Membrane Mimetic Environments Research Articles

Abstract 3238: The transmembrane domain of prostate-specific membrane antigen: Its role in oligomerization and activation in prostate cancer and nonprostatic neovasculature

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related deaths among American men. The high expression of the membrane protein called prostate specific membrane antigen (PSMA) is implicated in PCa invasiveness as well as neovasculature of nonprostatic solid tumors. Little is known about the exact role of PSMA in PCa progression albeit it is one of the most validated biomarker for the diagnosis and detection of PCa. The functional form of PSMA occurs as a non-covalent homodimer, which is a common motif among bitopic membrane proteins. Further, it is widely recognized that many membrane proteins participate in intracellular signal transduction through the lateral association of their transmembrane domains (TMDs), but this has not been directly demonstrated in PSMA. We hypothesize that PSMA TMD participates in oligomerization and subsequent activation of PSMA. Thus, to gain insight into the non-covalent assembly of PSMA, we aim to determine if the isolated PSMA TMD peptide forms a dimer and if the treatment of full-length PSMA with exogenous PSMA TMD peptide will perturb its properties. Studies using isolated TMDs have been widely used to probe protein-protein interactions and provide useful information on protein assembly under membrane mimetic systems. We identified the amino acid sequence and designed the isolated PSMA TMD peptide based on a known human PSMA sequence (UniProt accession code: Q04609). Two Lysine residues ware appended at both the N- and C-termini to increase the solubility of this highly hydrophobic peptide in polar solvents. Our initial studies revealed that the TMD has twenty-four residues and contains two repeat units of the Small-XXX-Small motif. This sequence is known to be crucial in inducing a strong self-assembly as observed in the bitopic protein Glycophorin A. We prepared the PSMA TMD peptide by microwave-assisted solid phase synthesis and purified by high performance liquid chromatography. The peptide was analyzed by MALDI-TOF mass spectrometry to establish homogeneity and by circular dichroism to examine its secondary structure in membrane mimetic environment. We investigated the oligomerization of the peptide using SDS-PAGE gel shift and fluorescence self-quenching assays. We tested the effect of the peptide against PSMA using an established assay. Herein we report on the oligomerization and PSMA binding properties of PSMA TMD. Our findings may shed light on the role of PSMA TMD in PSMA oligomerization and its potential as a model peptide that target transmembrane helices. It may also aid in the rational design and development of peptide probes for protein-protein interactions as well as therapeutics that target cancer- and clinically-relevant membrane proteins. Citation Format: Brianna S. Berg, Brandan M. Cook, Nathan Beattie, Cliff E. Berkman, Jonel P. Saludes. The transmembrane domain of prostate-specific membrane antigen: Its role in oligomerization and activation in prostate cancer and nonprostatic neovasculature. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3238. doi:10.1158/1538-7445.AM2014-3238

Polar interactions trump hydrophobicity in stabilizing the self-inserting membrane protein Mistic.

Canonical integral membrane proteins are attached to lipid bilayers through hydrophobic transmembrane helices, whose topogenesis requires sophisticated insertion machineries. By contrast, membrane proteins that, for evolutionary or functional reasons, cannot rely on these machineries need to resort to driving forces other than hydrophobicity. A striking example is the self-inserting Bacillus subtilis protein Mistic, which is involved in biofilm formation and has found application as a fusion tag supporting the recombinant production and bilayer insertion of other membrane proteins. Although this unusual protein contains numerous polar and charged residues and lacks characteristic membrane-interaction motifs, it is tightly bound to membranes in vivo and membrane-mimetic systems in vitro. Therefore, we set out to quantify the contributions from polar and nonpolar interactions to the coupled folding and insertion of Mistic. To this end, we defined conditions under which the protein can be unfolded completely and reversibly from various detergent micelles by urea in a two-state equilibrium and where the unfolded state is independent of the detergent used for solubilizing the folded state. This enabled equilibrium unfolding experiments previously used for soluble and β-barrel membrane proteins, revealing that polar interactions with ionic and zwitterionic headgroups and, presumably, the interfacial dipole potential stabilize the protein much more efficiently than nonpolar interactions with the micelle core. These findings unveil the forces that allow a protein to tightly interact with a membrane-mimetic environment without major hydrophobic contributions and rationalize the differential suitability of detergents for the extraction and solubilization of Mistic-tagged membrane proteins.

