Research Items (184)
The inner membrane-associated protein of 30 kDa (IM30), also known as the vesicle-inducing protein in plastids 1 (Vipp1), is found in the majority of photosynthetic organisms that use oxygen as an energy source, and its occurrence appears to be coupled to the existence of thylakoid membranes in cyanobacteria and chloroplasts. IM30 is most likely involved in thylakoid membrane biogenesis and/or maintenance, and has recently been shown to function as a membrane fusion protein in presence of Mg2+. However, the precise role of Mg2+ in this process and its impact on the structure and function of IM30 remains unknown. Here, we show that Mg2+ binds directly to IM30 with a binding affinity of approximately 1 mM. Mg2+-binding compacts the IM30 structure coupled with an increase in the thermodynamic stability of the proteins' secondary, tertiary and quaternary structure. Furthermore, the structural alterations trigger IM30 double ring formation in vitro due to increased exposure of hydrophobic surface regions. However, in vivo Mg2+-triggered exposure of hydrophobic surface regions most likely modulates membrane binding and induces membrane fusion.
Peptides and proteins with the N-terminal motifs NH2-Xxx-His and NH2-Xxx-Zzz-His form well-established copper(II) complexes with different coordination modes. Peptides with the sequence NH2-Xxx-His-His contain both motifs. This work shows that NH2-Ala-His-His indeed can access both Cu(II) coordination types in a pH dependent manner. Changes in the second coordination sphere can considerably impact the equilibrium between the two coordination modes. Thus, this motif is a chimera of XH and XZH with tunable properties.
The advancement of molecular electronics and spintronics requires novel hybrid materials with synergistic magnetic and electrical properties. The non-covalent functionalization of highly conductive graphene with magnetically bistable spin crossover (SCO) complex may yield such a multifunctional material. In this regard, a graphene-Fe(II) SCO complex hybrid (Gr-SCO) has been prepared by non-covalently anchoring of a pyrene decorated SCO complex with solution phase pre-exfoliated few layer graphene sheets. SQUID magnetometry revealed the preservation of SCO in the Gr-SCO hybrid material exhibiting more gradual spin state switching characteristics than in the bulk molecular complex. This persistence of SCO of a molecular Fe(II) complex upon anchoring on the graphene surface has consequences towards the realization of SCO based applications: in (i) reversible spin state dependent band gap tuning of graphene with an SCO complex analogous to chemical doping of graphene, and (ii) to probe the spin state dependence of electrical conductivity modulation by wiring the anchoring group (pyrene) tethered SCO complex between chemically robust few layer graphene electrodes.
Gene transfer using lentiviral vectors has therapeutic applications spanning from monogenic and infectious diseases to cancer. Such gene therapy has to be improved by enhancing the levels of viral infection of target cells and/or reducing the amount of lentivirus for greater safety and reduced costs. Vectofusin-1, a recently developed cationic amphipathic peptide with a pronounced capacity to enhance such viral transduction, strongly promotes the entry of several retroviral pseudotypes into target cells when added to the culture medium. To clarify the molecular basis of its action the peptide was investigated on a molecular and a supramolecular level by a variety of biophysical approaches. We show that in culture medium vectofusin-1 rapidly forms complexes in the 10 nm range that further assemble into annular and extended nanofibrils. These associate with viral particles allowing them to be easily pelleted for optimal virus-cell interaction. Thioflavin T fluorescence, circular dichroism and infrared spectroscopies indicate that these fibrils have a unique α-helical structure whereas most other viral transduction enhancers form β-amyloid fibrils. A vectofusin-1 derivative (LAH2-A4) is inefficient in biological assays and does not form nanofibrils, suggesting that supramolecular assembly is essential for transduction enhancement. Our observations define vectofusin-1 as a member of a new class of α-helical enhancers of lentiviral infection. Its fibril formation is reversible which bears considerable advantages in handling the peptide in conditions well-adapted to Good Manufacturing Practices and scalable gene therapy protocols.
Specialized infrared spectroscopic techniques have been developed that allow studying the secondary structure of membrane proteins and the influence of crucial parameters like lipid content and detergent. Here, we focus on an ATR-FTIR spectroscopic study of Af-Amt1 and the influence of LDAO/glycerol on its structural integrity. Our results clearly indicate that infrared spectroscopy can be used to identify the adapted sample conditions.
H/D exchange kinetics at the level of the amide proton in the mid infrared (1700–1500 cm⁻¹) make it possible to study the conformational flexibility of membrane proteins, independent of size or the presence of detergent or lipids. Slow, medium, and fast exchanging domains are distinguished, which reveal a different accessibility to the solvent. Whereas amide hydrogens undergo rapid exchange with solvent in an open structure, hydrogens experience much slower exchange when involved in H-bonded structures or when sterically inaccessible to the solvent. Here, we describe the protocol that was used to study the effect of phospholipids on the overall structure of the Na⁺ NQR from V. cholerae, a sodium pumping membrane protein.
Far infrared spectroscopy is a technique that allows probing the low frequency region of the vibrational spectrum and reveals, among others, vibrational modes of inter- and intramolecular hydrogen bonding. Due to their collective nature, these modes are highly sensitive to the conformational state of the molecule as well as to their interactions. Far infrared spectroscopy is thus an emerging technique for the characterization of the low frequency motions of complex molecules including polymers, peptides, proteins or ionic liquids. The technique is not limited by the molecules size and can be applied to solids and liquids. An overview on far infrared studies on complex structures and their interactions are given revealing the potential of the approach.
Molecular dynamics (MD) simulations and far-infrared (far-IR) spectroscopy were combined to study peptide binding by the second PDZ domain (PDZ1) of MAGI1, which has been identified as an important target for the Human Papilloma Virus. PDZ1 recognizes and binds to the C-terminal end of the E6 protein from high-risk Human Papilloma Virus. The far-IR spectra of two forms of the protein, an unbound APO form and a HOLO form (where the PDZ1 is bound to an 11-residue peptide derived from the C terminus of HPV16 E6), were obtained. MD simulations were used to determine the most representative structure of each form and these were used to compute their respective IR spectra by normal mode analysis. Far-UV circular dichroism spectroscopy was used to confirm the secondary structure content and the stability through temperature-dependent studies. Both the experimental and calculated far-IR spectra showed a red shift of the low-frequency peaks upon peptide binding. The calculations show that this is coincident with an increased number of hydrogen bonds formed as the peptide augments the protein β-sheet. We further identified the contribution of surface-bound water molecules to bands in the far-IR and, through the calculations, identified potential pathways for allosteric communication. Together, these results demonstrate the utility of combining far-IR experiments and MD studies to study peptide binding by proteins.
The structure-property relationships were compared for the iron and iron-copper complexes of two functional cytochrome c oxidase models, 1 and 2, both constructed upon a phenanthroline-strapped porphyrin bearing respectively pyridyl or picolinyl built-in proximal and distal ligands. The behavior of these heme models in the absence and in the presence of copper was studied by ¹H NMR, UV-visible absorption, EPR, Raman and FTIR spectroscopies, electrochemistry in solution and deposited on a rotating ring-disk graphite electrode. The distal binding site within the phenanthroline pocket of both 1 and 2 is available for the coordination of exogenic ligands, yet the oxygen binding affinity is higher for all complexes of 2 than for 1. Despite this difference, [1FeIICuI] more efficiently reproduced the electrocatalyzed reduction of oxygen to water than [2FeIICuI]. The oxygenated complexes of both iron(II)-copper(I) species mimic the ability of cytochrome c oxidase to reversibly bind O2, as shown by competitive binding studies in the presence of CO. Differences in the binding and electrocatalytic properties of these models stem from difference in rigidity of scaffolds upon binding of both the proximal and distal ligands, as well as from the bulkiness of the distal ligand.
Cytochrome c550, a diheme protein from the thermophilic bacterium Thermus thermophilus, is involved in an alternative respiration pathway allowing the detoxification of sulfite ions. It transfers the two electrons released from the oxidation of sulfite in a sulfite:cytochrome oxidoreductase (SOR) enzyme to heme/copper oxidases via the monoheme cytochrome c552. It consists of two conformationally independent and structurally different domains (the C- and N-terminal) connected by a flexible linker. Both domains harbor one heme moiety. We report here the redox properties of the full-length protein and the individual C- and N-terminal fragments. We show by UV/Vis and EPR potentiometric titrations that the two fragments exhibit very similar potentials, despite their different environments. In the full-length protein, however, the N-terminal heme is easier to reduce than the C-terminal one, due to cooperative interactions. This finding is consistent with the kinetic measurements which showed that the N-terminal domain only accepts electrons from the SOR. Cytochrome c552 is able to interact with its partners both through electrostatic and hydrophobic interactions as could be shown by measuring efficient electron transfer at gold electrodes modified with charged and hydrophobic groups, respectively. The coupling of electrochemistry with infrared spectroscopy allowed us to monitor the conformational changes induced by electron transfer to each heme separately and to both simultaneously.
