Stanley J Opella

University of California, San Diego, San Diego, California, United States

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Publications (291)1439.29 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The highly anisotropic environment of the lipid bilayer membrane imposes significant constraints on the structures and functions of membrane proteins. However, NMR structure calculations typically use a simple repulsive potential that neglects the effects of solvation and electrostatics, because explicit atomic representation of the solvent and lipid molecules is computationally expensive and impractical for routine NMR-restrained calculations that start from completely extended polypeptide templates. Here, we describe the extension of a previously described implicit solvation potential, eefxPot, to include a membrane model for NMR-restrained calculations of membrane protein structures in XPLOR-NIH. The key components of eefxPot are an energy term for solvation free energy that works together with other nonbonded energy functions, a dedicated force field for conformational and nonbonded protein interaction parameters, and a membrane function that modulates the solvation free energy and dielectric screening as a function of the atomic distance from the membrane center, relative to the membrane thickness. Initial results obtained for membrane proteins with structures determined experimentally in lipid bilayer membranes show that eefxPot affords significant improvements in structural quality, accuracy, and precision. Calculations with eefxPot are straightforward to implement and can be used to both fold and refine structures, as well as to run unrestrained molecular-dynamics simulations. The potential is entirely compatible with the full range of experimental restraints measured by various techniques. Overall, it provides a useful and practical way to calculate membrane protein structures in a physically realistic environment. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.
    Biophysical Journal 08/2015; 109(3):574-85. DOI:10.1016/j.bpj.2015.06.047 · 3.97 Impact Factor
  • Stanley J Opella
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    ABSTRACT: Many viruses express small hydrophobic membrane proteins. These proteins are often referred to as viroporins because they exhibit ion channel activity. However, the channel activity has not been definitively associated with a biological function in all cases. More generally, protein-protein and protein-phospholipid interactions have been associated with specific biological activities of these proteins. As research has progressed there is a decreased emphasis on potential roles of the channel activity, and increased research on multiple other biological functions. This being the case, it may be more appropriate to refer to them as 'viral membrane-spanning miniproteins'. Structural studies are illustrated with Vpu from HIV-1 and p7 from HCV. Copyright © 2015 Elsevier B.V. All rights reserved.
    Current Opinion in Virology 06/2015; 12. DOI:10.1016/j.coviro.2015.05.006 · 6.30 Impact Factor
  • Source
    Mary K Lewinski · Moein Jafari · Hua Zhang · Stanley J Opella · John Guatelli
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    ABSTRACT: The restriction factor BST2 (tetherin) prevents the release of enveloped viruses from the host cell and is counteracted by HIV-1 Vpu. Vpu and BST2 interact directly via their transmembrane domains. This interaction enables Vpu to induce the surface-downregulation and the degradation of BST2, but neither of these activities fully accounts for Vpu's ability to enhance virion release. During a study of naturally occurring Vpu proteins, we found that a tryptophan residue near the Vpu C-terminus is particularly important for enhancing virion release. Vpu proteins with a W76G polymorphism degraded and downregulated BST2 from the cell surface, yet they inefficiently stimulated virion release. Here we explore the mechanism of this anomaly. We find that W76 is critical for the ability of Vpu to displace BST2 from sites of viral assembly in the plane of the plasma membrane. This effect does not appear to involve a general reorganization of the membrane microdomains associated with virion assembly, but rather is a specific effect of Vpu on BST2. Using NMR spectroscopy, we find that the cytoplasmic domain of Vpu and W76 specifically interact with lipids. Moreover, paramagnetic relaxation enhancement studies show that W76 inserts into the lipid. These data are consistent with a model whereby W76 anchors the C-terminus of Vpu's cytoplasmic tail to the plasma membrane, enabling the movement of Vpu-bound BST2 away from viral assembly sites. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 03/2015; 290(17). DOI:10.1074/jbc.M114.630095 · 4.57 Impact Factor
  • Ye Tian · Charles Schwieters · Stanley Opella · Francesca Marassi
    Biophysical Journal 01/2015; 108(2):251a. DOI:10.1016/j.bpj.2014.11.1389 · 3.97 Impact Factor
  • Stanley J. Opella
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    ABSTRACT: The native environment for a membrane protein is a phospholipid bilayer. Because the protein is immobilized on NMR timescales by the interactions within a bilayer membrane, solid-state NMR methods are essential to obtain high-resolution spectra. Approaches have been developed for both unoriented and oriented samples, however, they all rest on the foundation of the most fundamental aspects solid-state NMR, and the chemical shift and homo- and hetero- nuclear dipole-dipole interactions. Solid-state NMR has advanced sufficiently to enable the structures of membrane proteins to be determined under near-native conditions in phospholipid bilayers.
