Membrane Mimetic Environments Research Articles

Amphipol-assisted folding of bacteriorhodopsin in the presence or absence of lipids: functional consequences

Amphipols are short amphipathic polymers designed to stabilize membrane proteins in aqueous solutions in the absence of detergent. Bacteriorhodopsin (BR), a light-driven proton pump, has been denatured, either by direct solubilization of the purple membrane in sodium dodecylsulfate (SDS) solution or by a procedure that involves delipidation with organic solvent followed by transfer to SDS, and renatured in amphipol A8-35. The effect of different renaturation procedures and of the presence or absence of lipids and the cofactor retinal have been investigated. The resulting samples have been characterized by absorbance spectroscopy, size-exclusion chromatography, thermostability measurements, and determination of photocycle kinetics. Transfer to A8-35 can be achieved by SDS precipitation, dilution, or dialysis, the first route resulting in the highest yield of refolding. Functional BR can be refolded whether in the presence or absence of lipids, higher yields being achieved in their presence. Retinal is not required for the protein to refold, but it stabilizes the refolded form and, thereby, improves folding yields. Lipids are not required for BR to perform its complete photocycle, but their presence speeds up the return to the ground state. Taken together, these data indicate that a membrane or membrane-mimetic environment is not required for correct decoding of the chemical information contained in the sequence of BR; functional folding is possible even in the highly foreign environment of lipid-free amphipols. BR interactions with lipids, however, contribute to an effective photocycle.

Dimerization of the Transmembrane Domain of Human Tetherin in Membrane Mimetic Environments

Tetherin/Bst-2 is a cell surface protein that can act as a restriction factor against a number of enveloped viruses, including HIV-1. It acts by tethering new virus particles to the host cell membrane, promoting their internalization and degradation. Tetherin is a type II membrane protein, with an N-terminal transmembrane domain, an extracellular coiled-coil domain, and a C-terminal GPI anchor. This double membrane anchor is important for anti-HIV activity, as is dimerization of the coiled-coil domain, but despite recent crystal structures of the coiled-coil ectodomains of human and mouse tetherin, the topology of tetherin with respect to host and viral membranes has yet to be determined. The tetherin transmembrane domain is also thought to mediate interactions with the HIV-1 encoded integral membrane protein Vpu, which is an antagonist of tetherin, through direct binding to the transmembrane region of Vpu. Using a combination of SDS-PAGE, size exclusion chromatography, and pyrene excimer fluorescence, we show that in the absence of the coiled-coil domain the transmembrane domain of human tetherin forms parallel homodimers in membrane mimetic environments. Transmembrane domain dimerization does not require disulfide bond formation and is favored in TFE, SDS micelles, and POPC liposomes. This observation has implications for functional models of tetherin, suggesting that both transmembrane domains in the dimeric molecule are inserted into the same lipid bilayer, rather than into opposing membranes.

Protein Structure in Membrane Domains

Of great interest to the academic and pharmaceutical research communities, helical transmembrane proteins are characterized by their ability to dissolve and fold in lipid bilayers--properties conferred by polypeptide spans termed transmembrane domains (TMDs). The apolar nature of TMDs necessitates the use of membrane-mimetic solvents for many structure and folding studies. This review examines the relationship between TMD structure and solvent environment, focusing on principles elucidated largely in membrane-mimetic environments with single-TMD protein and peptide models. Following a brief description of TMD sequence and conformational characteristics gleaned from the structural database, we present an overview of the conceptual models used to study folding in vitro. The impact of sequence and solvent context on the incorporation of TMDs into membranes, and its role in measurements of TMD self-assembly strengths, is then described. We conclude with a discussion of the nonspecific effects of membrane components on TMD stability.

