Depth Of Membrane Insertion Research Articles

Orientational Landscapes of Peptides in Membranes: Prediction of 2H NMR Couplings in a Dynamic Context

Unlike soluble proteins, membrane polypeptides face an anisotropic milieu. This imposes restraints on their orientation and provides a reference that makes structure prediction tractable by minimalistic thermodynamic models. Here we use this framework to build orientational distributions of monomeric membrane-bound peptides and to predict their expected solid-state (2)H NMR quadrupolar couplings when labeled at specific side chain positions. Using a complete rigid-body sampling of configurations relative to an implicit lipid membrane, peptide free energy landscapes are calculated. This allows us to obtain probability distributions of the peptide tilt, azimuthal rotation, and depth of membrane insertion. The orientational distributions are broad and originate from an interplay among the three relevant rigid-body degrees of freedom, which allows population of multiple states in shallow free energy minima. Remarkably, only when the orientational distributions are taken into account do we obtain a close correlation between predicted (2)H NMR splittings and values measured in experiments. Such a good correlation is not seen with splittings calculated from single configurations, being either the averaged or the lowest free energy state, showing there are distributions, rather than single structures, that best define the peptide-membrane systems. Moreover, we propose that these distributions contribute to the understanding of the rigid-body dynamics of the system.

A strong correlation between fusogenicity and membrane insertion depth of the HIV fusion peptide

Fusion between the membrane of HIV and the membrane of a host cell is a crucial step in HIV infection and is catalyzed by the binding of the fusion peptide domain (HFP) of the HIV gp41 protein to the host cell membrane. The HFP by itself induces vesicle fusion and is a useful model system to understand the fusion peptide/host cell membrane interaction. This article reports an experimental correlation between the membrane locations of different HFP constructs and their fusogenicities. The constructs were the HFP monomer with Val-2 to Glu-2 mutation (HFPmn_mut), wild type HFP monomer (HFPmn), and wild type HFP trimer (HFPtr). All constructs have predominant beta sheet structure in membranes with physiologically relevant cholesterol content. HFPmn_mut does not fuse vesicles, HFPmn has moderate fusion rate, and HFPtr has the putative oligomerization state of HIV gp41 and a very rapid fusion rate. The HFP membrane locations were probed with solid-state NMR measurements of distances between labeled carbonyl ((13)CO) nuclei in the HFP backbone and lipid nuclei in the surface or interior regions of the membrane bilayer. HFPmn_mut is located at the membrane surface, HFPmn is inserted into a single membrane leaflet, and HFPtr is the most deeply inserted construct with contact with the center of the membrane. These results show a clear positive correlation between the insertion depths and the fusion activities of the HFP constructs. Other disease-causing enveloped viruses contain fusion peptides and this correlation may be a general structure-function model for these peptides.

Structure and topology of monomeric phospholamban in lipid membranes determined by a hybrid solution and solid-state NMR approach

Phospholamban (PLN) is an essential regulator of cardiac muscle contractility. The homopentameric assembly of PLN is the reservoir for active monomers that, upon deoligomerization form 1:1 complexes with the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), thus modulating the rate of calcium uptake. In lipid bilayers and micelles, monomeric PLN exists in equilibrium between a bent (or resting) T state and a more dynamic (or active) R state. Here, we report the high-resolution structure and topology of the T state of a monomeric PLN mutant in lipid bilayers, using a hybrid of solution and solid-state NMR restraints together with molecular dynamics simulations in explicit lipid environments. Unlike the previous structural ensemble determined in micelles, this approach gives a complete picture of the PLN monomer structure in a lipid bilayer. This hybrid ensemble exemplifies the tilt, rotation, and depth of membrane insertion, revealing the interaction with the lipids for all protein domains. The N-terminal amphipathic helical domain Ia (residues 1-16) rests on the surface of the lipid membrane with the hydrophobic face of domain Ia embedded in the membrane bilayer interior. The helix comprised of domain Ib (residues 23-30) and transmembrane domain II (residues 31-52) traverses the bilayer with a tilt angle of approximately 24 degrees . The specific interactions between PLN and lipid membranes may represent an additional regulatory element of its inhibitory function. We propose this hybrid method for the simultaneous determination of structure and topology for membrane proteins with compact folds or proteins whose spatial arrangement is dictated by their specific interactions with lipid bilayers.

Functional and Structural Measurements of HIV gp41 Fusion Protein Constructs

The initial step of HIV infection is fusion between the viral and target cell membranes. Fusion is mediated by the HIV gp41 protein and its N-terminal “fusion peptide” (FP) which binds to target cell membranes. Shorter constructs of gp41 that contain the FP usually catalyze vesicle fusion and such fusion at physiological pH was measured for three different gp41 constructs which differed in their numbers of N-terminal gp41 residues. “FP34” and “N70” were respectively models of the FP and “pre-hairpin intermediate” gp41 conformation while “FP-hairpin” was a model of the final “six-helix-bundle” gp41 structure. N70 induced rapid fusion, FP34 induced moderate fusion, and FP-hairpin induced no fusion and even arrested fusion induced by FP34. The data therefore suggest that the six-helix bundle conformation stops membrane fusion. In related work, solid-state nuclear magnetic resonance measurements probed the membrane locations of three different FP constructs with very different fusion rates. There was a positive correlation between fusion rate and depth of membrane insertion for the FP in either helical or β strand conformation. The key determinant of fusion rate may therefore be FP membrane location rather than conformation.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Analysis of Tryptophan Residues in the Staphylococcal Multidrug Transporter QacA Reveals Long-Distance Functional Associations of Residues on Opposite Sides of the Membrane