Many biological functions involve the formation of protein-protein complexes. In the present study, we investigated the interaction of two proteins involved in electron transfer, adrenodoxin (Adx) and adrenodoxin reductase (AdR) by using Raman and infrared spectroscopies. Different shifts and splittings of the FeSb/t stretching vibrational modes upon interaction of the two proteins can be reported pointing towards major structural changes in the [2Fe2S] cluster. These changes may be necessary for optimizing electron transfer. The assignment of the shifted modes to the [2Fe2S] cluster was confirmed by ⁵⁴Fe labeling of the truncated Adx (4–108) as well as the investigation of mutants close to the interaction site and in the vicinity of the [2Fe2S] cluster. Electrochemically induced FTIR difference spectra revealed that the flavin cofactor in AdR also changes due to the interaction with [2Fe2S] cluster in the Adx/AdR electron transfer complex.
GHK and DAHK are biological peptides that bind both copper and zinc cations. Here we used infrared and Raman spectroscopies to study the coordination modes of both copper and zinc ions, at pH 6.8 and 8.9, correlating the data with the crystal structures that are only available for the copper-bound form. We found that Cu(II) binds to deprotonated backbone (amidate), the N-terminus and Nπ of the histidine side chain, in both GHK and DAHK, at pH 6.8 and 8.9. The data for the coordination of zinc at pH 6.8 points to two conformers including both nitrogens of a histidine residue. At pH 8.9, vibrational spectra of the ZnGHK complexes show that equilibria between monomers, oligomers exist, where deprotonated histidine residues as well as deprotonated amide nitrogen are involved in the coordination. A common feature is found: zinc cations coordinate to Nτ and/or Nπ of the His leading to the formation of GHK and DAHK multimers. In contrast, Cu(II) binds His via Nπ regardless of the peptide, in a pH-independent manner.
Lactose permease (LacY), a paradigm for the largest family of membrane transport proteins, catalyzes the coupled translocation of a galactoside and a H(+) across the cytoplasmic membrane of Escherichia coli (galactoside/H(+) symport). One of the most important aspects of the mechanism is the relationship between protonation and binding of the cargo galactopyranoside. In this regard, it has been shown that protonation is required for binding. Furthermore when galactoside affinity is measured as a function of pH, an apparent pK (pK(app)) of ∼10.5 is obtained. Strikingly, when Glu325, a residue long known to be involved in coupling between H(+) and sugar translocation, is replaced with a neutral side chain, the pH effect is abolished, and high-affinity binding is observed until LacY is destabilized at alkaline pH. In this paper, infrared spectroscopy is used to identify Glu325 in situ. Moreover, it is demonstrated that this residue exhibits a pKa of 10.5 ± 0.1 that is insensitive to the presence of galactopyranoside. Thus, it is apparent that protonation of Glu325 specifically is required for effective sugar binding to LacY.
Hemocyanins are large oligomeric respiratory proteins found in many arthropods and molluscs. Here we give infrared spectroscopic evidence of a high stability towards exposure to sub-zero temperatures for hemocyanins from the arthropods Limulus polyphemus and Eurypelma californicum at different pH values. Small but distinct temperature induced changes of the secondary structure were observed, but a stable core of at least 40% α-helical structure is preserved as identified in the infrared spectra obtained between 294 and 20 K. The structural changes differ in detail somewhat for the two hemocyanins, with overall fewer changes observed in the case of E. californicum. Notably, in both cases the overall changes in the α-helical content are found to be fully reversible. The small changes in the secondary structure and reversibility upon cold treatment seem to be a particular property of the two hemocyanins, since it was not observed for myoglobin studied in the same way.
Polymorphism is a common property of amyloid fibers that complicates their detailed structural and functional studies. Here we report experiments illustrating the chemical principles that enable the formation of amyloid polymorphs with distinct stoichiometric composition. Using appropriate covalent tethering we programmed self-assembly of a model peptide corresponding to the [20-41]-fragment of human β2-microglobulin into fibers with either trimeric or dimeric amyloid cores. Using a set of biophysical and biochemical methods we demonstrated their distinct structural, morphological and templating properties. Furthermore, we showed that supramolecular approaches in which the peptide is modified with bulky substituents can also be applied to modulate the formation of different fiber polymorphs. Such strategies when extended to disease-related peptides and proteins will greatly help in the evaluation of the biological properties of structurally distinct amyloids.
Cytochrome cbb3 (also known as C-type) oxidases belong to the family of heme-copper terminal oxidases which couple at the end of the respiratory chain the reduction of molecular oxygen into water and the pumping of protons across the membrane. They are expressed most often at low pressure of O2 and they exhibit a low homology of sequence with the cytochrome aa3 (A-type) oxidases found in mitochondria. Their binuclear active site comprises a high-spin heme b3 associated with a CuB center. The protein also contains one low-spin heme b and 3 hemes c. We address here the redox properties of cbb3 oxidases from three organisms, Rhodobacter sphaeroides, Vibrio cholerae and Pseudomonas stutzeri by means of electrochemical and spectroscopic techniques. We show that the redox potential of the heme b3 exhibits a relatively low midpoint potential, as in related cytochrome c-dependent nitric oxide reductases. Potential implications for the coupled electron transfer and proton uptake mechanism of C-type oxidases are discussed.
Actinobacteria are closely linked to human life as industrial producers of bioactive molecules and as human pathogens. Respiratory cytochrome bcc complex and cytochrome aa3 oxidase are key components of their aerobic energy metabolism. They form a supercomplex in Actinobacterium Corynebacterium glutamicum. With comprehensive bioinformatics and phylogenetic analysis we show that genes for cyt bcc-aa3 supercomplex are characteristic for Actinobacteria (Actinobacteria and Acidimicrobiia, except the anaerobic orders Actinomycetales and Bifidobacteriales). It is likely to be an obligatory supercomplex due to the lack of genes encoding alternative electron transfer partners such as mono-heme cyt c. Instead, subunit QcrC of bcc complex, here classified as short di-heme cyt c, will provide the exclusive electron transfer link between the complexes as in C. glutamicum. Purified to high homogeneity, the C. glutamicum bcc-aa3 supercomplex contained all subunits and cofactors as analyzed by SDS-PAGE, BN-PAGE, absorption and EPR spectroscopy. Highly uniform supercomplex particles in electron microscopy analysis support a distinct structural composition. The supercomplex possesses a dimeric stoichiometry with a ratio of a-type, b-type and c-type hemes close to 1:1:1. Redox titrations revealed a low potential bcc complex (EmISP = + 160 mV, EmbL = -291 mV, EmbH = -163 mV, Emcc = + 100 mV) fined-tuned for oxidation of menaquinol and a mixed potential aa3 oxidase (EmCuA = + 150 mV, Ema/a3 = + 143/+317 mV) mediating between low and high redox potential to accomplish dioxygen reduction. The generated molecular model supports a stable assembled supercomplex with defined architecture which permits energetically efficient coupling of menaquinol oxidation and dioxygen reduction in one supramolecular entity.
Richard Dluhy opened a general discussion of the paper by Duncan Graham: In your example of a heterogeneous solution-based assay for multicomponent analysis, what is the concentration of the target fungal ssPCR DNA that is used, and how do you manage the kinetics of the reaction such that the target reaches the probe in a time frame appropriate for a clinical assay?
Cytochrome bd oxidases are membrane proteins expressed by bacteria including a number of pathogens, which make them an attractive target for the discovery of new antibiotics. An electrochemical assay is developed to study the activity of these proteins and inhibition by quinone binding site tool compounds. The setup relies on their immobilization at electrodes specifically modified with gold nanoparticles, which allows achieving a direct electron transfer to/from the heme cofactors of this large enzyme. After optimization of the protein coverages, the assay shows at pH 7 a good reproducibility and readout stability over time, and it is thus suitable for further screening of small molecule collections.