    Journal of Magnetic Resonance 12/2014; 253. DOI:10.1016/j.jmr.2014.11.015 · 2.32 Impact Factor
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    ABSTRACT: The use of paramagnetic constraints in protein NMR is an active area of research because of the benefits of long-range distance measurements (>10 Å). One of the main issues in successful execution is the incorporation of a paramagnetic metal ion into diamagnetic proteins. The most common metal ion tags are relatively long aliphatic chains attached to the side chain of a selected cysteine residue with a chelating group at the end where it can undergo substantial internal motions, decreasing the accuracy of the method. An attractive alternative approach is to incorporate an unnatural amino acid that binds metal ions at a specific site on the protein using the methods of molecular biology. Here we describe the successful incorporation of the unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA) into two different membrane proteins by heterologous expression in E. coli. Fluorescence and NMR experiments demonstrate complete replacement of the natural amino acid with HQA and stable metal chelation by the mutated proteins. Evidence of site-specific intra- and inter-molecular PREs by NMR in micelle solutions sets the stage for the use of HQA incorporation in solid-state NMR structure determinations of membrane proteins in phospholipid bilayers.
    Journal of Biomolecular NMR 11/2014; 61(3-4). DOI:10.1007/s10858-014-9884-5 · 3.31 Impact Factor
  • Chin H. Wu · Anna A. De Angelis · Stanley J. Opella
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    ABSTRACT: The efficiency and selectivity of SPECIFIC-CP, a widely used method for selective double cross-polarization in triple-resonance magic angle spinning solid-state NMR, is improved by performing the tangential-shaped (13)C irradiation at an offset frequency that meets the Lee-Goldburg condition (LG-SPECIFIC-CP). This is demonstrated on polycrystalline samples of uniformly (13)C, (15)N labeled N-acetyl-leucine and N-formyl-Met-Leu-Phe-OH (MLF) at 700MHz and 900MHz (1)H resonance frequencies, respectively. For the single (13)Cα of N-acetyl-leucine, relative to conventional broad band cross-polarization, the SPECIFIC-CP signal has 47% of the intensity. Notably, the LG-SPECIFIC-CP signal has 72% of the intensity, essentially the theoretical maximum. There were no other changes in the experimental parameters. The three (13)Cα signals in MLF show some variation in intensities, reflecting the relatively narrow bandwidth of a frequency-offset procedure, and pointing to future developments for this class of experiment.
    Journal of Magnetic Resonance 09/2014; 246. DOI:10.1016/j.jmr.2014.06.012 · 2.32 Impact Factor
  • Source
    Ye Tian · George J Lu · Francesca M Marassi · Stanley J Opella
    Journal of Biomolecular NMR 08/2014; 60(1). DOI:10.1007/s10858-014-9852-0 · 3.31 Impact Factor
  • Bibhuti B. Das · Stanley J. Opella
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    ABSTRACT: Multiple acquisition spectroscopy (MACSY) experiments that enable multiple free induction decays to be recorded during individual experiments are demonstrated. In particular, the experiments incorporate separated local field spectroscopy into homonuclear and heteronuclear correlation spectroscopy. The measured heteronuclear dipolar couplings are valuable in structure determination as well as in enhancing resolution by providing an additional frequency axis. In one example four different three-dimensional spectra are obtained in a single experiment, demonstrating that substantial potential saving in experimental time is available when multiple multi-dimensional spectra are required as part of solid-state NMR studies.
    Journal of Magnetic Resonance 08/2014; 245. DOI:10.1016/j.jmr.2014.06.011 · 2.32 Impact Factor
  • Source
    Jasmina Radoicic · George J Lu · Stanley J Opella
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    ABSTRACT: Membrane proteins have always presented technical challenges for structural studies because of their requirement for a lipid environment. Multiple approaches exist including X-ray crystallography and electron microscopy that can give significant insights into their structure and function. However, nuclear magnetic resonance (NMR) is unique in that it offers the possibility of determining the structures of unmodified membrane proteins in their native environment of phospholipid bilayers under physiological conditions. Furthermore, NMR enables the characterization of the structure and dynamics of backbone and side chain sites of the proteins alone and in complexes with both small molecules and other biopolymers. The learning curve has been steep for the field as most initial studies were performed under non-native environments using modified proteins until ultimately progress in both techniques and instrumentation led to the possibility of examining unmodified membrane proteins in phospholipid bilayers under physiological conditions. This review aims to provide an overview of the development and application of NMR to membrane proteins. It highlights some of the most significant structural milestones that have been reached by NMR spectroscopy of membrane proteins, especially those accomplished with the proteins in phospholipid bilayer environments where they function.