The periplasmic domain of Escherichia coli outer membrane protein A can undergo a localized temperature dependent structural transition

Gram-negative bacteria such as Escherichia coli are surrounded by two membranes with a thin peptidoglycan (PG)-layer located in between them in the periplasmic space. The outer membrane protein A (OmpA) is a 325-residue protein and it is the major protein component of the outer membrane of E. coli. Previous structure determinations have focused on the N-terminal fragment (residues 1–171) of OmpA, which forms an eight stranded transmembrane β-barrel in the outer membrane. Consequently it was suggested that OmpA is composed of two independently folded domains in which the N-terminal β-barrel traverses the outer membrane and the C-terminal domain (residues 180–325) adopts a folded structure in the periplasmic space. However, some reports have proposed that full-length OmpA can instead refold in a temperature dependent manner into a single domain forming a larger transmembrane pore. Here, we have determined the NMR solution structure of the C-terminal periplasmic domain of E. coli OmpA (OmpA180–325). Our structure reveals that the C-terminal domain folds independently into a stable globular structure that is homologous to the previously reported PG-associated domain of Neisseria meningitides RmpM. Our results lend credence to the two domain structure model and a PG-binding function for OmpA, and we could indeed localize the PG-binding site on the protein through NMR chemical shift perturbation experiments. On the other hand, we found no evidence for binding of OmpA180–325 with the TonB protein. In addition, we have also expressed and purified full-length OmpA (OmpA1–325) to study the structure of the full-length protein in micelles and nanodiscs by NMR spectroscopy. In both membrane mimetic environments, the recombinant OmpA maintains its two domain structure that is connected through a flexible linker. A series of temperature-dependent HSQC experiments and relaxation dispersion NMR experiments detected structural destabilization in the bulge region of the periplasmic domain of OmpA above physiological temperatures, which may induce dimerization and play a role in triggering the previously reported larger pore formation.

Small expression tags enhance bacterial expression of the first three transmembrane segments of the apelin receptor.

G-protein coupled receptors (GPCRs) are inherently dynamic membrane protein modulators of various important cellular signaling cascades. The apelin receptor (AR or APJ) is a class A GPCR involved in numerous physiological processes, implicated in angiogenesis during tumour formation and as a CD4 co-receptor for entry of human immunodeficiency virus type 1 (HIV-1) to cells. Due to the lack of efficient methods to produce full-length GPCRs enriched with nuclear magnetic resonance (NMR) active (15)N, (13)C, and (or) (2)H isotopes, small GPCR fragments typically comprising 1-2 transmembrane segments are frequently studied using NMR spectroscopy. Here, we report successful overexpression of transmembrane segments 1-3 of AR (AR_TM1-3) in the C41(DE3) strain of Escherichia coli using an AT-rich gene tag previously reported to enhance cell-free expression yields. The resulting protein, with 6 additional N-terminal residues due to the expression tag, was purified using high-performance liquid chromatography (HPLC). Far UV circular dichroism spectropolarimetry demonstrates that AR_TM1-3 has the predicted ~40% α-helical character in membrane-mimetic environments. (1)H-(15)N HSQC NMR experiments imply amenability to high-resolution NMR structural characterization and stability in solution for weeks. Notably, this small expression tag approach may also be generally applicable to other membrane proteins that are difficult to express in E. coli.

Characterizing the structure of lipodisq nanoparticles for membrane protein spectroscopic studies