Far infrared spectra of complex molecular structures like lipid membranes or proteins show large and broad continuum modes that include contributions of the internal hydrogen bonding of the assembled structures. Here we corroborate the pH triggered structural rearrangement in pH sensitive liposomes with a clear shift of the far infrared mode from 170 to 159 cm-1. This spectral change was accompanied by the broadening of the hydrogen bonding signature by about 25 cm-1 and correlates with the well-known hydrogen bonding dependent shifts of the ν(PO2-)as vibration of the lipid head group in the mid infrared and with further shifts of functional group vibrations. Far infrared spectroscopy is thus a useful tool for the investigation of conformational changes in large molecular structures.
Insoluble amyloid fibers represent a pathological signature of many human diseases. To treat such diseases, inhibition of amyloid formation has been proposed as a possible therapeutic strategy. d-Peptides, which possess high proteolytic stability and lessened immunogenicity, are attractive candidates in this context. However, a molecular understanding of chiral recognition phenomena for d-peptides and l-amyloids is currently incomplete. Here we report experiments on amyloid growth of individual enantiomers and their mixtures for two distinct polypeptide systems of different length and structural organization: a 44-residue covalently-linked dimer derived from a peptide corresponding to the [20-41]-fragment of human β2-microglobulin (β2m) and the 99-residue full-length protein. For the dimeric [20-41]β2m construct, a combination of electron paramagnetic resonance of nitroxide-labeled constructs and (13) C-isotope edited FT-IR spectroscopy of (13) C-labeled preparations was used to show that racemic mixtures precipitate as intact homochiral fibers, i.e. undergo spontaneous Pasteur-like resolution into a mixture of left- and right-handed amyloids. In the case of full-length β2m, the presence of the mirror-image d-protein affords morphologically distinct amyloids that are composed largely of enantiopure domains. Removal of the l-component from hybrid amyloids by proteolytic digestion results in their rapid transformation into characteristic long straight d-β2m amyloids. Furthermore, the full-length d-enantiomer of β2m was found to be an efficient inhibitor of l-β2m amyloid growth. This observation highlights the potential of longer d-polypeptides for future development into inhibitors of amyloid propagation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
Although the architectures of several membrane proteins in respiration as well as the basic chemical reactions have been described, the interactions on molecular level, the high diversity and the high efficiency of mechanisms, are under discussion. Experiments have been developed which reveal how protons and other ions are drawn through proteins and how they are coupled to electron transfer.An electrochemical approach on proteins immobilized on gold nano particles was used to get insight into the redox properties in the presence and absence of substrate and inhibitors to the proton channels (Zn2+).1 Two approaches are presented to functionally probe the enzymes against substrates and inhibitors: i) The redox reaction was IR spectroscopically studied in solution and ii) the proteins are immobilized via their His-Tag on a gold layer, deposited on an ATR-crystal and studied by surface enhanced IR spectroscopy (SEIRAS). 2 The combination of the electrochemical and IR spectroscopic approach allowed the observation of protein action at the level of single functional groups within the large protein studied and thus provides essential knowledge's for the understanding of the mechanism of the studied enzymes.References1. Meyer, T.; Melin, F.; Xie, H.; von der Hocht, I.; Choi, S. K.; Noor, M. R.; Michel, H.; Gennis, R. B.: Soulimane, T.; Hellwig, P. (2014) J. Am. Chem. Soc., 136, 10854-10852. Kriegel, S., Uchida, T., Osawa, M., Friedrich, T., Hellwig, P. (2014) Biochemistry 53(40):6340-7.
The work aims to simulate in vitro the antioxidant activity of four phytoestrogen type isoflavones, whose effects are closer to those of estradiol (daidzein, formononetin, genistein, biochanin A) in biomimetic environments (in lecithin lipidic bi-layers and on silver nanoparticles), using the chemiluminescent system luminol–hydrogen peroxide, in phosphate buffer, pH 7.4. The contribution of a carrier protein, human serum albumin (HSA), to the antioxidant activity of these isoflavones has also been investigated. The rate of the fluorescence quenching of HSA by isoflavone as well as the binding constant of isoflavone to HSA, in both lecithin lipidic bi-layers and on silver nanoparticles, has also been investigated by fluorescence spectroscopy. The results are discussed with relevance to the oxidative stress process.
Abstract The combination of FTIR spectroscopy with electrochemical techniques for the study of the reaction mechanism of proteins on molecular level is presented. Different cell types combining transmission or reflection modes with surface-enhanced approaches are discussed. Examples are given on contributions to the understanding of the reaction mechanism of proteins, including cytochrome c, hydrogenases, cytochrome c oxidases, and photosystem.
Redox-dependent conformational changes are currently discussed to be a crucial part of the reaction mechanism of the respiratory complex I. Specialized difference Fourier transform infrared techniques allow the detection of side-chain movements and minute secondary structure changes. For complex I, (1)H/(2)H exchange kinetics of the amide modes revealed a better accessibility of the backbone in the presence of NADH and quinone. Interestingly, the presence of phospholipids, that is crucial for the catalytic activity of the isolated enzyme complex, changes the overall conformation. When comparing complex I samples from different species, very similar electrochemically induced FTIR difference spectra very similar rearrangements are reported. Finally, the information obtained with variants and from Zn(2+) inhibited samples for the conformational reorganization of complex I upon electron transfer are discussed in this review. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.
Proton transfer across membranes and membrane proteins is a central process in biological systems. Zn2+ ions are capable of binding to acidic residues, often found within such specific pathways, leading to a specific block. Here we probed the inhibition of the proton pumping NADH:ubiquinone oxidoreductase from Escherichia coli by means of electrochemically induced FTIR difference spectroscopy. Numerous conformational changes were identified including those that arise from the reorganization of the membrane arm upon electron transfer in the peripheral arm of the protein. Signals at very high frequencies at 1781 and 1756 cm-1 point towards the perturbation of acidic residues in a highly hydrophobic environment upon Zn2+ inhibition. In the D563NL variant, a residue that lacks part of the proton pumping activity and is located on the horizontal helix, the spectral signature of Zn2+ binding is changed. Our data support the functional role of this residue for proton translocation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
In chemical synthesis, the widely used Birch reduction of aromatic compounds to cyclic dienes requires alkali metals in ammonia as extremely low-potential electron donors. An analogous reaction is catalyzed by benzoyl-coenzyme A reductases (BCRs) that have a key role in the globally important bacterial degradation of aromatic compounds at anoxic sites. Because of the lack of structural information, the catalytic mechanism of enzymatic benzene ring reduction remained obscure. Here, we present the structural characterization of a dearomatizing BCR containing an unprecedented tungsten cofactor that transfers electrons to the benzene ring in an aprotic cavity. Substrate binding induces proton transfer from the bulk solvent to the active site by expelling a Zn(2+) that is crucial for active site encapsulation. Our results shed light on the structural basis of an electron transfer process at the negative redox potential limit in biology. They open the door for biological or biomimetic alternatives to a basic chemical synthetic tool.
Ground-state molecular vibrations can be hybridized through strong coupling with the vacuum field of a cavity optical mode in the infrared region, leading to the formation of two new coherent vibro-polariton states. The spontaneous Raman scattering from such hybridized light-matter states was studied, showing that the collective Rabi splitting occurs at the level of a single selected bond. Moreover, the coherent nature of the vibro-polariton states boosts the Raman scattering cross-section by two to three orders of magnitude, revealing a new enhancement mechanism as a result of vibrational strong coupling. This observation has fundamental consequences for the understanding of light-molecule strong coupling and for molecular science. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
Ground-state molecular vibrations can be hybridized through strong coupling with the vacuum field of a cavity optical mode in the infrared region, leading to the formation of two new coherent vibro-polariton states. The spontaneous Raman scattering from such hybridized light–matter states was studied, showing that the collective Rabi splitting occurs at the level of a single selected bond. Moreover, the coherent nature of the vibro-polariton states boosts the Raman scattering cross-section by two to three orders of magnitude, revealing a new enhancement mechanism as a result of vibrational strong coupling. This observation has fundamental consequences for the understanding of light-molecule strong coupling and for molecular science.