    Quarterly Reviews of Biophysics 07/2014; 47(03):1-35. DOI:10.1017/S0033583514000080 · 10.08 Impact Factor
  • Bibhuti B Das · Sang Ho Park · Stanley J Opella
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    ABSTRACT: The motional averaging of powder pattern lineshapes is one of the most fundamental aspects of sold-state NMR. Since membrane proteins in liquid crystalline phospholipid bilayers undergo fast rotational diffusion, all of the signals reflect the angles of the principal axes of their dipole-dipole and chemical shift tensors with respect to the axis defined by the bilayer normal. The frequency span and sign of the axially symmetric powder patterns that result from motional averaging about a common axis provide sufficient structural restraints for the calculation of the three-dimensional structure of a membrane protein in a phospholipid bilayer environment. The method is referred to as rotationally aligned (RA) solid-state NMR and demonstrated with results on full-length, unmodified membrane proteins with one, two, and seven trans-membrane helices. RA solid-state NMR is complementary to other MAS solid-state NMR methods, in particular oriented sample (OS) solid-state NMR of stationary, aligned samples. Structural distortions of membrane proteins from the truncations of terminal residues and other sequence modifications, and the use of detergent micelles instead of phospholipid bilayers have also been demonstrated. Thus, it is highly advantageous to determine the structures of unmodified membrane proteins in liquid crystalline phospholipid bilayers under physiological conditions. RA solid-state NMR provides a general method for obtaining accurate and precise structures of membrane proteins under near-native conditions. This article is part of a Special Issue entitled: NMR Spectroscopy for Atomistic Views of Biomembranes and Cell Surfaces.
    Biochimica et Biophysica Acta 04/2014; 1848(1). DOI:10.1016/j.bbamem.2014.04.002 · 4.66 Impact Factor
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    ABSTRACT: The benefits of protein structure refinement in water are well documented. However, performing structure refinement with explicit atomic representation of the solvent molecules is computationally expensive and impractical for NMR-restrained structure calculations that start from completely extended polypeptide templates. Here we describe a new implicit solvation potential, EEFx (Effective Energy Function for XPLOR-NIH), for NMR-restrained structure calculations of proteins in XPLOR-NIH. The key components of EEFx are an energy term for solvation energy that works together with other nonbonded energy functions, and a dedicated force field for conformational and nonbonded protein interaction parameters. The initial results obtained with EEFx show that significant improvements in structural quality can be obtained. EEFx is computationally efficient and can be used both to fold and refine structures. Overall, EEFx improves the quality of protein conformation and nonbonded atomic interactions. Moreover, such benefits are accompanied by enhanced structural precision and enhanced structural accuracy, reflected in improved agreement with the cross-validated dipolar coupling data. Finally, implementation of EEFx calculations is straightforward and computationally efficient. Overall, EEFx provides a useful method for the practical calculation of experimental protein structures in a physically realistic environment.
    Journal of Magnetic Resonance 04/2014; 243C:54-64. DOI:10.1016/j.jmr.2014.03.011 · 2.32 Impact Factor
  • Source
    George J Lu · Stanley J Opella
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    ABSTRACT: In the stationary, aligned samples used in oriented sample (OS) solid-state NMR, (1)H-(1)H homonuclear dipolar couplings are not attenuated as they are in magic angle spinning solid-state NMR; consequently, they are available for participation in dipolar coupling-based spin-exchange processes. Here we describe analytically the pathways of (15)N-(15)N spin-exchange mediated by (1)H-(1)H homonuclear dipolar couplings. The mixed-order proton-relay mechanism can be differentiated from the third spin assisted recoupling mechanism by setting the (1)H to an off-resonance frequency so that it is at the "magic angle" during the spin-exchange interval in the experiment, since the "magic angle" irradiation nearly quenches the former but only slightly attenuates the latter. Experimental spectra from a single crystal of N-acetyl leucine confirm that this proton-relay mechanism plays the dominant role in (15)N-(15)N dilute-spin-exchange in OS solid-state NMR in crystalline samples. Remarkably, the "forbidden" spin-exchange condition under "magic angle" irradiation results in (15)N-(15)N cross-peaks intensities that are comparable to those observed with on-resonance irradiation in applications to proteins. The mechanism of the proton relay in dilute-spin-exchange is crucial for the design of polarization transfer experiments.