The FKBP–Rapamycin Binding Domain of Human TOR Undergoes Strong Conformational Changes in the Presence of Membrane Mimetics with and without the Regulator Phosphatidic Acid

The Ser/Thr kinase target of rapamycin (TOR) is a central controller of cellular growth and metabolism. Misregulation of TOR signaling is involved in metabolic and neurological disorders and tumor formation. TOR can be inhibited by association of a complex of rapamycin and FKBP12 to the FKBP12-rapamycin binding (FRB) domain. This domain was further proposed to interact with phosphatidic acid (PA), a lipid second messenger present in cellular membranes. Because mammalian TOR has been localized at various cellular membranes and in the nucleus, the output of TOR signaling may depend on its localization, which is expected to be influenced by the interaction with complex partners and regulators in response to cellular signals. Here, we present a detailed characterization of the interaction of the FRB domain with PA and how it is influenced by the surrounding membrane environment. On the basis of nuclear magnetic resonance- and circular dichroism-monitored binding studies using different neutral and negatively charged lipids as well as different membrane mimetics (micelles, bicelles, and liposomes), the FRB domain may function as a conditional peripheral membrane protein. However, the data for the isolated domain just indicate an increased affinity for negatively charged lipids and membrane patches but no specific preference for PA or PA-enriched regions. The membrane-mimetic environment induces strong conformational changes that largely maintain the α-helical secondary structure content but presumably disperse the helices in the lipidic environment. Consistent with overlapping binding surfaces for different lipids and the FKBP12-rapamycin complex, binding of the inhibitor complex protects the FRB domain from interactions with membrane mimetics at lower lipid concentrations.

Efficiency of detergents at maintaining membrane protein structures in their biologically relevant forms

High-resolution structural analysis of membrane proteins by X-ray crystallography or solution NMR spectroscopy often requires their solubilization in the membrane-mimetic environments of detergents. Yet the choice of a detergent suitable for a given study remains largely empirical. In the present work, we considered the micelle-crystallized structures of lactose permease (LacY), the sodium/galactose symporter (vSGLT), the vitamin B12 transporter (BtuCD), and the arginine/agmatine antiporter (AdiC). Representative transmembrane (TM) segments were selected from these proteins based on their relative contact(s) with water, lipid, and/or within the protein, and were synthesized as Lys-tagged peptides. Each peptide was studied by circular dichroism and fluorescence spectroscopy in water, and in the presence of the detergents sodium dodecylsulfate (SDS, anionic); n-dodecyl phosphatidylcholine (DPC, zwitterionic); n-dodecyl-β-d-maltoside (DDM, neutral); and n-octyl-β-d-glucoside (OG, neutral, varying acyl tail length). We found that (i) the secondary structures of the TM segments were statistically indistinguishable in the four detergents studied; and (ii) a strong correlation exists between the extent of helical structure of each individual TM segment in detergents with its helicity level as it exists in the full-length protein, indicating that helix adoption is fundamentally the same in both environments. The denaturing properties of so-called ‘harsh’ detergents may thus largely be due to their interactions with non-membranous regions of proteins. Given the consistency of structural features observed for each TM segment in a variety of micellar media, the overall results suggest that the structure likely corresponds to its relevant biological form in the intact protein in its native lipid bilayer environment.

Initial Structural Studies of Mtb Membrane Protein “CrgA” in Membrane Mimetic Environment

Here we present initial NMR Spectroscopic studies of CrgA, one of the important putative inner membrane proteins in Mycobacterium tuberculosis (Mtb). CrgA helps in the cell division of Mtb contains 2 transmembrane helices (TM), 93 amino acid and is an important drug target. Our studies show that, CrgA interacts with FtsZ (involves in Zring formation during cell division) and Rv0008c (another putative membrane protein) in Mtb during the cell division. For NMR structural studies 15N, 13C uniform and amino acid specific isotope labeled CrgA reconstituted in POPC-POPG (4:1 mol/mol) liposomes was used for planar lipid bilayer oriented on glass slides and Magic Angle Spinning (MAS) correlation spectroscopy. The key behind these studies is to determine CrgA structure in membrane mimetic lipid bilayer environment. For oriented glass slide samples, 2D PISEMA experiments were performed which correlates 1H-15N dipolar coupling with the 15N chemical shift from the backbone resonances of 15N labeled protein in lipid bilayer. Each measured frequency reflects the orientation of the peptide plane of the protein w.r.t to the bilayer normal. Similarly the tilt angles of various parts of the protein (TM, N and C terminal) can also be obtained which is very useful for the structure calculation process. Moreover MAS experiments can measure distances between side chains of various residues. Altogether the aim of this research is to characterize the 3D backbone as well as the side chain structures of CrgA in native membrane like environment.

