Gabriel A Cook

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

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Publications (16)60.64 Total impact

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    ABSTRACT: Hepatitis C virus (HCV) protein p7 plays an important role in the assembly and release of mature virus particles. This small 63-residue membrane protein has been shown to induce channel activity, which may contribute to its functions. p7 is highly conserved throughout the entire range of HCV genotypes, which contributes to making p7 a potential target for antiviral drugs. The secondary structure of p7 from the J4 genotype and the tilt angles of the helices within bilayers have been previously characterized by nuclear magnetic resonance (NMR). Here we describe the three-dimensional structure of p7 in short chain phospholipid (1,2-dihexanoyl-sn-glycero-3-phosphocholine) micelles, which provide a reasonably effective membrane-mimicking environment that is compatible with solution NMR experiments. Using a combination of chemical shifts, residual dipolar couplings, and PREs, we determined the structure of p7 using an implicit membrane potential combining both CS-Rosetta decoys and Xplor-NIH refinement. The final set of structures has a backbone root-mean-square deviation of 2.18 Å. Molecular dynamics simulations in NAMD indicate that several side chain interactions might be taking place and that these could affect the dynamics of the protein. In addition to probing the dynamics of p7, we evaluated several drug–protein and protein–protein interactions. Established channel-blocking compounds such as amantadine, hexamethylene amiloride, and long alkyl chain iminosugar derivatives inhibit the ion channel activity of p7. It has also been shown that the protein interacts with HCV nonstructural protein 2 at the endoplasmic reticulum and that this interaction may be important for the infectivity of the virus. Changes in the chemical shift frequencies of solution NMR spectra identify the residues taking part in these interactions.
    Biochemistry 07/2013; 52(31):5295–5303. · 3.38 Impact Factor
  • Gabriel A. Cook, Lindsay Dawson, Stanley J. Opella
    Biophysical Journal 01/2012; 102(3):264-. · 3.67 Impact Factor
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    ABSTRACT: 'q-Titration' refers to the systematic comparison of signal intensities in solution NMR spectra of uniformly (15)N labeled membrane proteins solubilized in micelles and isotropic bicelles as a function of the molar ratios (q) of the long-chain lipids (typically DMPC) to short-chain lipids (typically DHPC). In general, as q increases, the protein resonances broaden and correspondingly have reduced intensities due to the overall slowing of protein reorientation. Since the protein backbone signals do not broaden uniformly, the differences in line widths (and intensities) enable the narrower (more intense) signals associated with mobile residues to be differentiated from the broader (less intense) signals associated with "structured" residues. For membrane proteins with between one and seven trans-membrane helices in isotropic bicelles, we have been able to find a value of q between 0.1 and 1.0 where only signals from mobile residues are observed in the spectra. The signals from the structured residues are broadened so much that they cannot be observed under standard solution NMR conditions. This q value corresponds to the ratio of DMPC:DHPC where the signals from the structured residues are "titrated out" of the spectrum. This q value is unique for each protein. In magnetically aligned bilayers (q>2.5) no signals are observed in solution NMR spectra of membrane proteins because the polypeptides are "immobilized" by their interactions with the phospholipid bilayers on the relevant NMR timescale (∼10(5)Hz). No signals are observed from proteins in liposomes (only long-chain lipids) either. We show that it is feasible to obtain complementary solution NMR and solid-state NMR spectra of the same membrane protein, where signals from the mobile residues are present in the solution NMR spectra, and signals from the structured residues are present in the solid-state NMR spectra. With assigned backbone amide resonances, these data are sufficient to describe major features of the secondary structure and basic topology of the protein. Even in the absence of assignments, this information can be used to help establish optimal experimental conditions.