Membrane protein spectroscopic studies are challenging due to the difficulty introduced in preparing homogenous and functional hydrophobic proteins incorporated into a lipid bilayer system. Traditional membrane mimics such as micelles or liposomes have proved to be powerful in solubilizing membrane proteins for biophysical studies, however, several drawbacks have limited their applications. Recently, a nanosized complex termed lipodisq nanoparticles was utilized as an alternative membrane mimic to overcome these caveats by providing a homogeneous lipid bilayer environment. Despite all the benefits that lipodisq nanoparticles could provide to enhance the biophysical studies of membrane proteins, structural characterization in different lipid compositions that closely mimic the native membrane environment is still lacking. In this study, the formation of lipodisq nanoparticles using different weight ratios of POPC/POPG lipids to SMA polymers was characterized via solid-state nuclear magnetic resonance (SSNMR) spectroscopy and dynamic light scattering (DLS). A critical weight ratio of (1/1.25) for the complete solubilization of POPC/POPG vesicles has been observed and POPC/POPG vesicles turned clear instantaneously upon the addition of the SMA polymer. The size of lipodisq nanoparticles formed from POPC/POPG lipids at this weight ratio of (1/1.25) was found to be about 30nm in radius. We also showed that upon the complete solubilization of POPC/POPG vesicles by SMA polymers, the average size of the lipodisq nanoparticles is weight ratio dependent, when more SMA polymers were introduced, smaller lipodisq nanoparticles were obtained. The results of this study will be helpful for a variety of biophysical experiments when specific size of lipid disc is required. Further, this study will provide a proper path for researchers working on membrane proteins to obtain pertinent structure and dynamic information in a physiologically relevant membrane mimetic environment. This article is part of a Special Issue entitled: NMR Spectroscopy for Atomistic Views of Biomembranes and Cell Surfaces. Guest Editors: Lynette Cegelski and David P. Weliky.

Binding interactions of bacterial lipopolysaccharide and the cationic amphiphilic peptides polymyxin B and WLBU2

Passage of blood through a sorbent device for removal of bacteria and endotoxin by specific binding with immobilized, membrane-active, bactericidal peptides holds promise for treating severe blood infections. Peptide insertion in the target membrane and rapid/strong binding is desirable, while membrane disruption and release of degradation products to the circulating blood is not. Here we describe interactions between bacterial endotoxin (lipopolysaccharide, LPS) and the membrane-active, bactericidal peptides WLBU2 and polymyxin B (PmB). Analysis of the interfacial behavior of mixtures of LPS and peptide using air–water interfacial tensiometry and optical waveguide lightmode spectroscopy strongly suggests insertion of intact LPS vesicles by the peptide WLBU2 without vesicle destabilization. In contrast, dynamic light scattering (DLS) studies show that LPS vesicles appear to undergo peptide-induced destabilization in the presence of PmB. Circular dichroism spectra further confirm that WLBU2, which shows disordered structure in aqueous solution and substantially helical structure in membrane-mimetic environments, is stably located within the LPS membrane in peptide-vesicle mixtures. We therefore expect that presentation of WLBU2 at an interface, if tethered in a fashion which preserves its mobility and solvent accessibility, will enable the capture of bacteria and endotoxin without promoting reintroduction of endotoxin to the circulating blood, thus minimizing adverse clinical outcomes. On the other hand, our results suggest no such favorable outcome of LPS interactions with polymyxin B.

A Minimal Hydrophobicity Is Needed To Employ Amphiphilic p(HPMA)-co-p(LMA) Random Copolymers in Membrane Research

Because a polymer environment might be milder than a detergent micelle, amphiphilic polymers have attracted attention as alternatives to detergents in membrane biochemistry. The polymer poly[N-(2-hydroxypropyl)-methacrylamid] [p(HPMA)] has recently been modified with hydrophobic lauryl methacrylate (LMA) moieties, resulting in the synthesis of amphiphilic p(HPMA)-co-p(LMA) polymers. p(HPMA)-co-p(LMA) polymers with a LMA content of 5 or 15% have unstable hydrophobic cores. This, on one hand, promotes interactions of the hydrophobic LMA moieties with membranes, resulting in membrane rupture, but at the same time prevents formation of a hydrophobic, membrane mimetic environment that is sufficiently stable for the incorporation of transmembrane proteins. On the other hand, the p(HPMA)-co-p(LMA) polymer with a LMA content of 25% forms a stable hydrophobic core structure, which prevents hydrophobic interactions with membrane lipids but allows stable incorporation of membrane proteins. On the basis of our data, it becomes obvious that amphiphilic polymers have to have threshold hydrophobicities should an application in membrane protein research be anticipated.