In this study, experimental far infrared measurements of l-serine, l-threonine, l-cysteine, and l-methionine are presented showing the spectra for the 1.0-13.0 pH range. In parallel, solid state DFT calculations were performed on the amino acid zwitterions in the crystalline form. We focused on the lowest frequency far infrared normal modes, which required the most precision and convergence of the calculations. Analysis of the computational results, which included the potential energy distribution of the vibrational modes, permitted a detailed and almost complete assignment of the experimental spectrum. In addition to characteristic signals of the two main acid-base couples, CO2H/CO2(-) and NH3(+)/NH2, specific side chain contributions for these amino acids, including CCO and CCS vibrational modes were analyzed. This study is in line with the growing application of FIR measurements to biomolecules. Copyright © 2015 Elsevier B.V. All rights reserved.
Drosophila melanogaster cryptochrome is one of the model proteins for animal blue-light photoreceptors. By using time-resolved and steady-state optical spectroscopy, we have studied the mechanism of light-induced radical-pair formation and decay, and the photoreduction of the FAD cofactor. Exact kinetics on a μs- to min-time scale could be extracted for the wild type protein using global analysis. The wild type exhibits a fast photoreduction reaction from the oxidized FAD to the FAD(•-) state with a very positive midpoint potential of ~ +125 mV, but no further reduction could be observed. We could also demonstrate that the terminal tryptophan of the conserved triad, W342, is directly involved in electron transfer; however, photoreduction could not be completely inhibited by a W342F mutant. The investigation of another mutant close to the FAD cofactor, C416N, rather unexpectedly reveals accumulation of a protonated flavin radical on a time scale of several seconds. The obtained data are critically discussed with the one obtained from another protein, E. coli photolyase, and we conclude that the amino acid opposite N(5) of the isoalloxazine moiety of FAD is able to (de)stabilize the protonated FAD radical, but not to significantly modulate the kinetics of any light-inducted reactions. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
Two different pathways had long been established for the access of protons in cytochrome c oxidase, operating during oxygen reduction from the mitochondrial matrix, or the bacterial cytoplasm, resp. Here, we follow oxygen reduction coupled to proton uptake by electrocatalytic current measurements with oxidase isolated from Paracoccus denitrificans. Wild type enzyme and site-specific mutants in both proton uptake pathways (K354M, D124N and K354M/D124N) are immobilized on gold nanoparticles, and oxygen reduction is probed electrochemically in the presence of varying concentrations of Zn2+ ions known to inhibit both the entry and the exit pathway(s) for protons in the enzyme. Our data suggest that under these conditions substrate protons gain access to the oxygen reduction site via the exit pathway.
In bioenergetic systems quinones play a central part in the coupling of electron and proton transfer. The specific function of each quinone binding site is based on the protein-quinone interaction that can be described by means of reaction induced FTIR difference spectroscopy, induced for example by light or electrochemically. The identification of sites in enzymes from the respiratory chain is presented together with the analysis of the accommodation of different types of quinones to the same enzyme and the possibility to monitor the interaction with inhibitors. Reaction induced FTIR difference spectroscopy is shown to give essential information on the general geometry of quinone binding sites, the conformation of the ring and of the substituents as well as essential structural information on the identity of the amino-acid residues lining this site. This article is part of a Special Issue entitled: Vibrational Spectroscopies in Molecular Bioenergetics.
The energy converting NADH:ubiquinone oxidoreductase, respiratory complex I, couples NADH oxidation and quinone reduction with the translocation of protons across the membrane. Complex I exhibits a unique L-shape with a peripheral arm extending in the aqueous phase and a membrane arm embedded in the lipid bilayer. Both arms have a length of about 180 Å. The electron transfer reaction is catalyzed by a series of cofactors in the peripheral arm, while the membrane arm catalyzes proton translocation. We used the inhibition of complex I by zinc to shed light on the coupling of the two processes, which is not yet understood. Enzyme kinetics revealed the presence of two high-affinity binding sites for Zn2+ that are attributed to the proton-translocation pathways in the membrane arm. Electrochemically induced FT-IR difference spectroscopy demonstrated that zinc binding involves at least two protonated acidic residues. EPR-spectroscopy showed that one of the cofactors is only partially reduced by NADH in the presence of Zn2+. We conclude that blocking the proton channels in the membrane arm leads to a partial block of the electron transfer in the peripheral arm indicating the long-range coupling between both processes.
In this study complex I was immobilized in a biomimetic environment on a gold layer deposited on an ATR-crystal in order to functionally probe the enzyme against substrates and inhibitors via Surface-Enhanced IR Absorption Spectroscopy (SEIRAS) and Cyclic Voltammetry (CV). To achieve this immobilization, two methods based on the generation of a high affinity Self-Assembled Monolayer (SAM) were probed. The first made use of the affinity of Ni-NTA towards a hexahistidine tag that was genetically engineered onto complex I and the second exploited the affinity of the enzyme towards its natural substrate NADH. Experiments were also performed with complex I reconstituted in lipids. Both approaches have found to be successful and electrochemically induced IR difference spectra of complex I were obtained.
Succinate: quinone reductases (SQRs) are the enzymes that couple the oxidation of succinate and the reduction of quinones in the respiratory chain of prokaryotes and eukaryotes. Herein, we compare the temperature-dependent activity and structural stability of two SQRs, the first from the mesophilic bacterium Escherichia coli and the second from the thermophilic bacterium Thermus thermophilus, using a combined electrochemical and infrared spectroscopy approach. Direct electron transfer was achieved with full membrane protein complexes at single-walled carbon nanotube (SWNT)-modified electrodes. The possible structural factors that contribute to the temperature-dependent activity of the enzymes and, in particular, to the thermostability of the Thermus thermophilus SQR are discussed.
Cytochrome aa3 from Paracoccus denitrificans and cytochrome ba3 from Thermus thermophilus, two distinct members of the heme–copper oxidase superfamily, were immobilized on electrodes modified with gold nanoparticles. This procedure allowed us to achieve direct electron transfer between the enzyme and the gold nanoparticles and to obtain evidence for different electrocatalytic properties of the two enzymes. The pH dependence and thermostability reveal that the enzymes are highly adapted to their native environments. These results suggest that evolution resulted in different solutions to the common problem of electron transfer to oxygen.
Na+-pumping NADH:ubiquinone oxidoreductase (Na+-NQR) is responsible for maintaining a sodium gradient across the inner bacterial membrane. This respiratory enzyme, which couples sodium pumping to the electron transfer between NADH and ubiquinone, is not present in eukaryotes and as such could be a target for antibiotics. In this paper it is shown that the site of ubiquinone reduction is conformationally coupled to the NqrB subunit, which also hosts the final cofactor in the electron transport chain, riboflavin. Previous work showed that mutations in conserved NqrB glycine residues 140 and 141 affect ubiquinone reduction and the proper functioning of the sodium pump. Surprisingly, these mutants did not affect the dissociation constant of ubiquinone or its analog HQNO (2-n-heptyl-4-hydroxyquinoline N-oxide) from Na+-NQR, which indicates that these residues do not participate directly in the ubiquinone binding site but probably control its accessibility. Indeed, redox-induced difference spectroscopy showed that these mutations prevented the conformational change involved in ubiquinone binding but did not modify the signals corresponding to bound ubiquinone. Moreover, data are presented that demonstrate the NqrA subunit is able to bind ubiquinone but with a low non-catalytically relevant affinity. It is also suggested that Na+-NQR contains a single catalytic ubiquinone binding site and a second site that can bind ubiquinone but is not active.
The 18-kDa translocator protein (TSPO) is evolutionarily conserved from bacteria to humans. TSPO expression has been observed in essentially all mammalian tissues, with a preferential localization in the outer mitochondrial membrane. TSPO is involved in various physiological functions. The evidence suggests that TSPO may function in different protein complexes. In mammalian cells, the best-characterized activity of TSPO is the transport of cholesterol from the cytosol to the mitochondrial matrix, where cholesterol is converted to a precursor of steroids or bile salts. No atomic structure of TSPO is currently available. TSPO does not belong to any known membrane protein structural family. It has five transmembrane domains containing α-helices that are involved in the transport of cholesterol. Cytosolic loops are involved in ligand binding and the activation of transport. It has been suggested that TSPO could form homopolymers within heteropolymer complexes. Because of the low native abundance of TSPO, production of recombinant TSPO is a first step for any structural study. In this chapter, we present the current understanding of TSPO overexpression studies in bacteria, purification of functional TSPO, and different approaches to solve TSPO structure.