    Journal of Magnetic Resonance 10/2011; 214(1):111-8. · 2.30 Impact Factor
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    ABSTRACT: It is challenging to find membrane mimics that stabilize the native structures, dynamics, and functions of membrane proteins. In a recent advance, nanodiscs have been shown to provide a bilayer environment compatible with solution NMR. We show that increasing the lipid to "belt" peptide ratio expands their diameter, slows their reorientation rate, and allows the protein-containing discs to be aligned in a magnetic field for oriented sample solid-state NMR. The spectroscopic properties of membrane proteins with one to seven transmembrane helices in q = 0.1 isotropic bicelles, ~10 nm diameter isotropic nanodiscs, ~30 nm diameter magnetically aligned macrodiscs, and q = 5 magnetically aligned bicelles are compared.
    Biochemistry 09/2011; 50(42):8983-5. · 3.38 Impact Factor
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    ABSTRACT: The design, synthesis, modeling and in vitro testing of channel-forming peptides derived from the cys-loop superfamily of ligand-gated ion channels are part of an ongoing research focus. Over 300 different sequences have been prepared based on the M2 transmembrane segment of the spinal cord glycine receptor α-subunit. A number of these sequences are water-soluble monomers that readily insert into biological membranes where they undergo supramolecular assembly, yielding channels with a range of selectivities and conductances. Selection of a sequence for further modifications to yield an optimal lead compound came down to a few key biophysical properties: low solution concentrations that yield channel activity, greater ensemble conductance, and enhanced ion selectivity. The sequence NK(4)-M2GlyR T19R, S22W (KKKKPARVGLGITTVLTMRTQW) addressed these criteria. The structure of this peptide has been analyzed by solution NMR as a monomer in detergent micelles, simulated as five-helix bundles in a membrane environment, modified by cysteine-scanning and studied for insertion efficiency in liposomes of selected lipid compositions. Taken together, these results define the structural and key biophysical properties of this sequence in a membrane. This model provides an initial scaffold from which rational substitutions can be proposed and tested to modulate anion selectivity. This article is part of a Special Issue entitled: Protein Folding in Membranes.
    Biochimica et Biophysica Acta 07/2011; 1818(4):1039-48. · 4.66 Impact Factor
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    Gabriel A Cook, Stanley J Opella
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    ABSTRACT: P7 is a small membrane protein that is essential for the infectivity of hepatitis C virus. Solution-state NMR experiments on p7 in DHPC micelles, including hydrogen/deuterium exchange, paramagnetic relaxation enhancement and bicelle 'q-titration,' demonstrate that the protein has a range of dynamic properties and distinct structural segments. These data along with residual dipolar couplings yield a secondary structure model of p7. We were able to confirm previous proposals that the protein has two transmembrane segments with a short interhelical loop containing the two basic residues K33 and R35. The 63-amino acid protein has a remarkably complex structure made up of seven identifiable sections, four of which are helical segments with different tilt angles and dynamics. A solid-state NMR two-dimensional separated local field spectrum of p7 aligned in phospholipid bilayers provided the tilt angles of two of these segments. A preliminary structural model of p7 derived from these NMR data is presented.
    Biochimica et Biophysica Acta 06/2011; 1808(6):1448-53. · 4.66 Impact Factor
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    ABSTRACT: The p7 protein from hepatitis C virus and the Vpu protein from HIV-1 are members of the viroporin family of small viral membrane proteins. It is essential to determine their structures in order to obtain an understanding of their molecular mechanisms and to develop new classes of anti-viral drugs. Because they are membrane proteins, it is challenging to study them in their native phospholipid bilayer environments by most experimental methods. Here we describe applications of NMR spectroscopy to both p7 and Vpu. Isotopically labeled p7 and Vpu samples were prepared by heterologous expression in bacteria, initial isolation as fusion proteins, and final purification by chromatography. The purified proteins were studied in the model membrane environments of micelles by solution NMR spectroscopy and in aligned phospholipid bilayers by solid-state NMR spectroscopy. The resulting structural findings enable comparisons to be made between the two proteins, demonstrating that they have quite different architectures. Most notably, Vpu has one trans-membrane helix and p7 has two trans-membrane helices; in addition, there are significant differences in the structures and dynamics of their internal loop and terminal regions.