Structural Insights for the Full Length Influenza A M2 Proton Channel in Native E. Coli Membranes

Membrane proteins are structurally characterized in membrane mimetic environments, environments that may or may not reflect the properties of native membrane proteins. This is critically important since the structure of membrane proteins is influenced by the protein's environment. Here we will show structural data from magic angle spinning solid state NMR of the full length M2 protein from Influenza A observed in E. coli membranes. This protein has never been exposed to the denaturing influence of detergents nor to the length isolation, purification and reconstitution protocols that typify membrane protein sample preparation for structural studies. The data will be compared to the purified full length protein in synthetic liposomes and to spectra of smaller constructs for which there is a high resolution structure in lipid bilayers.

Rhodopsin Simulations in Detergent Micelles Characterize Meta I and Meta II Photointermediates

G protein-coupled receptors (GPCRs) are prominent pharmaceutical targets due to their prevalence in the human genome. Membrane proteins such as GPCRs have been historically difficult to characterize by crystallographic methods, severely limiting development of therapeutical applications. A commonly used in vitro method for studying GPCRs is detergent solubilization, which can retain protein activity but affects thermodynamics and conformational flexibility. We used molecular dynamics (MD) to simulate the canonical GPCR rhodopsin in micelles containing the detergent DDM or CHAPS. The Meta I ↔ Meta II activation mechanism of rhodopsin is forward-shifted in a DDM micellar system [1], whereas it is back-shifted when solubilized in CHAPS. Previous MD simulations of rhodopsin could not reach Meta II due to the long timescale required (ms); in addition crystal structures of the activated state have been lacking [2-4]. However, detergents such as DDM accelerate Meta II kinetics to the s timescale [1], making it possible to reach the Meta II state in a DDM micelle. Using the recently published crystal structure of a putative Meta II state [5], we are able to show how the forward- and back-shifting of the Meta I ↔ Meta II equilibrium takes place in DDM and CHAPS detergent environments, respectively. These results provide important structural insights into rhodopsin activation, which can be extended to other GPCRs in a membrane mimetic environment, such as the A2A adenosine receptor. We have introduced a powerful tool which allows us to examine crystallization effects and isolate GPCR activated states.[1] S. Jager et al. (1997) Biochemistry 36:1999.[2] K. Martinez-Mayorga et al. (2006) JACS 128:16502.[3] P.-W. Lau et al. (2007) JMB 372:906.[4] T. Huber et al. (2004) Biophys. J. 86:2078.[5] H.-W. Choe et al. (2011) Nature 471:651.

Large multiple transmembrane domain fragments of a G protein-coupled receptor: biosynthesis, purification, and biophysical studies.

To conduct biophysical analyses on large domains of GPCRs, multimilligram quantities of highly homogeneous proteins are necessary. This communication discusses the biosynthesis of four transmembrane and five transmembrane-containing fragments of Ste2p, a GPCR recognizing the Saccharomyces cerevisiae tridecapeptide pheromone α-factor. The target fragments contained the predicted four N-terminal Ste2p[G(31) -A(198) ] (4TMN), four C-terminal Ste2p[T(155) -L(340) ] (4TMC), or five C-terminal Ste2p[I(120) -L(340) ] (5TMC) transmembrane segments of Ste2p. 4TMN was expressed as a fusion protein using a modified pMMHa vector in L-arabinose-induced Escherichia coli BL21-AI, and cleaved with cyanogen bromide. 4TMC and 5TMC were obtained by direct expression using a pET21a vector in IPTG-induced E. coli BL21(DE3) cells. 4TMC and 5TMC were biosynthesized on a preparative scale, isolated in multimilligram amounts, characterized by MS and investigated by biophysical methods. CD spectroscopy indicated the expected highly α-helical content for 4TMC and 5TMC in membrane mimetic environments. Tryptophan fluorescence showed that 5TMC integrated into the nonpolar region of 1-stearoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol) micelles. HSQC-TROSY investigations revealed that [(15) N]-labeled 5TMC in 50% trifluoroethanol-d(2) /H(2) O/0.05%-trifluoroacetic acid was stable enough to conduct long multidimensional NMR measurements. The entire Ste2p GPCR was not readily reconstituted from the first two and last five or first three and last four transmembrane domains.