    The Journal of Chemical Physics 03/2014; 140(12):124201. DOI:10.1063/1.4869345 · 3.12 Impact Factor
  • Bibhuti B Das · Hua Zhang · Stanley J Opella
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    ABSTRACT: A method for making resonance assignments in magic angle spinning solid-state NMR spectra of membrane proteins that utilizes the range of heteronuclear dipolar coupling frequencies in combination with conventional chemical shift based assignment methods is demonstrated. The Dipolar Assisted Assignment Protocol (DAAP) takes advantage of the rotational alignment of the membrane proteins in liquid crystalline phospholipid bilayers. Improved resolution is obtained by combining the magnetically inequivalent heteronuclear dipolar frequencies with isotropic chemical shift frequencies. Spectra with both dipolar and chemical shift frequency axes assist with resonance assignments. DAAP can be readily extended to three- and four-dimensional experiments and to include both backbone and side chain sites in proteins.
    Journal of Magnetic Resonance 03/2014; 242C:224-232. DOI:10.1016/j.jmr.2014.02.018 · 2.32 Impact Factor
  • Source
    Ye Tian · Charles Schwieters · Stanley J. Opella · Francesca M. Marassi
    Biophysical Journal 01/2014; 106(2):462a. DOI:10.1016/j.bpj.2013.11.2617 · 3.97 Impact Factor
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    Biophysical Journal 01/2014; 106(2):293a. DOI:10.1016/j.bpj.2013.11.1712 · 3.97 Impact Factor
  • Gabriel A. Cook · Lindsay A. Dawson · Bibhuti B. Das · Stanley J. Opella
    Biophysical Journal 01/2014; 106(2):47a. DOI:10.1016/j.bpj.2013.11.340 · 3.97 Impact Factor
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    ABSTRACT: While antimicrobial peptides (AMPs) have been widely investigated as potential therapeutics, high resolution structures obtained under biologically relevant conditions are lacking. Here, the high-resolution structures of the homologous 22-residue long AMPs piscidin 1 (p1) and piscidin 3 (p3) are determined in fluid-phase 3:1 phosphatidylcholine/phosphatidylglycerol (PC/PG) and 1:1 phosphatidylethanolamine/phosphatidylglycerol (PE/PG) bilayers to identify molecular features important for membrane destabilization in bacterial cell membrane mimics. Structural refinement of (1)H-(15)N dipolar couplings and (15)N chemical shifts measured by oriented sample solid-state NMR and all-atom molecular dynamics (MD) simulations provide structural and orientational information of high precision and accuracy about these interfacially-bound α-helical peptides. The tilt of the helical axis, τ, is between 83° and 93° with respect to the bilayer normal for all systems and analysis methods. The average azimuthal rotation, ρ, is 235°, which results in burial of hydrophobic residues in the bilayer. The refined NMR and MD structures reveal a slight kink at G13 that delineates two helical segments characterized by a small difference in their τ angles (<10°) and significant difference in their ρ angles (~25°). Remarkably, the kink, at the end of a G(X)4G motif highly conserved among members of the piscidin family, allows p1 and p3 to adopt ρ angles that maximize their hydrophobic moments. Two structural features differentiate the more potent p1 from p3: p1 has a larger ρ angle and less N-terminal fraying. The peptides have comparable depths of insertion in PC/PG but p3 is 2Å more deeply inserted than p1 in PE/PG. In contrast to the ideal α-helical structures typically assumed in mechanistic models of AMPs, p1 and p3 adopt disrupted α-helical backbones that correct for differences in the amphipathicity of their N- and C-ends, and their centers of mass lie approximately 1.2-3.6 Å below the plane defined by the C2 atoms of the lipid acyl chains.