The NADH:ubiquinone oxidoreductase (complex I) couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. It was proposed that the electron transfer involves quinoid groups localized at the end of the electron transfer chain. In order to identify these groups, fluorescence excitation and emission spectra of the Escherichia coli complex I and its fragments, namely the NADH dehydrogenase fragment containing the FMN and six iron-sulfur (Fe/S)-clusters, and the quinone reductase fragment containing three Fe/S clusters were measured. Signals sensitive to reduction by either NADH or dithionite were detected within the complex and the quinone reductase fragment and attributed to the redox transition of protonated ubiquinone radicals. A fluorescence spectroscopic electrochemical redox titration revealed a midpoint potential of -37 and- 235 mV (vs. SHE) for the redox transitions of the quinone radicals in complex I with an absorption around 325 nm and a fluorescence emission at 460/475 nm. The role of these cofactor(s) for electron transfer is discussed.
A practically simple top-down process for the exfoliation of graphene (GN) and few-layer graphene (FLG) from graphite is described. We have discovered that a biocompatible amphiphilic pyrene-based hexahistidine peptide is able to exfoliate, functionalize, and dissolve few layer graphene flakes in pure water under exceptionally mild, sustainable and virtually innocuous low intensity cavitation conditions. Large area functionalized graphene flakes with the hexahistidine oligopeptide (His6-TagGN = His6@GN) have been produced efficiently at room temperature and characterized by TEM, Raman, and UV spectroscopy. Conductivity experiments carried out on His6-TagGN samples revealed superior electric performances as compared to reduced graphene oxide (rGO) and non-functionalized graphene, demonstrating the non-invasive features of our non-covalent functionalization process. We postulated a rational exfoliation mechanism based on the intercalation of the peptide amphiphile under cavitational chemistry. We also demonstrated the ability of His6-TagGN nanoassemblies to self-assemble spontaneously with inorganic iron oxide nanoparticles generating magnetic two-dimensional (2D) His6-TagGN/Fe3O4 nanocomposites under mild and non-hydrothermal conditions. The set of original experiments described here open novel perspectives in the facile production of water dispersible high quality GN and FLG sheets that will improve and facilitate the interfacing, processing and manipulation of graphene for promising applications in catalysis, nanocomposite construction, integrated nanoelectronic devices and bionanotechnology.
The Na+-pumping NADH-quinone oxidoreductase (Na+-NQR) is a unique respiratory enzyme that conserves energy by translocating Na+ through the plasma membrane. Found only in prokaryotes, the enzyme serves as the entry point for electrons into the respiratory chain in many pathogens including Vibrio cholerae and Yersinia pestis. In this study a combined electrochemical and FTIR spectroscopic approach revealed that Na+-NQR undergoes significant conformational changes upon oxidoreduction, dependent on the monovalent cation present (Na+, Li+, K+ or Rb+). In the presence of the inhibitor Rb+ additional conformational changes are evident, indicating a changed accessibility of the sodium binding sites. In electrochemically-induced FTIR difference spectra the involvement of deprotonated acid residues in the binding of cations could be identified, together with the spectral features, that point towards a monodentate binding mode for these acid residues in the oxidized form of the enzyme and bidentate binding in the reduced form. The measurements confirmed that NqrB-D397 is one of the acid residues involved in Na+ and Li+ binding. In the NqrB-D397E mutant, the spectral features characteristic of (COO-) groups are shifted, and a weakening of the hydrogen binding of the ion-binding cluster is concluded. Finally, H/D exchange kinetics of amide protons confirmed that Na+-NQR adopts different conformations, with different accessibilities to the aqueous environment, depending on the cation present, which contributes to the selectivity mechanism of ion translocation.
Abstract Integral membrane proteins are encountered in fundamental natural processes, such as photosynthesis and respiration. The relation between the structure of the proteins and their function and dynamics are still not clear in most cases. Once fully understood, these processes could ultimately help researchers to develop alternative methods for producing energy, either from light or biomass. They could also lead to more efficient antibiotics, which would selectively inhibit a specific membrane protein of pathogenic bacteria. Since the chemical reactions involved in both photosynthesis and respiration are redox reactions, electrochemical methods can play a considerable role in uncovering their mechanisms. The electrochemical characterization of membrane proteins is, however, quite challenging. An overview on the techniques used for the characterisation of membrane proteins, including classical approaches like voltammetry and spectroelectrochemistry and recent developments like the combination to surface enhanced techniques is given.
New membrane-protein based electrodes were prepared incorporating cytochrome bo(3) from E. coli and gold nanoparticles. Direct electron transfer between the electrode and the immobilized enzymes was achieved, resulting in an electrocatalytic activity in presence of O(2). The size of the gold nanoparticles was shown to be important and smaller particles were shown to reduce the overpotential of the process.
This article provides a review about the most common techniques used to study the interaction of metal ions with amyloidogenic peptides. It is addressed to researchers, who want to know what kind of techniques exists, the information which can be obtained, their advantages, limits and the demands concerning sample preparation etc. It is not addressed to specialists of these techniques as the physical principles of the techniques are nor given. First, a general overview is given about sample preparation and treatment, with focus on the metal ions Zn(II), Cu(I/II) and Fe(II/III) and the peptides amyloid-β (but also α-synuclein, prion etc.). Then the methods NMR, EPR, electrochemistry, optical spectroscopy, isothermal titration calorimetry, FTIR, X-ray absorption and mass spectrometry are treated.
We describe the specific spectral signature of different phospholipids and sphingolipids in the far infrared. Three specific spectral domains have been found: the head group contributions (600 and 480 cm−1); the modes of the torsion motion of the hydrocarbon chains and of the skeleton vibration (460 to 180 cm−1); and the hydrogen-bonding continuum (below 300 cm−1). Marker bands for individual phospholipids are distinguished.
The Na+-pumping NADH:quinone oxidoreductase (Na+-NQR) is the main entrance for electrons into the respiratory chain of many marine and pathogenic bacteria. The enzyme accepts electrons from NADH and donates them to ubiquinone, and the free energy released by this redox reaction is used to create an electrochemical gradient of sodium across the cell membrane. Here we report the role of glycine 140 and glycine 141 of the NqrB subunit in the functional binding of ubiquinone. Mutations at these residues altered the affinity of the enzyme for ubiquinol. Moreover, mutations in residue NqrB-G140 almost completely abolished the electron transfer to ubiquinone. Thus, NqrB-G140 and -G141 are critical for the binding and reaction of Na+-NQR with its electron acceptor, ubiquinone.
The Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR) is a fundamental enzyme of the oxidative phosphorylation metabolism and ionic homeostasis in several pathogenic and marine bacteria. To understand the mechanism that couples electron transfer with sodium translocation in Na(+)-NQR, the ion dependence of the redox potential of the individual cofactors was studied using a spectroelectrochemical approach. The redox potential of one of the FMN cofactors increased 90 mV in the presence of Na(+) or Li(+), compared to the redox potentials measured in the presence of other cations that are not transported by the enzyme, such as K(+), Rb(+), and NH(4)(+). This shift in redox potential of one FMN confirms the crucial role of the FMN anionic radicals in the Na(+) pumping mechanism and demonstrates that the control of the electron transfer rate has both kinetic (via conformational changes) and thermodynamic components.
The energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, couples the electron transfer from NADH to ubiquinone with a proton translocation across the membrane. Electron microscopy revealed the two-part structure of the enzyme complex. A peripheral arm, composed of globular subunits, extends into the aqueous phase. The arm contains the cofactors for the electron transfer reaction, namely one flavin mononucleotide and up to ten iron-sulfur (Fe/S) clusters. The other arm, the membrane arm, is embedded in the lipid bilayer and thus necessarily involved in proton translocation. The (ubi)quinone binding site is most likely located at the interface of the two arms. The oxidation of one NADH is coupled with the translocation of four protons (current consensus value). In this chapter, the binding of the substrates NADH and (ubi)quinone, the role of individual Fe/S clusters and the mechanism of proton translocation are discussed in the light of recent data obtained from our laboratories. We propose a model for the respiratory complex I, in which the electron transfer is coupled with the translocation of two protons by the (ubi)quinone redox chemistry and the residual two protons by conformational changes within the membrane arm. © 2012 Springer Science+Business Media Dordrecht. All rights are reserved.
The hydrophobically guided complex formation between the Cu(A) fragment from Thermus thermophilus ba(3) terminal oxidase and its electron transfer substrate, cytochrome c(552), was investigated electrochemically. In the presence of the purified Cu(A) fragment, a clear downshift of the c(552) redox potential from 171 to 111mV±10mV vs SHE' was found. Interestingly, this potential change fully matches complex formation with this electron acceptor site in other oxidases guided by electrostatic or covalent interactions. Redox induced FTIR difference spectra revealed conformational changes associated with complex formation and indicated the involvement of heme propionates. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).