Antimicrobial peptides against Pseudomonas syringae pv. actinidiae and Erwinia amylovora: Chemical synthesis, secondary structure, efficacy, and mechanistic investigations

We report on structurally modified dodecapeptide amides (KYKLFKKILKFL-NH2) and two analogs of a hexapeptide amide (WRWYCR-NH2) with antibacterial activity against the Gram negative pathogens Pseudomonas syringae pv. actinidiae (Psa) and Erwinia amylovora (Ea). Dodecapeptide minimal inhibitory concentrations (MICs) ranged from 3.2 to 15.4 µM, with the unmodified peptide being the most potent against both pathogens. The unmodified dodecapeptide also had 32-58% α-helicity in membrane mimetic environments (50% v/v trifluoroethanol and 30 mM SDS micelles). Structural modifications which included branching, acylation, and conjugation with 5-nitro-2-furaldehyde (NFA) proved detrimental to both antimicrobial activity and α-helicity. Scanning electron microscopy (SEM) revealed distinct morphological changes to bacterial cells treated with the different peptides, leading to blistering of the membrane and cell lysis. MICs of the hexapeptide amide were 3.9-7.7 µM against both pathogens. The hexapeptide acid did not show anti-bacterial activity against either pathogen. However, the NFA conjugated hexapeptide acid was more active than the parent peptide or NFA alone with MICs of 1.6-3.2 µM against the pathogens. SEM analysis revealed shriveling and collapse of bacterial cells treated with the hexapeptide, whereas shortening and compactness on exposure to streptomycin. A colorimetric assay demonstrated that the dodecapeptides were likely to act by targeting the bacterial membrane, whereas the hexapeptides, streptomycin, and NFA were not, thereby supporting the morphological changes observed during SEM. To the best of our knowledge, this appears to be the first report of antimicrobial peptide activity against Psa, a pathogen that is currently devastating the kiwifruit industry internationally.

Tuning Function of the Light-Driven Proteorhodopsin Proton Pump by Formation of Oligomeric and Surfactant-Based Synthetic Complexes

The Proteorhodopsin (PR) membrane protein equips marine bacteria worldwide with the capability of converting light energy into chemical energy. The proton pump absorbs light via an intrinsic retinal molecule, followed by subsequent conformational changes that store energy in the form of a proton gradient across the bacterial cell membrane. Because PR exists in the complex membrane environment, a multitude of factors could contribute to its light absorption and energy-transducing properties, including protein-protein, protein-lipid, and electrostatic interactions. Here we demonstrate by steady-state and time-resolved optical absorption spectroscopy that there are profound functional effects (e.g. altered activity and photocycle kinetics) modulated by the association of PR with other PR molecules to form oligomeric complexes, and by reconstitution in membrane-mimetic surfactant environments with varying chemical and physical characteristics.We study the molecular-level properties accompanying these functional consequences of environment with magnetic resonance techniques, specifically amino acid side-chain dynamics probed by Electron Paramagnetic Resonance (EPR) and local hydration dynamics by Overhauser Dynamic Nuclear Polarization (ODNP). Such information lends insight into the mechanisms tuning PR function and can be applied towards the design of function-enhancing synthetic environments. For example, surface water diffusion is faster in a model lipid bilayer than in surfactant micelles, concurrent with faster conformational motion, and possibly efficiency (Hussain, et.al, Angew. Chem. 52, 2013). We further have developed a synthetic platform for potential device applications by the incorporation of active PR into surfactant-directed mesostructured silica films, in both its hexameric and monomeric forms, and using different surfactants. This provides a unique opportunity to apply results from the biophysical characterization of PR to the design of an optimized solar energy-harvesting biomaterial, tuning molecular interactions to match the desired functional properties.

Solid-State NMR Characterization of S31N M2 Transmembrane Domain Bound to Novel Adamantanes with Persistent In Vitro Efficacy