Unchanged Transmembrane Segment Kink and Topology Despite Variation in Membrane-Mimetic Environment

Membrane mimetics such as micelles, bicelles or bilayers provide an essential tool for studying membrane proteins and their fragments. Following the “divide and conquer” strategy, we have determined the structure of the N-terminal tail and first transmembrane segment of the human apelin receptor (residues 1-55, AR55). This was solved using solution-state nuclear magnetic resonance (NMR) spectroscopy in sodium dodecylsuphate (SDS), dodecylphosphocholine (DPC) and 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) (LPPG) micelles as well as in a solution of 50% H2O and 50% hexafluoroisopropanol (HFIP). This high-resolution structure of AR55 consists of a helix-kink-helix motif in the micelle-spanning transmembrane region in all of the membrane mimetics studied. The N-terminal tail of AR55 has a similar structure in all three micelle conditions and has a much higher helical content in HFIP. Dynamics along the polypeptide backbone from 15N nuclear spin relaxation measurements of AR55 are correlated to solvent accessibility determined by paramagnetic relaxation enhancement, allowing definitive comparison of the manner of embedding of the protein. We show that AR55 adopts a highly similar conformation and topology in SDS, DPC and LPPG micelles despite the dramatic differences between these micelles in both headgroup and tailgroup. The transmembrane domain conformation of AR55 in HFIP is largely similar, including at the kink, despite the induction of helical character in the solution exposed N-terminal tail.

Trichogin GA IV: A versatile template for the synthesis of novel peptaibiotics

Trichogin GA IV, isolated from the fungus Trichoderma longibrachiatum, is the prototype of lipopeptaibols, the sub-class of short-length peptaibiotics exhibiting membrane-modifying properties. This peptaibol is predominantly folded in a mixed 3(10)-/α- helical conformation with a clear, albeit modest, amphiphilic character, which is likely to be responsible for its capability to perturb bacterial membranes and to induce cell death. In previous papers, we reported on the interesting biological properties of trichogin GA IV, namely its good activity against Gram positive bacteria, in particular methicillin-resistant S. aureus strains, its stability towards proteolytic degradation, and its low hemolytic activity. Aiming at broadening the antimicrobial activity spectrum by increasing the peptide helical amphiphilicity, in this work we synthesized, by solution and solid-phase methodologies, purified and fully characterized a set of trichogin GA IV analogs in which the four Gly residues at positions 2, 5, 6, 9, lying in the poorly hydrophilic face of the helical structure, are substituted by one (position 2, 5, 6 or 9), two (positions 5 and 6), three (positions 2, 5, and 9), and four (positions 2, 5, 6, and 9) Lys residues. The conformational preferences of the Lys-containing analogs were assessed by FT-IR absorption, CD and 2D-NMR techniques in aqueous, organic, and membrane-mimetic environments. Interestingly, it turns out that the presence of charged residues induces a transition of the helical conformation adopted by the peptaibols (from 3(10)- to α-helix) as a function of pH in a reversible process. The role played in the analogs by the markedly increased amphiphilicity was further tested by fluorescence leakage experiments in model membranes, protease resistance, antibacterial and antifungal activities, cytotoxicity, and hemolysis. Taken together, our biological results provide evidence that some of the least substituted among these analogs are good candidates for the development of new membrane-active antimicrobial agents.