Specific amino acid side chains are crucial to the stability of proteins structure and in the catalysis of enzymatic reactions. An important role is granted to Tyr residues and Tyr derivatives originating from posttranslational modifications. An example for such a modified Tyr is found in cytochrome c oxidase, where a covalent linkage between a Tyr and a His was found. The role of this linkage for the catalytic mechanism was explored by a number of research groups, several of the studies including model compound studies. Electrochemical and spectroscopic studies gave evidence to the influence of this linkage on the electron transfer ability of the Tyr and its function within the catalytic mechanism.
Alzheimer's disease is a neurodegenerative disorder in which the formation of amyloid-β (Aβ) aggregates plays a causative role. There is ample evidence that Cu(II) can bind to Aβ and modulate its aggregation. Moreover, Cu(II) bound to Aβ might be involved in the production of reactive oxygen species, a process supposed to be involved in the Alzheimer's disease. The native Aβ40 contains a high affinity binding site for Cu(II), which is comprised in the N-terminal portion. Thus, Aβ16 (amino acid 1-16 of Aβ) has often been used as a model for Cu(II)-binding to monomeric Aβ. The Cu(II)-binding to Aβ is pH dependent and at pH 7.4, two different type of Cu(II) coordinations exist in equilibrium. These two forms are predominant at pH 6.5 and pH 9.0. In either form, a variety of studies show that the N-terminal Asp and the three His play a key role in the coordination, although the exact binding of these amino acids has not been addressed. Therefore, we studied the coordination modes of Cu(II) at pH 6.5 and 9.0 with the help of Fourier transform infrared (FTIR) spectroscopy. Combined with isotopic labeling of the amino acids involved in the coordination sphere, the data points toward the coordination of Cu(II) via the carboxylate of Asp1 at both pH values in a pseudobridging monovalent fashion. At low pH, His6 binds copper via Nτ, while His13 and His14 are bound via Nπ. At high pH, direct evidence is given on the coordination of Cu(II) via the Nτ atom of His6. Additionally, this study clearly shows the effect of Cu(II) binding on the protonation state of the His residues where a proton displacement takes places on the nitrogen atoms of the imidazole ring.
This study compares the behavior as cytochrome c oxidase (CcO) functional and structural models of a series of reported and unreported ligands that provide either a binding site for copper without a built-in proximal base, or both a flexible binding site for copper and a built-in proximal base, or a fixed binding site for copper with a built-in proximal base. The comparisons of the models show that the relative position of the two metal sites is not only a crucial parameter in the control of the catalytic behavior but also essential in mimicking other features of the enzyme such as CO exchange between the ferrous heme a(3) and the cuprous Cu(B) center.
The Cytochrome bo3 ubiquinol oxidase (QOX) from Escherichia coli (E. coli) contains a redox-active quinone, the so-called "high-affinity" QH quinone. The location of this cofactor and its binding site has yet to be accurately determined by X-ray crystallographic studies. Based on site-directed mutagenesis studies, a putative quinone binding site in the protein has been proposed. The exact binding partner of this cofactor and also whether it is stabilised as an anionic semiquinone or as a neutral radical species is a matter of some speculation. Both Hyperfine Sub-level Correlation (HYSCORE) and Double Nuclear Coherence Transfer Spectroscopy (DONUT-HYSCORE) spectroscopy as well as density functional theory (DFT) have been applied to investigate the QH binding site in detail to resolve these issues. Use is made of site-directed variants as well as globally 15N/14N-exchanged protein. Comparison of computed and experimental 13C hyperfine tensors provides strong support for the binding of the semiquinone radical in an anionic rather than a neutral protonated form. These results are compared with the corresponding information available on other protein binding sites and/or on model systems and are discussed with regard to the location and potential function of QH in the overall mechanism of function of this family of haem copper oxidases.
Herein, we present the development of a far-infrared spectroscopic approach for studying metalloenzyme active sites in a redox-dependent manner. An electrochemical cell with 5 mm path and based on silicon windows was found to be appropriate for the measurement of aqueous solutions down to 200 cm(-1) . The cell was probed with the infrared redox signature of the metal-ligand vibrations of different iron-sulfur proteins. Each Fe-S cluster type was found to show a specific spectral signature. As a common feature, a downshift of the frequency of the Fe-S vibrations was seen upon reduction, in line with the increase of the Fe-S bond. This downshift was found to be fully reversible. Electrochemically induced FTIR difference spectroscopy in the far infrared is now possible, opening new perspectives on the understanding of metalloproteins in function of the redox state.
The electrochemical behavior of three proteins fragments from the respiratory chain of the extremophilic bacterium Thermus thermophilus , namely, cytochrome c(1) (Cyt-c(1)), cytochrome c(552) (Cyt-c(552)), and Cu(A), immobilized on three-dimensional gold nanoparticles electrodes was investigated by cyclic voltammetry. The gold nanoparticles were modified by either dithiobissuccinimidyl propionate (DTSP) or a mixed self-assembled monolayer of 6-mercaptohexan-1-ol and hexanethiol, depending on the surface of the protein. High surface coverages with enzymes and good electron transfer rates were achieved in the case of Cyt-c(1) immobilized on DTSP-modified gold nanoparticles and Cyt-c(552) or Cu(A) immobilized on mixed SAMs-modified gold nanoparticles. Interestingly, high surface coverages with Cu(A) were also observed on DTSP-modified gold nanoparticles, but a slower electron transfer rate was determined in this case. The gold nanoparticle/protein assemblies were characterized by surface-enhanced IR spectroscopy and transmission electron microscopy.
The cytochrome (cyt) bc(1) complex (cyt bc(1)) plays a major role in the electrogenic extrusion of protons across the membrane responsible for the proton motive force to produce ATP. Proton-coupled electron transfer underlying the catalysis of cyt bc(1) is generally accepted, but the molecular basis of coupling and associated proton efflux pathway(s) remains unclear. Herein we studied Zn(2+)-induced inhibition of Rhodobacter capsulatus cyt bc(1) using enzyme kinetics, isothermal titration calorimetry (ITC), and electrochemically induced Fourier transform infrared (FTIR) difference spectroscopy with the purpose of understanding the Zn(2+) binding mechanism and its inhibitory effect on cyt bc(1) function. Analogous studies were conducted with a mutant of cyt b, E295, a residue previously proposed to bind Zn(2+) on the basis of extended X-ray absorption fine-structure spectroscopy. ITC analysis indicated that mutation of E295 to valine, a noncoordinating residue, results in a decrease in Zn(2+) binding affinity. The kinetic study showed that wild-type cyt bc(1) and its E295V mutant have similar levels of apparent K(m) values for decylbenzohydroquinone as a substrate (4.9 ± 0.2 and 3.1 ± 0.4 μM, respectively), whereas their K(I) values for Zn(2+) are 8.3 and 38.5 μM, respectively. The calorimetry-based K(D) values for the high-affinity site of cyt bc(1) are on the same order of magnitude as the K(I) values derived from the kinetic analysis. Furthermore, the FTIR signal of protonated acidic residues was perturbed in the presence of Zn(2+), whereas the E295V mutant exhibited no significant change in electrochemically induced FTIR difference spectra measured in the presence and absence of Zn(2+). Our overall results indicate that the proton-active E295 residue near the Q(o) site of cyt bc(1) can bind directly to Zn(2+), resulting in a decrease in the electron transferring activity without changing drastically the redox potentials of the cofactors of the enzyme. We conclude that E295 is involved in proton efflux coupled to electron transfer at the Q(o) site of cyt bc(1).
In this work we analyzed the specificity of the amide VI band for different types of secondary structure elements in protein structures. This band involves the bending motion of the CO group of the peptide chain that is typically observed in the spectral region from 590 to 490 cm−1. The infrared absorbance spectra of a set of polypeptide model compounds of well known secondary structure was obtained at defined pH, including poly (l-lysine), poly (l-tyrosine), poly (l-alanine) and poly (l-histidine). In addition spectra of membrane proteins from the respiratory chain, namely the NADH:ubiquinone oxidoreductase, the cytochrome c oxidase and its CuA fragment, the cytochrome bc1 complex, a Rieske-type protein and in addition myoglobin, have been comparatively investigated. The systematic analysis of the amide VI band of the polypeptides and the proteins allowed correlating the signal appearing at ∼525 cm−1 to α-helical structures and signals at ∼545 cm−1 to β-sheet contributions. Random coils have been found to contribute at ∼535 cm−1 while the β-turns were observed at ∼560 cm−1.
