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Influence of High pH and Cholesterol on Single Arginine-Containing Transmembrane Peptide Helices.

ABSTRACT: An essential component of mammalian cells, cholesterol exerts significant influence on the physical properties of the cell membrane and in turn its constituents, including membrane proteins. Although sparse, polar amino acid residues are highly conserved in membrane proteins and play pivotal roles in determining specific structural and functional properties. To improve our understanding of particular polar residues in the membrane environment, we have examined two specific "guest" Arg residues within a well-defined and deuterium-labeled "host" framework provided by the transmembrane helical peptide GWALP23 (acetyl-GGALWLALALALALALALWLAGA-amide). Solid-state 2H nuclear magnetic resonance (NMR) spectra from aligned bilayer membrane samples effectively report changes in the host helix properties because of the incorporation of the guest residues. The focus of this work is two-pronged. First, GWALP23-R14 was examined over a pH range of 2-13 in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) ester- or ether-linked bilayer membranes. Our findings indicate that the Arg guanidinium side chain remains charged over this entire range, in agreement with numerous molecular dynamics simulations. Second, GWALP23-R12 and GWALP23-R14 peptides were characterized in DOPC bilayers with varying cholesterol content. Our findings suggest that 10 or 20% cholesterol content has minimal impact on the orientation of the R14 peptide. Although the NMR signals are broader and weaker in the presence of 20% cholesterol, the deuterium quadrupolar splittings for [2H]Ala residues in GWALP23-R14 change very little. Conversely, cholesterol appears to modulate the multistate behavior of GWALP23-R12 and to favor a major interfacial state for the helix, bound at the bilayer surface. These results indicate a conditional sensitivity of a complex multistate transmembrane Arg-containing peptide helix to the presence of cholesterol.

SUBMITTER: Thibado JK

PROVIDER: S-EPMC5266483 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Influence of High pH and Cholesterol on Single Arginine-Containing Transmembrane Peptide Helices.

Thibado Jordana K JK Martfeld Ashley N AN Greathouse Denise V DV Koeppe Roger E RE

Biochemistry 20161104 45

An essential component of mammalian cells, cholesterol exerts significant influence on the physical properties of the cell membrane and in turn its constituents, including membrane proteins. Although sparse, polar amino acid residues are highly conserved in membrane proteins and play pivotal roles in determining specific structural and functional properties. To improve our understanding of particular polar residues in the membrane environment, we have examined two specific "guest" Arg residues w ...[more]

PMID: 27782382

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Project description:Human immunodeficiency virus (HIV) is membrane-enveloped, and an initial infection step is joining/fusion of viral and cell membranes. This step is catalyzed by gp41, which is a single-pass integral viral membrane protein. The protein contains an ∼170-residue ectodomain located outside the virus that is important for fusion and includes the fusion peptide (FP), N-helix, loop, C-helix, and viral membrane-proximal external region (MPER). The virion initially has noncovalent complexes between three gp41 ectodomains and three gp120 proteins. A gp120 contains ∼500 residues and functions to identify target T-cells and macrophages via binding to specific protein receptors of the target cell membrane. gp120 moves away from the gp41 ectodomain, and the ectodomain is thought to bind to the target cell membrane and mediate membrane fusion. The secondary and tertiary structures of the ectodomain are different in the initial complex with gp120 and the final state without gp120. There is not yet imaging of gp41 during fusion, so the temporal relationship between the gp41 and membrane structures is not known. This study describes biophysical and functional characterization of large gp41 constructs that include the ectodomain and transmembrane domain (TM). Significant fusion is observed of both neutral and anionic vesicles at neutral pH, which reflects the expected conditions of HIV/cell fusion. Fusion is enhanced by the FP, which in HIV/cell fusion likely contacts the host membrane, and the MPER and TM, which respectively interfacially contact and traverse the HIV membrane. Initial contact with vesicles is made by protein trimers that are in a native oligomeric state that reflects the initial complex with gp120 and also is commonly observed for the ectodomain without gp120. Circular dichroism data support helical structure for the N-helix, C-helix, and MPER and nonhelical structure for the FP and loop. Distributions of monomer, trimer, and hexamer states are observed by size-exclusion chromatography (SEC), with dependences on solubilizing detergent and construct. These SEC and other data are integrated into a refined working model of HIV/cell fusion that includes dissociation of the ectodomain into gp41 monomers followed by folding into hairpins that appose the two membranes, and subsequent fusion catalysis by trimers and hexamers of hairpins. The monomer and oligomer gp41 states may therefore satisfy dual requirements for HIV entry of membrane apposition and fusion.

Dataset Information

Influence of High pH and Cholesterol on Single Arginine-Containing Transmembrane Peptide Helices.

Publications

Influence of High pH and Cholesterol on Single Arginine-Containing Transmembrane Peptide Helices.

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