    Biochimica et Biophysica Acta 02/2011; 1808(2):554-60. · 4.66 Impact Factor
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    Gabriel A Cook, Susanne Stefer, Stanley J Opella
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    ABSTRACT: A small 63-residue membrane protein, p7, has essential roles in the infectivity of the hepatitis C virus in humans. This hydrophobic membrane protein forms homo-oligomeric ion channels in bilayers, which can be blocked by known channel-blocking compounds. To perform structural studies of p7 by nuclear magnetic resonance (NMR) spectroscopy, it is necessary to produce milligram quantities of isotopically labeled protein; as is the case for most membrane-associated proteins, this is challenging. We describe the successful expression of full-length p7 and two truncated constructs in Escherichia coli using a fusion partner that directs the overexpressed protein to inclusion bodies. Following isolation of the fusion proteins by affinity chromatography, they were chemically cleaved with cyanogen bromide. The p7-polypeptides were purified by size-exclusion chromatography. Solution NMR two-dimensional heteronuclear single quantum coherence spectra of uniformly (15) N-labeled p7-polypeptides in 1,2-dihexyl-1-sn-glycero-3-phosphocholine isotropic micelles are fully resolved, with a single resonance for each amide site. The solid-state NMR spectra of the same polypeptides in magnetically aligned 14-O-PC/6-O-PC bicelles demonstrate their reconstitution into planar phospholipid bilayers.
    Biopolymers 01/2011; 96(1):32-40. · 2.88 Impact Factor
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    ABSTRACT: Synthetic channel-forming peptides that can restore chloride conductance across epithelial membranes could provide a novel treatment of channelopathies such as cystic fibrosis. Among a series of 22-residue peptides derived from the second transmembrane segment of the glycine receptor alpha(1)-subunit (M2GlyR), p22-S22W (KKKKP ARVGL GITTV LTMTT QW) is particularly promising with robust membrane insertion and assembly. The concentration to reach one-half maximal short circuit current is reduced to 45 +/- 6 microM from that of 210 +/- 70 microM of peptide p22 (KKKKP ARVGL GITTV LTMTT QS). However, this is accompanied with nearly 50% reduction in conductance. Toward obtaining a molecular level understanding of the channel activities, we combine information from solution NMR, existing biophysical data, and molecular modeling to construct atomistic models of the putative pentameric channels of p22 and p22-S22W. Simulations in membrane bilayers demonstrate that these structural models, even though highly flexible, are stable and remain adequately open for ion conductance. The membrane-anchoring tryptophan residues not only rigidify the whole channel, suggesting increased stability, but also lead to global changes in the pore profile. Specifically, the p22-S22W pore has a smaller opening on average, consistent with lower measured conductance. Direct observation of several incidences of chloride transport suggests several qualitative features of how these channels might selectively conduct anions. The current study thus helps to rationalize the functional consequences of introducing a single C-terminal tryptophan. Availability of these structural models also paves the way for future work to rationally modify and improve M2GlyR-derived peptides toward potential peptide-based channel replacement therapy.
    Proteins Structure Function and Bioinformatics 08/2010; 78(10):2238-50. · 3.34 Impact Factor
  • Gabriel A Cook, Stanley J Opella
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    ABSTRACT: Nuclear magnetic resonance studies of membrane proteins yield valuable insights into their structure and topology. For example, the tilt angle and rotation of the helices in an ion channel can be determined by solid-state NMR spectroscopy in aligned lipid bilayers. Details about the structure of the protein in aligned phospholipids environments are immediately apparent from inspection of the SAMMY spectrum and the data can be further used for the determination of atomic resolution three-dimensional structures. SAR by NMR is a technique that is well suited for the field of membrane transporter proteins. The experiments on protein/phospholipid samples provide a unique insight into the interaction of drugs and the functional proteins.The advances required to transform solid-state NMR from a spectroscopic technique to a generally applicable method for determining molecular structures included multiple-pulse sequences, double-resonance methods, and separated local field spectroscopy. It also required improvements in instrumentation, especially the use of high-field magnets and efficient probes capable of high-power radio-frequency irradiations at high frequencies. The pace of development is accelerating and the local field is being utilized in an increasing number of ways in spectroscopic investigations of molecular structure and dynamics. Applications to many helical membrane proteins are underway and promise to add to our understanding of membrane proteins in health and disease.