The catalytic activity of the respiratory NADH:ubiquinone oxidoreductase (complex I) is based on conformational reorganizations. Herein we probe the effect of substrates on the conformational flexibility of complex I by means of (1)H/(2)H exchange kinetics at the level of the amide proton in the mid-infrared spectral range (1700-1500 cm(-1)). Slow, medium, and fast exchanging domains are distinguished that reveal different accessibilities to the solvent. Whereas amide hydrogens undergo rapid exchange with the solvent in an open structure, hydrogens experience much slower exchange when they are involved in H-bonded structures or when they are sterically inaccessible for the solvent. The results indicate a structure that is more open in the presence of both NADH and quinon. Complementary information on the overall internal hydrogen bonding of the protein was probed in the far infrared (300-30 cm(-1)), a spectral range that includes a continuum mode of the hydrogen bonding signature.
Spectroscopic techniques that use the low frequency region are strongly emerging for the study of biological molecules. Far infrared and far Raman spectroscopies, THz time domain approaches and inelastic neutron scattering reveal the presence of vibrational modes involving inter-and intramolecular hydrogen bonding. Due to their collective nature, such modes are highly sensitive to the conformational state of the molecule. Here the influence of the secondary structure on these vibrational features in the far infrared for model compounds and proteins of well known structure are described. Since temperature has a large effect on hydrogen bonding, the development of the signature of poly-L-lysine between 14 and 294 K is presented. The data does not only reveal the increase of the number of hydrogen bonds with temperature, but also the reorganizations within the structures.
A new way to study the electrochemical properties of proteins by coupling front-face fluorescence spectroscopy with an optically transparent thin-layer electrochemical cell is presented. First, the approach was examined on the basis of the redox-dependent conformational changes in tryptophans in cytochrome c, and its redox potential was successfully determined. Second, an electrochemically induced fluorescence analysis of periplasmic thiol-disulfide oxidoreductases SoxS and SoxW was performed. SoxS is essential for maintaining chemotrophic sulfur oxidation of Paracoccus pantotrophus active in vivo, while SoxW is not essential. According to the potentiometric redox titration of tryptophan fluorescence, the midpoint potential of SoxS was -342 ± 8 mV versus the standard hydrogen electrode (SHE') and that of SoxW was -256 ± 10 mV versus the SHE'. The fluorescence properties of the thioredoxins are presented and discussed together with the intrinsic fluorescence contribution of the tyrosines.
The use of the far-infrared spectral range presents a novel approach for analysis of the hydrogen bonding in proteins. Here it is presented for the analysis of Fe--S vibrations (500-200 cm(-1)) and of the intra- and intermolecular hydrogen bonding signature (300-50 cm(-1)) in the Rieske protein from Thermus thermophilus as a function of temperature and pH. Three pH values were adequately chosen in order to study all the possible protonation states of the coordinating histidines. The Fe--S vibrations showed pH-dependent shifts in the FIR spectra in line with the change of protonation state of the histidines coordinating the [2Fe--2S] cluster. Measurements of the low-frequency signals between 300 and 30 K demonstrated the presence of a distinct overall hydrogen bonding network and a more rigid structure for a pH higher than 10. To further support the analysis, the redox-dependent shifts of the secondary structure were investigated by means of an electrochemically induced FTIR difference spectroscopic approach in the mid infrared. The results confirmed a clear pH dependency and an influence of the immediate environment of the cluster on the secondary structure. The results support the hypothesis that structure-mediated changes in the environment of iron--sulfur centers play a critical role in regulating enzymatic catalysis. The data point towards the role of the overall internal hydrogen bonding organization for the geometry and the electronic properties of the cluster.
Cytochrome oxidases are perfect model substrates for analyzing the assembly of multisubunit complexes because the need for cofactor incorporation adds an additional level of complexity to their assembly. cbb3-type cytochrome c oxidases (cbb3-Cox) consist of the catalytic subunit CcoN, the membrane-bound c-type cytochrome subunits CcoO and CcoP, and the CcoQ subunit, which is required for cbb3-Cox stability. Biogenesis of cbb3-Cox proceeds via CcoQP and CcoNO subcomplexes, which assemble into the active cbb3-Cox. Most bacteria expressing cbb3-Cox also contain the ccoGHIS genes, which encode putative cbb3-Cox assembly factors. Their exact function, however, has remained unknown. Here we analyzed the role of CcoH in cbb3-Cox assembly and showed that CcoH is a single spanning-membrane protein with an N-terminus-out-C-terminus-in (Nout-Cin) topology. In its absence, neither the fully assembled cbb3-Cox nor the CcoQP or CcoNO subcomplex was detectable. By chemical cross-linking, we demonstrated that CcoH binds primarily via its transmembrane domain to the CcoP subunit of cbb3-Cox. A second hydrophobic stretch, which is located at the C terminus of CcoH, appears not to be required for contacting CcoP, but deleting it prevents the formation of the active cbb3-Cox. This suggests that the second hydrophobic domain is required for merging the CcoNO and CcoPQ subcomplexes into the active cbb3-Cox. Surprisingly, CcoH does not seem to interact only transiently with the cbb3-Cox but appears to stay tightly associated with the active, fully assembled complex. Thus, CcoH behaves more like a bona fide subunit of the cbb3-Cox than an assembly factor per se.
The proton-pumping NADH:ubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Electron microscopy revealed the two-part structure of the complex with a peripheral arm involved in electron transfer and a membrane arm most likely involved in proton translocation. It was proposed that the quinone binding site is located at the joint of the two arms. Most likely, proton translocation in the membrane arm is enabled by the energy of the electron transfer reaction in the peripheral arm transmitted by conformational changes. For the detection of the conformational changes and the localization of the quinone binding site, we set up a combination of site-directed spin labeling and EPR spectroscopy. Cysteine residues were introduced to the surface of the Escherichia coli complex I. The spin label (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfonate (MTSL) was exclusively bound to the engineered positions. Neither the mutation nor the labeling had an effect on the NADH:decyl-ubiquinone oxidoreductase activity. The characteristic signals of the spin label were detected by EPR spectroscopy, which did not change by reducing the preparation with NADH. A decyl-ubiquinone derivative with the spin label covalently attached to the alkyl chain was synthesized in order to localize the quinone binding site. The distance between a MTSL labeled complex I variant and the bound quinone was determined by continuous-wave (cw) EPR allowing an inference on the location of the quinone binding site. The distances between the labeled quinone and other complex I variants will be determined in future experiments to receive further geometry information by triangulation.
The use of the far IR spectral range presents a novel approach for analysis of proteins. Here it is presented for the analysis of Fe-S vibrations (500-200 cm-1) and of the intra-and intermolecular H-bonding signature (300-50 cm-1) in Rieske proteins and lipids in function of T, redox state and pH.
The proton-pumping NADH:ubiquinone oxidoreductase couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. This process is suggested to be accompanied by conformational changes of the enzyme that may be monitored by redox-induced FT-IR difference spectroscopy. Signals observed in the amide I range are partially attributed to local rearrangements that occur as an electrostatic response to the redox reactions of the FeS clusters. In addition, conformational changes can be reported that depend on pH and at the same time can be perturbed by site-directed mutagenesis of residue E67 on subunit B (the bacterial homologue of the mitochondrial PSST subunit). This residue is located in the vicinity of the cluster N2. Re-evaluating these previous data we here discuss a mechanism, by which the redox reaction of N2 induces conformational changes possibly leading to proton translocation.
In the genome of the untypical cyanobacterium Gloeobacter violaceus PCC 7421 two potential cytochrome b (6) proteins PetB1 and PetB2 are encoded. Such a situation has not been observed in cyanobacteria, algae and higher plants before, and both proteins are not characterized at all yet. Here, we show that both apo-proteins bind heme with high affinity and the spectroscopic characteristics of both holo-proteins are distinctive for cytochrome b (6) proteins. However, while in PetB2 one histidine residue, which corresponds to H100 and serves as an axial ligand for heme b (H) in PetB1, is mutated, both PetB proteins bind two heme molecules with different midpoint potentials. To recreate the canonical heme b (H) binding cavity in PetB2 we introduced a histidine residue at the position corresponding to H100 in PetB1 and subsequently characterized the generated protein variant. The presented data indicate that two bona fide cytochrome b (6) proteins are encoded in Gloeobacter violaceus. Furthermore, the two petB genes of Gloeobacter violaceus are each organized in an operon together with a petD gene. Potential causes and consequences of the petB and petD gene heterogeneity are discussed.
