images of vermiculite, a non-beam-sensitive mineral, when treated identically. Secondly, although charging might still be a problem with paraffin, when images are taken with the objective aperture in the column, a procedure which is normally expected to reduce charging, no improvement in contrast is found. Thus we think that the use of the small beam minimizes the effect of specimen motion on image contrast by minimizing the specimen area exposed at each instant and therefore the resultant image blurring. Further improvements with even smaller illumination beam diameters might be expected.
The absolute direction of the retinal chromophore of bacterio-rhodopsin relative to the membrane plane is investigated by using an optical second-harmonic interference technique. Compared with the known adsorbed geometry of free retinylidene Schiff base on a glass substrate, our data indicate the beta-ionone ring of the chromophore of bacteriorhodopsin points away from the cytoplasmic surface of the purple membrane. The implication of this finding is discussed in light of other chemical and structural results on bacteriorhodopsin.
Transmembrane location of the retinal chromophore, either native or reduced in situ to a fluorescent derivative, of the purple membrane of Halobacterium halobium was investigated with fluorescence energy transfer techniques. Single sheets of purple membrane, either native or reduced with borohydride, were adsorbed on polylysine-coated glass; the orientation, whether the exposed surfaces were cytoplasmic or extracellular, was controlled by adjusting the pH of the membrane suspension before the adsorption. On the exposed surface of the reduced membrane, a layer of cytochrome c, hemoglobin, or ferritin was deposited. The rate of excitation energy transfer from the fluorescent chromophore in the membrane to the colored protein was greater when the protein was on the cytoplasmic surface of the membrane than when it was on the extracellular surface. Analysis in which uniform distribution of the protein on the surface was assumed showed that the reduced chromophore is situated at a depth of <1.5 nm from the cytoplasmic surface. The location of the native retinal chromophore was examined by depositing a small amount of tris(2,2'-bipyridyl)ruthenium(II) complex on the native membrane adsorbed on the glass. Energy transfer from the luminescent complex to the retinal chromosphore was more efficient on the cytoplasmic surface than on the extracellular surface, suggesting that the native chromophore is also on the cytoplasmic side. From these and previous results we conclude that the chromophore, whether native or reduced, of bacteriorhodopsin is located at a depth of 1.0 +/- 0.3 nm from the cytoplasmic surface of purple membrane.
Purple membranes were prepared in which bacteriorhodopsin was labeled at lysine 41 with phenylisothiocyanate (PITC) and with perdeuterated PITC. The in-plane position of this small label containing only five deuterons was determined from the differences between the neutron diffraction intensities of the two samples. At 8.7-A resolution the Fourier difference map revealed a well-defined site between helices 3 and 4. This position was confirmed by a refinement procedure in reciprocal space. Model calculations showed that the observed difference density had the right amplitude for the label. Thus it is possible to locate a small group in a large protein structure by replacing as few as five hydrogens by deuterium. The observed location of PITC restricts the number of possibilities for the assignment of helix B in the sequence (to which lysine 41 is attached) to one of the seven helices of the structure. Taking into account the size of the label and the length of the lysine side chain our result excludes helices 1, 2, and 7 as candidates for B.
The axial projection of the glutamine synthetase molecule has been reconstructed from electron micrographs of a stained preparation by using a new method of correlation search and averaging. The average over 50 individual molecules appears as a radial pattern with sixfold symmetry. The handedness evident in the average is attributed to nonuniformity of the negative stain.
An X-ray diffraction analysis of oriented specimens of the purple membrane from Halobacterium halobium shows that the protein and lipid components are packed in a P3 hexagonal lattice, with one protein molecule per asymmetric unit. The structure is made up of a single layer of the protein molecules, oriented vectorially in the same direction across the membrane.The presence of strong diffraction peaks equatorially centred at 10 Å, and axially at 5 Å and 1.5 Å, show that the protein molecules, which make up most of the mass of the membrane, are composed to a considerable extent of α-helices, 25 to 35 Å long, arranged roughly perpendicular to the plane of the membrane to form superhelical groupings of the “coiled-coil” type.The surface of the membrane is flat, with no bumps or dimples large enough to affect the X-ray pattern when the electron density of the suspending medium is altered. The phospholipids may be less exactly positioned in the lattice than the protein, since the presence of uranyl acetate, which is expected to co-ordinate with the acidic phosphate groups, produces intensity changes only at low resolution.
The direction of orientation of the protein bacteriorhodopsin within the purple membrane of Halobacterium halobium has been determined by selected-area electron diffraction of membranes preferentially oriented by adsorption to polylysine. Purple membrane is known to adsorb preferentially to polylysine by its cytoplasmic surface at neutral pH and by its extracellular surface at low pH. To maintain the adsorbed membranes in a well-ordered state in the electron microscope, an improved technique of preparing frozen specimens was developed. Large areas of frozen-hydrated specimens, devoid of bulk water, were obtainable after the specimen was passed through a Ca stearate film at an air-water interface. High-resolution microscopy was used to relate the orientation observed in the electron diffraction patterns to the orientation of the projected structure that is obtained from images. We have found that the three-dimensional structure determined by Henderson and and Unwin [Henderson, R. & Unwin, P.N.T. (1975) Nature 257, 28--32] is oriented with the cytoplasmic side uppermost--i.e., the helices fan outward on the cytoplasmic side of the membrane.