The M2 proton channel from Influenza A virus is essential for the viral lifecycle and is an important drug target. Amino acid mutations in the residues lining the pore of the channel have abrogated clinical efficacy of the previously FDA-approved antiviral agents, amantadine and rimantadine. More than 95% of the circulating strains isolated from human clinical cases bear S31N mutation and a subsequent resistance to the licensed small molecule inhibitors. Efforts in rational drug design targeting S31N M2 channel have been impeded by the limited number of experimental techniques with capabilities for structural characterization of the protein-ligand interaction in native-like membrane mimetic environments. Solution and Solid State Nuclear Magnetic Resonance (NMR) investigations of the recently introduced adamantane analogues suggest multiple orientations of the inhibitor molecules bound in the pore. Position of the substituent moiety in the channel varies for different compounds, unlike the fixed orientation in the wild type channel. Here we report a Solid State NMR investigation of the transmembrane domain of S31N M2 (TMD) proton channel bound to the novel inhibitor molecules, while also incorporated into a lipid bilayer environment. Oriented sample solid state NMR experiments of S31N M2 TMD indicate that the channel is sampling two states with helical tilts of 28° and 33° relative to the bilayer normal. The kink in the monomer between two helical fragments, observed in the wild type M2 TMD bound with amantadine, is absent for the S31N M2 TMD bound to all inhibitor molecules tested to date. Stabilizing interactions are further investigated with Rotational Echo Double Resonance Magic Angle Spinning (REDOR MAS) experiments for measuring distances from the inhibitor to the protein.

Characterizing the Structure of Lipodisq for Membrane Protein Spectroscopic Studies

Membrane protein spectroscopic studies are challenging due to the difficulty introduced in preparing homogenous and functional protein incorporated lipid system. Traditional membrane mimetics such as micelles or vesicles are proved to be powerful. Though, they all possess their own drawbacks. The lack of lipid bilayers of micelles could result in the increasing dynamics of membrane proteins and obtaining homogenous vesicles are not easy in the real applications. Recently, a nano-sized particle lipodisq was utilized to serve as a better membrane mimetic, it provides a lipid bilayer environment and homogenous samples. Furthermore, unlike nanodisc, lipodisq won't interfere the absorbance property of membrane proteins. Though lipodisq shows a high potential to become a good membrane mimetic to enhance the biophysical studies of membrane proteins, there is still lack of structural characterization of lipodisq in different lipid compositions that close to native lipid environment of membrane proteins. In this study, the formation of lipodisq nanoparticles using different weight ratio of 3:1 SMA polymer to POPC/POPG lipid was characterized using dynamic light scattering (DLS) and solid state nuclear magnetic resonance (SSNMR) spectroscopy. We achieved a physiologically relevant size (10nm) of lipodisq nanoparticles complex at weight ratio of 2.25:1 (3:1 SMA polymer : POPC/POPG lipid) and the transition phase from vesicle to lipodisq was characterized. These data were also compared with the corresponding data obtained for bicelles and micelles. This study will provide a proper path for the researcher working on membrane protein system to obtain pertinent structure and dynamic information on physiologically relevant membrane mimetic environment.

High-resolution paramagnetically enhanced solid-state NMR spectroscopy of membrane proteins at fast magic angle spinning

Magic angle spinning nuclear magnetic resonance (MAS NMR) is well suited for the study of membrane proteins in membrane mimetic and native membrane environments. These experiments often suffer from low sensitivity, due in part to the long recycle delays required for magnetization and probe recovery, as well as detection of low gamma nuclei. In ultrafast MAS experiments sensitivity can be enhanced through the use of low power sequences combined with paramagnetically enhanced relaxation times to reduce recycle delays, as well as proton detected experiments. In this work we investigate the sensitivity of (13)C and (1)H detected experiments applied to 27 kDa membrane proteins reconstituted in lipids and packed in small 1.3 mm MAS NMR rotors. We demonstrate that spin diffusion is sufficient to uniformly distribute paramagnetic relaxation enhancement provided by either covalently bound or dissolved CuEDTA over 7TM alpha helical membrane proteins. Using paramagnetic enhancement and low power decoupling in carbon detected experiments we can recycle experiments ~13 times faster than under traditional conditions. However, due to the small sample volume the overall sensitivity per unit time is still lower than that seen in the 3.2 mm probe. Proton detected experiments, however, showed increased efficiency and it was found that the 1.3 mm probe could achieve sensitivity comparable to that of the 3.2 mm in a given amount of time. This is an attractive prospect for samples of limited quantity, as this allows for a reduction in the amount of protein that needs to be produced without the necessity for increased experimental time.