    Methods in molecular biology (Clifton, N.J.) 01/2010; 637:263-75. · 1.29 Impact Factor
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    Gabriel A Cook, Stanley J Opella
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    ABSTRACT: The p7 protein of hepatitis C virus (HCV) plays an important role in the viral lifecycle. Like other members of the viroporin family of small membrane proteins, the amino acid sequence of p7 is largely conserved over the entire range of genotypes, and it forms ion channels that can be blocked by a number of established channel-blocking compounds. Its characteristics as a membrane protein make it difficult to study by most structural techniques, since it requires the presence of lipids to fold and function properly. Purified p7 can be incorporated into phospholipid bilayers and micelles. Initial solid-state nuclear magnetic resonance (NMR) studies of p7 in 14-O-PC/6-O-PC bicelles indicate that the protein contains helical segments that are tilted approximately 10 degrees and 25 degrees relative to the bilayer normal. A truncated construct corresponding to the second transmembrane domain of p7 is shown to have properties similar to those of the full-length protein, and was used to determine that the helix segment tilted at 10 degrees is in the C-terminal portion of the protein. The addition of the channel blocker amantadine to the full-length protein resulted in selective chemical shift changes, demonstrating that NMR has a potential role in the development of drugs targeted to p7.
    Biophysics of Structure and Mechanism 10/2009; 39(7):1097-104. · 2.44 Impact Factor
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    ABSTRACT: A strip-shield inserted between a high inductance double-tuned solenoid coil and the glass tube containing the sample improves the efficiency of probes used for high-field solid-state NMR experiments on lossy aqueous samples of proteins and other biopolymers. A strip-shield is a coil liner consisting of thin copper strips layered on a PTFE (polytetrafluoroethylene) insulator. With lossy samples, the shift in tuning frequency is smaller, the reduction in Q, and RF-induced heating are all significantly reduced when the strip-shield is present. The performance of 800MHz (1)H/(15)N and (1)H/(13)C double-resonance probes is demonstrated on aqueous samples of membrane proteins in phospholipid bilayers.
    Journal of Magnetic Resonance 07/2009; 200(1):74-80. · 2.30 Impact Factor
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    ABSTRACT: Three series of 22-residue peptides derived from the transmembrane M2 segment of the glycine receptor alpha1-subunit (M2GlyR) have been designed, synthesized, and tested to determine the plasticity of a channel-forming sequence and to define whether channel pores with enhanced conductive properties could be created. Sixteen sequences were examined for aqueous solubility, solution-association tendency, secondary structure, and half-maximal concentration for supramolecular assembly, channel activity, and ion transport properties across epithelial monolayers. All peptides interact strongly with membranes: associating with, inserting across, and assembling to form homooligomeric bundles when in micromolar concentrations. Single and double amino acid replacements involving arginine and/or aromatic amino acids within the final five C-terminal residues of the peptide cause dramatic effects on the concentration dependence, yielding a range of K1/2 values from 36 +/- 5 to 390 +/- 220 microM for transport activity. New water/lipid interfacial boundaries were established for the transmembrane segment using charged or aromatic amino acids, thus limiting the peptides' ability to move perpendicularly to the plane of the bilayer. Formation of discrete water/lipid interfacial boundaries appears to be necessary for efficient supramolecular assembly and high anion transport activity. A peptide sequence is identified that may show efficacy in channel replacement therapy for channelopathies such as cystic fibrosis.