    Journal of the American Chemical Society 01/2014; 136(9). DOI:10.1021/ja411119m · 11.44 Impact Factor
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    ABSTRACT: Acyl carrier protein (ACP) transports the growing fatty acid chain between enzymatic domains of fatty acid synthase (FAS) during biosynthesis. Because FAS enzymes operate on ACP-bound acyl groups, ACP must stabilize and transport the growing lipid chain. ACPs have a central role in transporting starting materials and intermediates throughout the fatty acid biosynthetic pathway. The transient nature of ACP-enzyme interactions impose major obstacles to obtaining high-resolution structural information about fatty acid biosynthesis, and a new strategy is required to study protein-protein interactions effectively. Here we describe the application of a mechanism-based probe that allows active site-selective covalent crosslinking of AcpP to FabA, the Escherichia coli ACP and fatty acid 3-hydroxyacyl-ACP dehydratase, respectively. We report the 1.9 Å crystal structure of the crosslinked AcpP-FabA complex as a homodimer in which AcpP exhibits two different conformations, representing probable snapshots of ACP in action: the 4'-phosphopantetheine group of AcpP first binds an arginine-rich groove of FabA, then an AcpP helical conformational change locks AcpP and FabA in place. Residues at the interface of AcpP and FabA are identified and validated by solution nuclear magnetic resonance techniques, including chemical shift perturbations and residual dipolar coupling measurements. These not only support our interpretation of the crystal structures but also provide an animated view of ACP in action during fatty acid dehydration. These techniques, in combination with molecular dynamics simulations, show for the first time that FabA extrudes the sequestered acyl chain from the ACP binding pocket before dehydration by repositioning helix III. Extensive sequence conservation among carrier proteins suggests that the mechanistic insights gleaned from our studies may be broadly applicable to fatty acid, polyketide and non-ribosomal biosynthesis. Here the foundation is laid for defining the dynamic action of carrier-protein activity in primary and secondary metabolism, providing insight into pathways that can have major roles in the treatment of cancer, obesity and infectious disease.
    Nature 12/2013; 505(7483). DOI:10.1038/nature12810 · 42.35 Impact Factor
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    George J Lu · Stanley J Opella
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    ABSTRACT: Oriented sample solid-state NMR spectroscopy can be used to determine the three-dimensional structures of membrane proteins in magnetically or mechanically aligned lipid bilayers. The bottleneck for applying this technique to larger and more challenging proteins is making resonance assignments, which is conventionally accomplished through the preparation of multiple selectively isotopically labeled samples and performing an analysis of residues in regular secondary structure based on Polarity Index Slant Angle (PISA) Wheels and Dipolar Waves. Here we report the complete resonance assignment of the full-length mercury transporter, MerF, an 81-residue protein, which is challenging because of overlapping PISA Wheel patterns from its two trans-membrane helices, by using a combination of solid-state NMR techniques that improve the spectral resolution and provide correlations between residues and resonances. These techniques include experiments that take advantage of the improved resolution of the MSHOT4-Pi4/Pi pulse sequence; the transfer of resonance assignments through frequency alignment of heteronuclear dipolar couplings, or through dipolar coupling correlated isotropic chemical shift analysis; (15)N/(15)N dilute spin exchange experiments; and the use of the proton-evolved local field experiment with isotropic shift analysis to assign the irregular terminal and loop regions of the protein, which is the major "blind spot" of the PISA Wheel/Dipolar Wave method.
    Journal of Biomolecular NMR 12/2013; 58(1). DOI:10.1007/s10858-013-9806-y · 3.31 Impact Factor

Publication Stats

11k Citations
1,439.29 Total Impact Points


  • 2001–2015
    • University of California, San Diego
      • Department of Chemistry and Biochemistry
      San Diego, California, United States
  • 2013
    • Sanford-Burnham Medical Research Institute
      La Jolla, California, United States
  • 2011
    • Hamilton College
      • Department of Chemistry
      Клинтон, New York, United States
    • University of Arkansas
      • Department of Chemistry and Biochemistry
      Fayetteville, Arkansas, United States
  • 1979–2008
    • University of Pennsylvania
      • Department of Chemistry
      Philadelphia, PA, United States
  • 2005
    • University of Massachusetts Amherst
      Amherst Center, Massachusetts, United States
  • 2004
    • University of San Diego
      • Department of Chemistry and Biochemistry
      San Diego, California, United States
  • 2003
    • National University (California)
      San Diego, California, United States
  • 1983–2003
    • William Penn University
      Filadelfia, Pennsylvania, United States
  • 1998–2000
    • Wistar Institute
      Philadelphia, Pennsylvania, United States
  • 1999
    • Noguchi Institute
      Edo, Tōkyō, Japan
  • 1988
    • University of Delaware
      • Department of Chemistry and Biochemistry
      Ньюарк, Delaware, United States
  • 1980–1986
    • Philadelphia University
      • Department of Chemistry
      Filadelfia, Pennsylvania, United States
  • 1977
    • Massachusetts Institute of Technology
      • Department of Chemistry
      Cambridge, MA, United States