High-Resolution Conformation of Gramicidin A in a Lipid Bilayer by Solid-State NMR

Ketchem, Randal R.; Hu, Wei‐Xiao; Cross, Timothy A.

doi:10.1126/science.7690158

Cited by 665 publications

(658 citation statements)

References 33 publications

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“…Recent developments in bacterial expression systems for the preparation of recombinant isotopically labeled membrane proteins, methods for sample preparation, pulse sequences for high-resolution spectroscopy, and structural indices that guide the structure assembly process, have greatly extended the capabilities of these techniques, and the structures of a variety of helical membrane proteins have been determined by NMR in micelles and in bilayers [3][4][5][6][7][8][9][10][11].…”

Section: Introductionmentioning

confidence: 99%

NMR of membrane proteins in micelles and bilayers: The FXYD family proteins

Franzin

Gong

Thai

et al. 2007

Methods

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Determining the atomic resolution structures of membrane proteins is of particular interest in contemporary structural biology. Helical membrane proteins constitute one-third of the expressed proteins encoded in a genome, many drugs have membrane-bound proteins as their receptors, and mutations in membrane proteins result in human diseases. Although integral membrane proteins provide daunting technical challenges for all methods of protein structure determination, nuclear magnetic resonance (NMR) spectroscopy can be an extremely versatile and powerful method for determining their structures and characterizing their dynamics, in lipid environments that closely mimic the cell membranes. Once milligram amounts of isotopically labeled protein are expressed and purified, micelle samples can be prepared for solution NMR analysis, and lipid bilayer samples can be prepared for solid-state NMR analysis. The two approaches are complementary and can provide detailed structural and dynamic information. This paper describes the steps for membrane protein structure determination using solution and solid-state NMR. The methods for protein expression and purification, sample preparation and NMR experiments are described and illustrated with examples from the FXYD proteins, a family of regulatory subunits of the Na, KATPase.

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Section: Introductionmentioning

confidence: 99%

NMR of membrane proteins in micelles and bilayers: The FXYD family proteins

Franzin

Gong

Thai

et al. 2007

Methods

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“…The use of lipid bilayers provides a medium that closely resembles the biological environment, and sample orientation serves as a mechanism for resonance line narrowing, and also preserves the membrane-defined protein topology. The structures of gramicidin, the M2 transmembrane segment from the acetylcholine receptor, the transmembrane segment of the M2 H + channel protein from influenza virus, and the membrane bound fd coat protein (PDB files 1MAG, 1CEK, 1MP6, 1MZT), have been determined from solid-state NMR orientational restraints using this approach [2][3][4][5].…”

Section: Introductionmentioning

confidence: 99%

Hydration-optimized oriented phospholipid bilayer samples for solid-state NMR structural studies of membrane proteins

Marassi

Crowell

2003

Journal of Magnetic Resonance

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The preparation of oriented, hydration-optimized lipid bilayer samples, for NMR structure determination of membrane proteins, is described. The samples consist of planar phospholipid bilayers, containing membrane proteins, that are oriented on single pairs of glass slides, and are placed in the coil of the NMR probe with the bilayer plane perpendicular to the direction of the magnetic field. Lipid bilayers provide a medium that closely resembles the biological membrane, and sample orientation both preserves the intrinsic membrane-defined directional quality of membrane proteins, and provides the mechanism for resonance line narrowing. The hydrationoptimized samples overcome some of the difficulties associated with multi-dimensional, highresolution, solid-state NMR spectroscopy of membrane proteins. These samples have greater stability over the course of multidimensional NMR experiments, they have lower sample conductance for greater rf power efficiency, and enable greater rf coil filling factors to be obtained for improved experimental sensitivity. Sample preparation is illustrated for the membrane protein CHIF (channel inducing factor), a member of the FXYD family of ion transport regulators.

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“…In gramicidin, a 15-residue antibiotic peptide secreted by Bacillus brevis, the alternating pattern of D and L amino acids yields a unique secondary structure termed the ␤-helix (Ketchm, Hu & Cross, 1993;Langs et al, 1991). Monomeric peptides form head-to-head dimers in membranes to create a large intra-helix pore which permits ions to transverse the lipid bilayer.…”

Section: Introductionmentioning

confidence: 99%

Structure of the Transmembrane Cysteine Residues in Phospholamban

Arkin

Adams

Brünger

et al. 1997

Journal of Membrane Biology

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Abstract. Phospholamban, a 52-residue membrane protein, associates to form a pentameric complex of five long ␣-helices traversing the sarcoplasmic reticulum membrane of cardiac muscle cells. The transmembrane domain of the protein is largely hydrophobic, with only three cysteine residues having polar side chains, yet it functions as a Ca 2+ -selective ion channel. In this report, infrared spectroscopy is used to probe the conformation of the three cysteine side chains and to establish whether the free S-H groups form intrahelical hydrogen bonds in the pentameric complex. Vibrational spectra of a transmembrane peptide were obtained which corresponded to the transmembrane domain of wild-type phospholamban and three peptides each containing a cysteine ⇒ alanine substitution. The observed S-H frequencies argue that each of the sulfhydryl groups is hydrogen-bonded to an i-4 backbone carbonyl oxygen. Electrostatic calculations on a model of phospholamban based on molecular dynamics and mutagenesis studies, show that the sulfhydryl groups may significantly contribute to the electrostatic potential field of the protein.

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High-Resolution Conformation of Gramicidin A in a Lipid Bilayer by Solid-State NMR

Cited by 665 publications

References 33 publications

NMR of membrane proteins in micelles and bilayers: The FXYD family proteins

NMR of membrane proteins in micelles and bilayers: The FXYD family proteins

Hydration-optimized oriented phospholipid bilayer samples for solid-state NMR structural studies of membrane proteins

Structure of the Transmembrane Cysteine Residues in Phospholamban

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