Conversion of an Alpha-Helical to a Beta-Sheet Membrane Protein Fold through Pro-Gly Mutations in the Loop of a Human CFTR Transmembrane Hairpin

Membrane proteins adopt two fundamental types of fold in nature. Eukaryotic membranes harbor only α-helical bundles composed of series of interacting hydrophobic α-helices separated by aqueous-located loops of varying length, while β-barrel structures are found in the outer membrane of gram-negative bacteria, mitochondria and chloroplasts. Their interwoven β-sheet structures are comprised of pairs of membrane-spanning β-strand hairpins with a characteristic alternating hydrophobic/small residue sequence pattern, connected by a short intervening loop. We report that mutations in the loop of a eukaryotic α-helical hairpin peptide from the cystic fibrosis transmembrane conductance regulator (CFTR) can induce the peptide to adopt a β-sheet structure in a membrane-mimetic environment. We characterized in sodium dodecylsulfate (SDS), sodium perfluorooctanoate (SPFO) and dodecyl maltoside (DDM) micelles several constructs of the CFTR transmembrane 3 and 4 (TM3-loop-TM4) ‘hairpins’ in which Pro-Gly residues have been either inserted or substituted to promote a β-turn within the putative loop region. Suitable positioning of the Pro-Gly couplet caused the formation of a ladder of discrete oligomers on SDS-PAGE and SPFO-PAGE. Circular dichroism spectroscopy showed that mutants displaying oligomeric patterns adopt a stable β-sheet structure in detergents. In addition, size-exclusion chromatography and pyrene excimer fluorescence analysis evidenced that β-sheet oligomers are not proliferative and/or random aggregates. Such a facile change from an α-helical to an oligomeric β-hairpin structure may have implications into the evolutionary connection between α-helical and β-barrel membrane proteins. Indeed, considering that the first membrane proteins were helical hairpins (1), these results could reproduce an early differentiation event in the evolution of membrane proteins.1. Frishman, D. (2010) Structural bioinformatics of membrane proteins, 1st ed., pp xi, 281 p. Springer, New York.

Structure of Transmembrane Domain and Dimerization Mechanism of Amyloid Precursor Protein

Alzheimer's disease (AD) is the most prevalent form of dementia that affects senior people regardless of religious conviction, nationality or color of skin. The central role in understanding the underlying mechanisms of AD plays biosynthesis of amyloid-β peptide (Aβ). Aβ is a product of sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretase. More than half of mutations that cause so called familial forms of AD are situated in membrane domain of APP and in presenilin-1/presenilin-2 that are part of γ-secretase complex and cleavage APP within the membrane. The pathogenic mutations presumably affect the structure and dynamic properties of the APP transmembrane domain by changing its conformational stability, lateral mobility and /or dimerization. In the present study, the structure of right-handed dimer and dynamics properties of the recombinant peptide corresponding to the APP fragment, Gln686-Lys726, which comprises the APP transmembrane domain with an adjacent N-terminal juxtamembrane sequence, were determined. The structure was solved in the membrane mimetic environment composed of dodecylphosphocholine micelles using NMR spectroscopy. The conformation of transmembrane region each subunit does not alter from published monomer structure. Residues GLY709 and ALA713 form classical GxxxA motif for helix-helix interaction. Juxtamembrane part of investigated peptide does not form tight secondary structure. In this research for the first time dimerization mechanism of APP in atomic resolution is described.

Reconstitution of integral membrane proteins into isotropic bicelles with improved sample stability and expanded lipid composition profile

Reconstitution of integral membrane proteins into membrane mimetic environments suitable for biophysical and structural studies has long been a challenge. Isotropic bicelles promise the best of both worlds—keeping a membrane protein surrounded by a small patch of bilayer-forming lipids while remaining small enough to tumble isotropically and yield good solution NMR spectra. However, traditional methods for the reconstitution of membrane proteins into isotropic bicelles expose the proteins to potentially destabilizing environments. Reconstituting the protein into liposomes and then adding short-chain lipid to this mixture produces bicelle samples while minimizing protein exposure to unfavorable environments. The result is higher yield of protein reconstituted into bicelles and improved long-term stability, homogeneity, and sample-to-sample reproducibility. This suggests better preservation of protein structure during the reconstitution procedure and leads to decreased cost per sample, production of fewer samples, and reduction of the NMR time needed to collect a high quality spectrum. Furthermore, this approach enabled reconstitution of protein into isotropic bicelles with a wider range of lipid compositions. These results are demonstrated with the small multidrug resistance transporter EmrE, a protein known to be highly sensitive to its environment.