Investigation of Homeodomain Membrane Translocation Properties: Insights from the Structure Determination of Engrailed-2 Homeodomain in Aqueous and Membrane-Mimetic Environments

In addition to their well-known DNA-binding properties, homeodomains have the ability to efficiently translocate across biological membranes through still poorly-characterized mechanisms. To date, most biophysical studies addressing the mechanisms of internalization have focused on small synthetic peptides rather than full-length globular homeodomains. In this work, we characterized the conformational properties of chicken Engrailed 2 homeodomain (En2HD) in aqueous solution and in membrane mimetic environments using circular dichroism, Trp fluorescence, and NMR spectroscopy. En2HD adopts a well-defined three-helical bundle fold in aqueous solution. The Trp-48 residue, which is critical for internalization, is fully buried in the hydrophobic core. Circular dichroism and fluorescence reveal that a conformational transition occurs in anionic lipid vesicles and in micelles. En2HD loses its native three-dimensional structure in micellar environments but, remarkably, near-native helical secondary structures are maintained. Long-range interactions could be detected using site-directed spin labels, indicating that the three helices do not adopt extended orientations. Noncovalent paramagnetic probes yielded information about helix positioning and unveiled the burial of critical aromatic and basic residues within the micelles. Our results suggest that electrostatic interactions with membranes may be determinant in inducing a conformational change enabling Trp-48 to insert into membranes.

The Structure of the Mercury Transporter MerF in Phospholipid Bilayers: A Large Conformational Rearrangement Results from N-Terminal Truncation

The three-dimensional structure of the 81-residue mercury transporter MerF determined in liquid crystalline phospholipid bilayers under physiological conditions by Rotationally Aligned (RA) solid-state NMR has two long helices, which extend well beyond the bilayer, with a well-defined interhelical loop. Truncation of the N-terminal 12 residues, which are mobile and unstructured when the protein is solubilized in micelles, results in a large structural rearrangement of the protein in bilayers. In the full-length protein, the N-terminal helix is aligned nearly parallel to the membrane normal and forms an extension of the first transmembrane helix. By contrast, this helix adopts a perpendicular orientation in the truncated protein. The close spatial proximity of the two Cys-containing metal binding sites in the three-dimensional structure of full-length MerF provides insights into possible transport mechanisms. These results demonstrate that major changes in protein structure can result from differences in amino acid sequence (e.g., full-length vs truncated proteins) as well as the use of a non-native membrane mimetic environment (e.g., micelles) vs liquid crystalline phospholipid bilayers. They provide further evidence of the importance of studying unmodified membrane proteins in near-native bilayer environments in order to obtain accurate structures that can be related to their functions.

Conformation of gramicidin A in Triton X‐100 micelles from CD and FTIR data: a clean example of antiparallel double β5.6 helix formation

The linear peptide gramicidin A (gA) forms prototypical ion channels specific for monovalent cations and has been extensively used to study the organization and dynamics of membrane channels. This polymorphic peptide can adopt two different types of structures, the helical dimer β6.3 ('channel state') and the double helical structure with two intertwined monomers. The structure of gA in micelles of detergent Triton X-100 has been studied using CD, Fourier transform infrared, and fluorescence spectroscopy. The results obtained demonstrate that only one thermodynamically stable gA structure, the antiparallel left-handed double helix β5.6, is formed in this membrane-mimetic environment. The position of the tryptophan fluorescence maximum at 332 nm is the same as that in phospholipid membranes. The causative factors governing the double helix formation in the micellar medium are discussed on the basis of known physicochemical properties of Triton X-100.

Spectroscopically Labeled Peptaibiotics. Synthesis and Properties of Selected Trichogin GA IV Analogs Bearing a Side‐Chain‐Monofluorinated Aromatic Amino Acid for19F‐NMR Analysis

We prepared by solid-phase methods, chromatographically purified, and characterized three analogs of the ten-amino acid-residue, membrane-active, lipopeptaibiotic trichogin GA IV, each containing a single (4-fluorophenyl)alanine in position 3, 7, or 10, where it replaces the hydrophobic residue Leu(3) , Leu(7) , or Ile(10) , respectively. We incorporated the fluorine probe based on the observation that the (19) F-NMR technique has been extensively utilized to analyze peptidemembrane interactions in biological systems. A detailed conformational investigation in solution, including a membrane-mimetic environment, was performed on these compounds using FT-IR absorption, CD, and 2D-NMR, combined with molecular-dynamics calculations. The experimentally observed, mixed 310 /α-helical structures unequivocally show that the principal conformational features of trichogin GA IV are preserved in all three analogs. Analogies and differences between the behavior of the natural lipopeptaibiotic and those of the peptides characterized by the side-chain monofluorinated aromatic amino acid were found in membrane-permeabilization experiments and antimicrobial assays. The results of a preliminary solution (19) F-NMR study support the view that the (19) F label is an excellent reporter for changes in the helical environment of the peptide.