Far infrared spectra of zwitterionic, cationic, and anionic forms of aliphatic amino acids in solid state have been studied experimentally. Measurements were done on glycine, L-alanine, L-valine, L-leucine, and L-isoleucine powder samples and film samples obtained from dried solutions prepared at pH ranging from 1 to 13. Solid state density functional theory calculations were also performed, and detailed potential energy distributions were obtained from normal mode results. A good correspondence between experimental and simulated spectra was achieved and this allowed us to propose an almost complete band assignment for the far infrared spectra of zwitterionic forms. In the 700-50 cm(-1) range, three regions were identified, each corresponding to a characteristic set of normal modes. A first region between 700 and 450 cm(-1) mainly contained the carboxylate bending, rocking, and wagging modes as well as the ammonium torsional mode. The 450-250 cm(-1) region was representative of backbone and sidechain skeletal bending modes. At last, the low wavenumber zone, below 250 cm(-1), was characteristic of carboxylate and skeletal torsional modes and of lattice modes. Assignments are also proposed for glycine cationic and anionic forms, but could not be obtained for all aliphatic amino acids due to the lack of structural data. This work is intended to provide fundamental information for the understanding of peptides vibrational properties.
The basic motions and the conformational flexibility of a protein have a strong impact on its molecular recognition properties and ultimately on its function. In the far infrared (or THz) spectral range the breathing of the hydrogen bonds can be monitored, providing essential information on local dynamics and mechanism. The use of this spectral range is rapidly evolving and a number of IR synchrotron beamlines are available for this research. Here we present a study on the I-domain of the integrin LFA-1, an allosteric receptor that transmits signals across the plasma membrane in a bidirectional way. The I-domain contains the principal binding site for extracellular ligands and thus crucial for the signaling and the integrin-mediated cell adhesion. We measured the temperature dependence of the conformational dynamics of the I-domain bound to four different divalent metal ions (Mg2+, Ca2+, Mn2+ and Fe2+) in the range 10-300 K. The H-bonding vibrations show distinct temperature dependences for the different samples.
Phospholipids are studied by means of Fourier transform infrared (FTIR) spectroscopy in the mid- and far-infrared spectral ranges, thereby establishing the hydrogen-bonding continuum as a function of the temperature. The well-known mid-infrared spectrum of the phospholipid layer clearly shows a temperature-dependent phase transition. In the far-infrared region (from 300 to 50 cm(-1)), an alternation of the interaction between the phospholipids and water molecules is found. The hydrogen-bonding network ensemble and bound water molecules can be monitored in this spectral region. The lipid structure is found to strongly influence the intermolecular hydrogen-bonding interplay. Thus, studies in the far-infrared region provide significant information--at the molecular level--about the intermolecular hydrogen-bonding signature of self-assembled phospholipids.
Papillomavirus E6 oncoproteins bind and often provoke the degradation of many cellular proteins important for the control of cell proliferation and/or cell death. Structural studies on E6 proteins have long been hindered by the difficulties of obtaining highly concentrated samples of recombinant E6. Here, we show that recombinant E6 proteins from eight human papillomavirus strains and one bovine papillomavirus strain exist as oligomeric and multimeric species. These species were characterized using a variety of biochemical and biophysical techniques, including analytical gel filtration, activity assays, surface plasmon resonance, electron microscopy and Fourier transform infrared spectroscopy. The characterization of E6 oligomers is facilitated by the fusion to the maltose binding protein, which slows the formation of higher-order multimeric species. The proportion of each oligomeric form varies depending on the viral strain considered. Oligomers appear to consist of folded units, which, in the case of high-risk mucosal human papillomavirus E6, retain binding to the ubiquitin ligase E6-associated protein and the capacity to degrade the proapoptotic protein p53. In addition to the small-size oligomers, E6 proteins spontaneously assemble into large organized multimeric structures, a process that is accompanied by a significant increase in the beta-sheet secondary structure content. Finally, co-localisation experiments using E6 equipped with different tags further demonstrate the occurrence of E6 self-association in eukaryotic cells. The ensemble of these data suggests that self-association is a general property of E6 proteins that occurs both in vitro and in vivo and might therefore be functionally relevant.
2'-(1-Imidazolyl)-4-methylphenol (C-N bonding in the ortho' position at the phenyl group), a model compound for a tyrosine-histidine covalent linkage, was studied with a combined electrochemical and UV-vis/IR spectroscopic approach. Electrochemical analysis of the 2'-(1-imidazolyl)-4-methylphenol model compound by the means of cyclic voltammetry yielded a potential of 0.48 vs ferrocene (1.15 V vs NHE) for the oxidation of the deprotonated form, the reaction being kinetically irreversible. A tentative assignment of the electrochemically induced Fourier transform infrared (FTIR) difference infrared spectra is presented that indicates the deprotonation of the tyrosine before oxidation and importantly the strong influence of the solvent on the spectral properties and on the mechanism of radical formation. Fluorescence lifetimes and pre-exponential factors of the tyrosine-histidine model compounds are presented and discussed in comparison to tyrosine. The tyrosine-histidine fluorescence lifetime is found to be solvent dependent. The influence of the solvent on the reaction mechanism is proposed with regard to the mechanism of electron coupled proton transfer in proteins that include covalently linked amino acid side chains, like the cytochrome c oxidase.
Specific protein-lipid interactions have been identified in X-ray structures of membrane proteins. The role of specifically bound lipid molecules in protein function remains elusive. In the current study, we investigated how phospholipids influence catalytic, spectral and electrochemical properties of the yeast respiratory cytochrome bc(1) complex and how disruption of a specific cardiolipin binding site in cytochrome c(1) alters respiratory supercomplex formation in mitochondrial membranes. Purified yeast cytochrome bc(1) complex was treated with phospholipase A(2). The lipid-depleted enzyme was stable but nearly catalytically inactive. The absorption maxima of the reduced b-hemes were blue-shifted. The midpoint potentials of the b-hemes of the delipidated complex were shifted from -52 to -82 mV (heme b(L)) and from +113 to -2 mV (heme b(H)). These alterations could be reversed by reconstitution of the delipidated enzyme with a mixture of asolectin and cardiolipin, whereas addition of the single components could not reverse the alterations. We further analyzed the role of a specific cardiolipin binding site (CL(i)) in supercomplex formation by site-directed mutagenesis and BN-PAGE. The results suggested that cardiolipin stabilizes respiratory supercomplex formation by neutralizing the charges of lysine residues in the vicinity of the presumed interaction domain between cytochrome bc(1) complex and cytochrome c oxidase. Overall, the study supports the idea, that enzyme-bound phospholipids can play an important role in the regulation of protein function and protein-protein interaction.
Biochemical studies have shown that cardiolipin is essential for the integrity and activity of the cytochrome bc(1) complex and many other membrane proteins. Recently the direct involvement of a bound cardiolipin molecule (CL) for proton uptake at center N, the site of quinone reduction, was suggested on the basis of a crystallographic study. In the study presented here, we probe the low frequency infrared spectroscopy region as a technique suitable to detect the involvement of the lipids in redox induced reactions of the protein. First the individual infrared spectroscopic features of lipids, typically present in the yeast membrane, have been monitored for different pH values in micelles and vesicles. The pK(a) values for cardiolipin molecule have been observed at 4.7+/-0.3 and 7.9+/-1.3, respectively. Lipid contributions in the electrochemically induced FTIR spectra of the bc(1) complex from yeast have been identified by comparing the spectra of the as isolated form, with samples where the lipids were digested by lipase-A(2). Overall, a noteworthy perturbation in the spectral region typical for the protein backbone can be reported. Interestingly, signals at 1159, 1113, 1039 and 980 cm(-1) have shifted, indicating the perturbation of the protonation state of cardiolipin coupled to the reduction of the hemes. Additional shifts are found and are proposed to reflect lipids reorganizing due to a change in their direct environment upon the redox reaction of the hemes. In addition a small shift in the alpha band from 559 to 556 nm can be seen after lipid depletion, reflecting the interaction with heme b(H) and heme c. Thus, our work highlights the role of lipids in enzyme reactivity and structure.