Structural Characterization of Hsp12, the Heat Shock Protein from Saccharomyces cerevisiae, in Aqueous Solution Where It Is Intrinsically Disordered and in Detergent Micelles Where It Is Locally α-Helical

Hsp12 (heat shock protein 12) belongs to the small heat shock protein family, partially characterized as a stress response, stationary phase entry, late embryonic abundant-like protein located at the plasma membrane to protect membrane from desiccation. Here, we report the structural characterization of Hsp12 by NMR and biophysical techniques. The protein was labeled uniformly with nitrogen-15 and carbon-13 so that its conformation could be determined in detail both in aqueous solution and in two membrane-mimetic environments, SDS and dodecylphosphocholine (DPC) micelles. Secondary structural elements determined from assigned chemical shifts indicated that Hsp12 is dynamically disordered in aqueous solution, whereas it gains four helical stretches in the presence of SDS micelles and a single helix in presence of DPC. These conclusions were reinforced by circular dichroism spectra of the protein in all three environments. The lack of long range interactions in NOESY spectra indicated that the helices present in SDS micelles do not pack together. R(1) and R(2), relaxation and heteronuclear NOE measurements showed that the protein is disordered in aqueous solution but becomes more ordered in presence of detergent micelles. NMR spectra collected in presence of paramagnetic spin relaxation agents (5DSA, 16DSA, and Gd(DTPA-BMA)) indicated that the amphipathic α-helices of Hsp12 in SDS micelles lie on the membrane surface. These observations are in agreement with studies suggesting that Hsp12 functions to protect the membrane from desiccation.

Lipid and Membrane Mimetic Environments Modulate Spin Label Side Chain Configuration in the Outer Membrane Protein A

In the present work, the factors that determine EPR line shapes from spin labels at the protein-hydrocarbon interface of a β-barrel membrane protein are examined. The EPR spectra from hydrocarbon facing sites in the outer membrane protein A (OmpA) are highly dependent upon the detergent or lipid into which OmpA is reconstituted. In general, line shapes at these sites are correlated with the solvent accessibility in the supporting amphiphile. A notable exception is CHAPS, which yields rigid limit EPR line shapes for labels at every position along a transmembrane β-strand in OmpA. EPR line shapes from the surface of OmpA are not strongly influenced by steric interference with neighboring side chains, but are modulated by solutes that should interact with hydrophobic surfaces. These results suggest that differences in EPR spectra in different supporting environments are not the result of differences in protein dynamics but are a result of different configurations or rotameric states that are assumed by the label. This conclusion is supported by distance measurements across the OmpA β-barrel, which indicate that labels yielding more motionally restricted line shapes interact more closely with the protein surface. These results have implications for the use of spin-label-derived distance constraints in protein structure determination and demonstrate that spin labels on membrane proteins provide a highly sensitive probe for the environment surrounding a membrane protein.

The structure and assembly model of the third transmembrane domain of Slc11a1 in SDS micelles revealed by NMR study of the Leu‐substituted peptide

Slc11a1 is an integral membrane protein with 12 putative transmembrane domains (TMDs) and functions as a pH-coupled divalent metal cation transporter. The conservation of three negatively charged residues in the TMD3 of Slc11 protein family implies the important role of this domain in the function of the proteins. However, aggregation of the transmembrane peptide in micelles prevents structural study of the peptide in these membrane-mimetic environments by NMR spectroscopy. Here, we characterized the structure, position, and assembly model of Slc11a1-TMD3 (Lys128-Ile151) in SDS micelles by the NMR study of its Leu-substituted peptide. It was found that the two-site substitutions of Ala for Leu residues at positions 136 and 140 of TMD3 disrupt the aggregation without altering the secondary structure of the peptide. The Leu-substituted peptide folds as an α-helix spanning from Leu133 to Gly144 and embedded in the micelles. A Leu zipper is suggested to account for the self-assembly of the wild-type peptide in SDS micelles.