Modeling the membrane environment has implications for membrane protein structure and function: Influenza A M2 protein

The M2 protein, a proton channel, from Influenza A has been structurally characterized by X-ray diffraction and by solution and solid-state NMR spectroscopy in a variety of membrane mimetic environments. These structures show substantial backbone differences even though they all present a left-handed tetrameric helical bundle for the transmembrane domain. Variations in the helix tilt influence drug binding and the chemistry of the histidine tetrad responsible for acid activation, proton selectivity and transport. Some of the major structural differences do not arise from the lack of precision, but instead can be traced to the influences of the membrane mimetic environments. The structure in lipid bilayers displays unique chemistry for the histidine tetrad, which binds two protons cooperatively to form a pair of imidazole-imidazolium dimers. The resulting interhistidine hydrogen bonds contribute to a three orders of magnitude enhancement in tetramer stability. Integration with computation has provided detailed understanding of the functional mechanism for proton selectivity, conductance and gating of this important drug target.

Influences of Membrane Mimetic Environments on Membrane Protein Structures

The number of membrane protein structures in the Protein Data Bank is becoming significant and growing. Here, the transmembrane domain structures of the helical membrane proteins are evaluated to assess the influences of the membrane mimetic environments. Toward this goal, many of the biophysical properties of membranes are discussed and contrasted with those of the membrane mimetics commonly used for structure determination. Although the mimetic environments can perturb the protein structures to an extent that potentially gives rise to misinterpretation of functional mechanisms, there are also many structures that have a native-like appearance. From this assessment, an initial set of guidelines is proposed for distinguishing native-like from nonnative-like membrane protein structures. With experimental techniques for validation and computational methods for refinement and quality assessment and enhancement, there are good prospects for achieving native-like structures for these very important proteins.

Deracemization and the first CD spectrum of a 310-helical peptide made of achiral α-amino-isobutyric acid residues in a chiral membrane mimetic environment

Interaction of the racemic helical homo-octapeptide made by the achiral C(α)-methyl alanine (Aib) amino acid with a chiral enantiopure micellar aggregate made of N-dodecylproline led to the deracemization of the helical Aib sequence thus allowing us to obtain for the first time the CD signature in water of a 310 helix devoid of the contribution of any chiral amino acid.

Antibacterial and leishmanicidal activities of temporin-SHd, a 17-residue long membrane-damaging peptide

Temporins are a family of short antimicrobial peptides (8-17 residues) that mostly show potent activity against Gram-positive bacteria. Herein, we demonstrate that temporin-SHd, a 17-residue peptide with a net charge of +2 (FLPAALAGIGGILGKLF(amide)), expressed a broad spectrum of antimicrobial activity. This peptide displayed potent antibacterial activities against Gram-negative and Gram-positive bacteria, including multi-drug resistant Staphylococcus aureus strains, as well as antiparasitic activity against promastigote and the intracellular stage (amastigote) of Leishmania infantum, at concentration not toxic for the macrophages. Temporin-SHd that is structured in a non-amphipathic α-helix in anionic membrane-mimetic environments, strongly and selectively perturbs anionic bilayer membranes by interacting with the polar head groups and acyl region of the phospholipids, with formation of regions of two coexisting phases: one phase rich in peptide and the other lipid-rich. The disruption of lipid packing within the bilayer may lead to the formation of transient pores and membrane permeation/disruption once a threshold peptide accumulation is reached. To our knowledge, Temporin-SHd represents the first known 17-residue long temporin expressing such broad spectrum of antimicrobial activity including members of the trypanosomatidae family. Additionally, since only a few shorter members (13 residues) of the temporin family are known to display antileishmanial activity (temporins-TA, -TB and -SHa), SHd is an interesting tool to analyze the antiparasitic mechanism of action of temporins.