Tryptophan residues can possess a multitude of functions within a multidrug transport protein, e.g., mediating interactions with substrates or distal parts of the protein, or fulfilling a structural requirement, such as guiding the depth of membrane insertion. In this study, the nine tryptophan residues of the staphylococcal QacA multidrug efflux protein were individually mutated to alanine and phenylalanine, and the functional consequences of these changes were determined. Phenylalanine substitutions for each tryptophan residue were functionally tolerated. However, alanine modifications revealed an important functional role for three tryptophan residues, W58, W149, and W173, each of which is well conserved among QacA-related transport proteins in the major facilitator superfamily. The most functionally compromising mutation, an alanine substitution for W58, likely to be located at the extracellular interface of transmembrane segment 2, abolished all detectable QacA-mediated resistance and transport function. Second-site suppressor analyses identified several mutations that rescued the function of the W58A QacA mutant. Remarkably, all of these suppressor mutations were shown to be located in cytoplasmic loops between transmembrane helices 2 and 3 or 12 and 13, demonstrating novel functional associations between amino acid positions on opposite sides of the membrane and in distal N- and C-terminal regions of the QacA protein.

Protein Folding in Membranes: Insights from Neutron Diffraction Studies of a Membrane β-Sheet Oligomer

Studies of the assembly of the hexapeptide Acetyl-Trp-Leu 5 (AcWL 5) into β-sheets in membranes have provided insights into membrane protein folding. Yet, the exact structure of the oligomer in the lipid bilayer is unknown. Here we use neutron diffraction to study the disposition of the peptides in bilayers. We find that pairs of adjacent deuterium-labeled leucines have no well-defined peak or dip in the transmembrane distribution profiles, indicative of heterogeneity in the depth of membrane insertion. At the same time, the monomeric homolog AcWL 4 exhibits a homogeneous, well-defined, interfacial location in neutron diffraction experiments. Thus, although the bilayer location of monomeric AcWL 4 is determined by hydrophobicity matching or complementarity within the bilayer, the AcWL 5 molecules in the oligomer are positioned at different depths within the bilayer because they assemble into a staggered transmembrane β-sheet. The AcWL 5 assembly is dominated by protein-protein interactions rather than hydrophobic complementarity. These results have implications for the structure and folding of proteins in their native membrane environment and highlight the importance of the interplay between hydrophobic complementarity and protein-protein interactions in determining the structure of membrane proteins.

Effects of Lipid Phase Transition and Membrane Surface Charge on the Interfacial Activation of Phospholipase A2

Phospholipase A2 (PLA2) enzymes act at the membrane-water interface to access their phospholipid substrate from the membrane. They are regulated by diverse factors, including the membrane charge, fluidity, mode of membrane binding (insertion, orientation), and allosteric conformational effects. Relative contributions of these factors to the complex kinetics of PLA2 activation are not well understood. Here we examine the effects of thermal phase transitions and the surface charge of phospholipid membranes on the activation of human pancreatic PLA2. The temperature dependence of the initial catalytic rate of PLA2 peaks around the lipid phase transition temperature (Tm) when Tm is not too far from physiological temperatures (30-40 degrees C), and the peak is higher in the presence of anionic membranes. High PLA2 activity can be induced by thermal perturbations of the membrane. Temperature-dependent fluorescence quenching experiments show that despite dramatic effects of the lipid phase transition on PLA2 activity, the membrane insertion depth of PLA2 increases only modestly above Tm. The data show that membrane structural disorder, and not the depth of membrane insertion, plays a major role in PLA2 activity.

Isoform-Specific Membrane Insertion of Secretory Phospholipase A2and Functional Implications

Despite increasing evidence that the membrane-binding mode of interfacial enzymes including the depth of membrane insertion is crucial for their function, the membrane insertion of phospholipase A(2) (PLA(2)) enzymes has not been studied systematically. Here, we analyze the membrane insertion of human group IB PLA(2) (hIBPLA(2)) and compare it with that of a structurally homologous V3W mutant of human group IIA PLA(2) (V3W-hIIAPLA(2)) and with a structurally divergent group III bee venom PLA(2) (bvPLA(2)). Increasing the anionic charge of membranes results in a blue shift of the fluorescence of Trp(3) of hIBPLA(2), a decrease in quenching by acrylamide, and an increase in enzyme activity, reflecting an enhancement in the membrane binding of PLA(2). Fluorescence quenching by brominated lipids indicates significant penetration of Trp(3) into fluid POPC/POPG membranes but little insertion into the solid DPPC/DPPG membranes. Increased membrane fluidity also supports hIBPLA(2) activity, suggesting that membrane insertion of hIBPLA(2) is controlled by membrane fluidity and is necessary for the full activity of the enzyme. Trp fluorescence quenching of the V3W-hIIAPLA(2) and bvPLA(2) by water- and membrane-soluble quenchers indicates substantial membrane insertion of Trp(3) of V3W-hIIAPLA(2), similar to that found for hIBPLA(2), and no insertion of tryptophans of bvPLA(2). Our results provide evidence that (a) structurally similar group IB and IIA PLA(2)s, but not structurally diverse group III PLA(2), significantly penetrate into membranes; (b) membrane insertion is controlled by membrane fluidity and facilitates activation of IB and IIA PLA(2)s; and (c) structurally distinct PLA(2) isoforms may employ different tactics of substrate accession/product release during lipid hydrolysis.