Caveolin-1 hydrophobic segment peptides insertion into membrane mimetic systems: Role of Proline residue

Caveolin-1 has a segment of hydrophobic amino acids comprising approximately residues 103–122 that are anchored to the membrane with cholesterol-rich domains. Previously, we reported that changing the Pro110 residue to Ala (the P110A mutant) prevents not only the localization of the protein into lipid rafts but also the formation and functioning of caveolae. The conformational state of caveolin-1 can be shifted toward the transmembrane arrangement by this single amino acid mutation. To model the conformation, and extent of membrane insertion of this segment into membrane-mimetic environments, we have prepared a peptide corresponding to this hydrophobic segment of caveolin-1 having the sequence KKKKLSTIFGIPMALIWGIYFAILKKKKK-amide and the mutated version, KKKKLSTIFGIAMALIWGIYFAILKKKKK-amide. These peptides contain flanking Lys residues to facilitate purification and handling of the peptide. Circular dichroism measurements demonstrated that the mutated peptide has increased helical content compared with the wild type both in the presence and absence of lipid. The fluorescence emission from the Trp residues in the peptide showed significant blue shifts in the presence of liposomes, however the presence of cholesterol in hydrated vesicle bilayers decreases its helical content. Our overall findings support our studies with the intact protein in cells and suggest that the peptide of WT caveolin-1 hydrophobic segment has an intrinsic preference not to maintain its conformation as a rigid transmembrane helix. Substituting the Pro residue with an Ala allows the peptide to exist in a more hydrophobic environment likely as a consequence of a change in its conformation to a straight hydrophobic helix that traverses the membrane.

Probing ground and excited states of phospholamban in model and native lipid membranes by magic angle spinning NMR spectroscopy

In this paper, we analyzed the ground and excited states of phospholamban (PLN), a membrane protein that regulates sarcoplasmic reticulum calcium ATPase (SERCA), in different membrane mimetic environments. Previously, we proposed that the conformational equilibria of PLN are central to SERCA regulation. Here, we show that these equilibria detected in micelles and bicelles are also present in native sarcoplasmic reticulum lipid membranes as probed by MAS solid-state NMR. Importantly, we found that the kinetics of conformational exchange and the extent of ground and excited states in detergent micelles and lipid bilayers are different, revealing a possible role of the membrane composition on the allosteric regulation of SERCA. Since the extent of excited states is directly correlated to SERCA inhibition, these findings open up the exciting possibility that calcium transport in the heart can be controlled by the lipid bilayer composition. This article is part of a Special Issue entitled: Membrane protein structure and function.

Structural Framework for the Modulation of the Activity of the Hybrid Antibiotic Peptide Cecropin A‐Melittin [CA(1–7)M(2–9)] by Nε‐Lysine Trimethylation

The 3D structures of six linear pentadecapeptides derived from the cecropin A-melittin antimicrobial peptide CA(1-7)M(2-9) [KWKLFKKIGAVLKVL-NH(2)] have been studied. These analogues are modified by ε-NH(2) trimethylation of one or more lysine residues and showed variation in both antimicrobial and cytotoxic activities, depending on the number and position of modified lysines. Since it is expected that these peptides will display a strong conformational ordering when in contact with membranes, we have investigated their structure on the basis of the data extracted from NMR experiments performed in membrane-mimetic environments. We show that inclusion of N(ε)-trimethylated lysine residues induces a certain degree of structural flexibility, while preserving to a variable extent a largely α-helical structure. In addition, peptide orientation with respect to SDS micelles has been explored by detection of the intensity changes of peptide NMR signals upon addition of a paramagnetic probe (Mn(2+) ions).