The orientation of the 568 nm transition dipole moment of the retinal chromophore of bacteriorhodopsin has been determined in purple membranes from Halobacterium halobium and in reconstituted vesicles. The angle between the 568 nm transition dipole moment and the normal to the plane of the membrane was measured in two different ways.In the first method the angle was obtained from transient dichroism measurements on bacteriorhodopsin incorporated into large phosphatidylcholine vesicles. Following flash excitation with linearly polarized light, the anisotropy of the 568 nm ground-state depletion signal first decays but then reaches a time-independent value. This result, obtained above the lipid phase transition, is interpreted as arising from rotational motion of bacteriorhodopsin which is confined to an axis normal to the plane of the membrane. It is shown that the relative amplitude of the time-independent component depends on the orientation of the 568 nm transition dipole moment. From the data an angle of 78 ° ± 3 ° is determined.In the second method the linear dichroism was measured as a function of the angle of tilt between the oriented purple membranes and the direction of the light beam. The results were corrected for the angular distribution of the membranes within the oriented samples, which was determined from the mosaic spread of the first-order lamellar neutron diffraction peak. In substantial agreement with the results of the transient dichroism method, linear dichroism measurements on oriented samples lead to an angle of 71 ° ± 4 °.No significant wavelength dependence of the dichroic ratio across the 568 nm band was observed, implying that the exciton splitting in this band must be substantially smaller than the recently suggested value of 20 nm (Ebrey et al., 1977).The orientation of the 568 nm transition dipole moment, which coincides with the direction of the all-trans polyene chain of retinal, is not only of interest in connection with models for the proton pump, but can also be used to calculate the inter-chromophore distances in the purple membrane.
MITCHELL'S hypothesis of chemiosmotic coupling between redox reactions and ATP synthesis in membranes1 is supported by the finding of a light-driven proton pump in the purple membrane of Halobacterium halobium2-4. The purple membrane contains the protein bacteriorhodopsin in a crystalline array, with retinal as chromophore5,6. We propose here, on the basis of quantumchemical arguments and experimental observations, that the H. halobium proton pump may involve proton translocation through photoisomerisation of retinal about its 14-15 single bond.
The ferritin/avidin/biotin labelling procedure of Heitzmann & Richards (1974) has been applied to purple membrane. Two biotin reagents of differing specificity have been used and the biotin then visualised in the electron microscope using avidin/ferritin conjugate. With biotin N-hydroxy succinimide ester only the protein molecules are labelled; with biotin hydrazide, using periodate-oxidised membranes, only the lipid molecules are labelled; for both of these reagents, the distribution of biotin on each side of the membrane has been correlated with previous structural studies using electron diffraction and freeze-fracture electron microscopy.The above experiments enable three deductions to be made. 1.(1) Biotin labelling of the protein, almost certainly at an accessible lysine residue, occurs only on the extracellular surface of the membrane.2.(2) Biotin labelling of lipid, almost certainly glycolipid, also occurs only on the extracellular surface of the membrane.3.(3) The bottom of the model of purple membrane obtained by electron microscopy (Henderson and Unwin, 1975) corresponds to the extracellular surface of the membrane.
We have used electron microscopy and image processing by computer to study the surface layers from the cell walls of two strains of Clostridia. The glycoprotein subunits in these layers form regular periodic arrays, one with hexagonal symmetry, the other with tetragonal symmetry. Because the lattices tend to be curved or otherwise distorted, a computer procedure for image averaging to reduce noise was developed, which takes account of and corrects the spatial distortions prior to averaging. The average images so obtained show subunits of a size which can be correlated with the known molecular weight of the glycoprotein. The subunits are joined by fine bridges, which provide a good covering of the underlying cell wall.
The purple membrane of Halobacterium halobium contains the protein bacteriorhodopsin which resembles the visual pigment, rhodopsin, in many aspects. The isomeric configurations of its chromophore, retinal, were studied by a combination of methylene chloride extraction and analysis by high-pressure liquid chromatography. The light-adapted form bR570LA yields solely all-trans-retinal, while the dark-adapted form of bacteriorhodopsin, bR560DA, yields a mixture of 13-cis and all-trans with a ratio of similar to 1;1. The photointermediate M412 in a membrane modified by ether at high NaCl concentration also yields an approximately 1:1 mixture of 13-cis-and all-trans-retinals, while a similar M405 species produced by illumination in 2 M guanidine hydrochloride at high pH yields mainly 13-cis-retinal. These results indicate that the photochemical cycle of bR570LA may involve an isomerization of the retinal chromophore from the all-trans to the 13-cis form.
Resolution tests on amorphous carbon foils were carried out in an electron microscope with a superconducting system containing 4 lenses including a shielding lens at 200 kV beam voltage. Due to the mechanical and electrical stability of the system and the absence of contamination of the specimen the highest space frequencies transferred at vertically incident beam were 6 nm-1 corresponding to a resolution of 0.17 nm, a value which approaches the theoretical resolving power of the electron optical system. It should also be feasible to apply such a lens system for microprobe analysis without strongly reducing the theoretical resolution limit, if the construction of the shielding lens is slightly changed.