    Biophysical Journal 04/2006; 90(6):2138-50. · 3.67 Impact Factor
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    ABSTRACT: The synthetic peptide (C(18)H(37))(2)NCOCH(2)OCH(2)CON-(Gly)(3)-Pro-(Gly)(3)-OCH(2)Ph forms chloride-selective channels in liposomes and exhibits voltage-gating properties in planar phospholipid bilayers. The peptide fragment of the channel is based on a conserved motif in naturally occurring chloride transporters. Membrane-anchoring residues at the N- and C-terminal ends augment the peptide. NMR spectra (1D and 2D) of the channel in CDCl(3) showed significant variation in the absence and presence of stoichiometric tetrabutylammonium chloride (Bu(4)NCl). One-dimensional solution-state NMR titration studies combined with computational molecular simulation studies indicate that the peptide interacts with the salt as an ion pair and H-bonds chloride. To our knowledge, this is the first structural analysis of any synthetic anion-channel salt complex.
    Journal of the American Chemical Society 03/2006; 128(5):1633-8. · 10.68 Impact Factor
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    ABSTRACT: Ion channel-forming peptides enable us to study the conformational dynamics of a transmembrane helix as a function of sequence and environment. Molecular dynamics simulations are used to study the conformation and dynamics of three 22-residue peptides derived from the second transmembrane domain of the glycine receptor (NK4-M2GlyR-p22). Simulations are performed on the peptide in four different environments: trifluoroethanol/water; SDS micelles; DPC micelles; and a DMPC bilayer. A hierarchy of alpha-helix stabilization between the different environments is observed such that TFE/water < micelles < bilayers. Local clustering of trifluoroethanol molecules around the peptide appears to help stabilize an alpha-helical conformation. Single (S22W) and double (S22W,T19R) substitutions at the C-terminus of NK4-M2GlyR-p22 help to stabilize a helical conformation in the micelle and bilayer environments. This correlates with the ability of the W22 and R19 side chains to form H-bonds with the headgroups of lipid or detergent molecules. This study provides a first atomic resolution comparison of the structure and dynamics of NK4-M2GlyR-p22 peptides in membrane and membrane-mimetic environments, paralleling NMR and functional studies of these peptides.
    Biophysical Journal 03/2006; 90(6):1855-64. · 3.67 Impact Factor
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    ABSTRACT: A number of channel-forming peptides derived from the second transmembrane (TM) segment (M2) of the glycine receptor alpha(1) subunit (M2GlyR), including the 22-residue sequence NK(4)-M2GlyR p22 wild type (WT) (KKKKPARVGLGITTVLTMTTQS), induce anion permeation across epithelial cell monolayers. In vitro assays suggest that this peptide or related sequences might function as a candidate for ion channel replacement therapy in treating channelopathies such as cystic fibrosis (CF). The wild-type sequence forms soluble associations in water that diminish its efficacy. Introduction of a single substitution S22W at the C-terminus, NK(4)-M2GlyR p22 S22W, eliminates the formation of higher molecular weight associations in solution. The S22W peptide also reduces the concentration of peptide required for half-maximal anion transport induced across Madin-Darby canine kidney cells (MDCK) monolayers. A combination of 2D double quantum filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and rotating frame nuclear Overhauser effect spectroscopy (ROESY) data were recorded for both the associating WT and nonassociating S22W peptides and used to compare the primary structures and to assign the secondary structures. High-resolution structural studies were recorded in the solvent system (40% 2,2,2-Trifluoroethanol (TFE)/water), which gave the largest structural difference between the two peptides. Nuclear Overhauser effect crosspeak intensity provided interproton distances and the torsion angles were measured by spin-spin coupling constants. These constraints were put into the DYANA modeling program to generate a group of structures. These studies yielded energy-minimized structures for this mixed solvent environment. Structure for both peptides is confined to the 15-residue transmembrane segments. The energy-minimized structure for the WT peptide shows a partially helical extended structure. The S22W peptide adopts a bent conformation forming a hydrophobic pocket by hydrophobic interactions.
    Biophysical Journal 04/2004; 86(3):1424-35. · 3.67 Impact Factor

Publication Stats

174 Citations
60.64 Total Impact Points

Institutions

  • 2009–2011
    • University of California, San Diego
      • Department of Chemistry and Biochemistry
      San Diego, CA, United States
  • 2004–2006
    • Kansas State University
      • Department of Biochemistry and Molecular Biophysics
      Kansas, United States