FTIR spectroscopic studies of the conformation and amide hydrogen exchange of a peptide model of the hydrophobic transmembrane alpha-helices of membrane proteins.

The conformation and amide hydrogen exchangeability of the hydrophobic peptide Lys2-Gly-Leu24-Lys2-Ala-amide were studied by Fourier transform infrared spectroscopy. In these studies information on the secondary structure of the peptide was obtained from an examination of the contours of both the amide I and amide II absorption bands. The conformationally sensitive amide I and amide II regions of the infrared spectra suggest that the peptide is predominantly alpha-helical and that it contains some non-alpha-helical structures which are probably in an extended conformation. Studies of the exchangeability of the amide protons of the peptide indicate that there are two populations of amide protons which differ markedly with respect to their exchangeability with the bulk solvent phase, whether the peptide is dissolved in methanol or dispersed in hydrated lipid bilayers. One population of amide protons is very readily exchangeable, and our data suggest that it arises primarily but not exclusively from the extended regions of the peptide. The other population exchanges very slowly with the bulk solvent and appears to originate entirely from the alpha-helical domain of the peptide. This latter population is virtually unexchangeable when the peptide is dispersed in hydrated phosphatidylcholine bilayers but can be largely exchanged when the peptide is solubilized with methanol. We suggest that this slowly exchanging population of amide protons arises from the central part of the hydrophobic polyleucine core which forms a very stable alpha-helix that would be deeply buried in the hydrophobic domain of hydrated lipid bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

The fusogenic properties of the cytotoxins of cobra venom in a model membrane system

By the methods of EPR spinal probes, energy migration of triplet excitation and NMR spectroscopy, the structural changes on hydrocarbon region of membranes, the changes in dynamic state of water of lipid hydrate jacket, the intermembrane lipid material exchange and fusion of membranes induced by cytotoxins of cobra venom have been studied. The sequence of events preceded the membrane fusion is suggested. The probability of membrane fusion has been shown not to be determined by fusogenic agent structure only, but much it depends on lipid composition of membranes.     

Distribution of hydrophobic residues is crucial for the fusogenic properties of the Ebola virus GP2 fusion peptide

The lipid-destabilizing properties of the N-terminal domain of the GP2 of Ebola virus were investigated. Our results suggest that the domain of Ebola virus needed for fusion is shorter than that previously reported. The fusogenic properties of this domain are related to its oblique orientation at the lipid/water interface owing to an asymmetric distribution of the hydrophobic residues when helical.     

Structure-function study on a de novo synthetic hydrophobic ion channel.

Ion conduction properties of a de novo synthesized channel, formed from cyclic octa-peptides consisting of four alternate L-alanine (Ala) and N'-acylated 3-aminobenzoic acid (Aba) moieties, were studied in bilayer membranes. The single-channel conductance was 9 pS in symmetrical 500 mM KCl. The channel favored permeation of cations over anions with a permeability ratio (PCl-/PK+) of 0.15. The selectivity sequence among monovalent cations based on permeability ratio (PX+/PK+) fell into an order: NH4+(1.4) > Cs+(1. 1) >/= K+(1.0) > Na+(0.4) >> Li+(0). The conductance-activity relationship of the channel in K+ solutions followed simple Michaelis-Menten kinetics with a half-maximal saturating activity of 8 mM and a maximal conductance of 9 pS. The permeability ratio PNa+/PK+ remained constant ( approximately 0.40) under biionic concentrations from 10 to 500 mM. These results suggests that the channel is a one-ion channel. The pore diameter probed by a set of organic cations was approximately 6 A. The single-channel current was blocked by Ca2+ in a dose-dependent manner that followed a single-site titration curve with a voltage-dependent dissociation constant of 0.6 mM at 100 mV. The electric distance of the binding site for Ca2+ was 0.07 from both entrances of the channel, indicating the presence of two symmetrical binding sites in each vicinity of the channel entrance. Correlations between conduction properties and structural aspects of the channel are discussed in terms of a three-barrier and two-binding-site (3B2S) model of Eyring rate theory. All available structural information supported an idea that the channel was formed from a tail-to-tail associated dimer of the molecule, the pore of which was lined with hydrophobic acyl chains. This is the first report to have made a systematic analysis of ion permeation through a hydrophobic pore.     

De novo design of the hydrophobic core of ubiquitin.

We have previously reported the development and evaluation of a computational program to assist in the design of hydrophobic cores of proteins. In an effort to investigate the role of core packing in protein structure, we have used this program, referred to as Repacking of Cores (ROC), to design several variants of the protein ubiquitin. Nine ubiquitin variants containing from three to eight hydrophobic core mutations were constructed, purified, and characterized in terms of their stability and their ability to adopt a uniquely folded native-like conformation. In general, designed ubiquitin variants are more stable than control variants in which the hydrophobic core was chosen randomly. However, in contrast to previous results with 434 cro, all designs are destabilized relative to the wild-type (WT) protein. This raises the possibility that beta-sheet structures have more stringent packing requirements than alpha-helical proteins. A more striking observation is that all variants, including random controls, adopt fairly well-defined conformations, regardless of their stability. This result supports conclusions from the cro studies that non-core residues contribute significantly to the conformational uniqueness of these proteins while core packing largely affects protein stability and has less impact on the nature or uniqueness of the fold. Concurrent with the above work, we used stability data on the nine ubiquitin variants to evaluate and improve the predictive ability of our core packing algorithm. Additional versions of the program were generated that differ in potential function parameters and sampling of side chain conformers. Reasonable correlations between experimental and predicted stabilities suggest the program will be useful in future studies to design variants with stabilities closer to that of the native protein. Taken together, the present study provides further clarification of the role of specific packing interactions in protein structure and stability, and demonstrates the benefit of using systematic computational methods to predict core packing arrangements for the design of proteins.     

Solid-phase synthesis and purification of a set of uniformly 13C, 15N labelled de novo designed membrane fusogenic peptides.

The transmembrane segments of soluble N-ethylmaleimide-sensitive factor (SNARE) proteins or viral envelope proteins drive membrane fusion, which suggests that simple synthetic biology constructs for fusion exist and can be evaluated. We describe the high-yield synthesis of a set of de novo designed fusogenic peptides for use in functional investigations, which are highly enriched in 13C and 15N using three equivalents of labelled amino acids and optimized reaction conditions minimizing aggregation. The biomimetic peptides have a high purity >90% and show reproducible and fusogenic activity that correlates well with the intended functional design characteristics, from strongly fusogenic to almost non-fusogenic.     

Interaction of a peptide model of a hydrophobic transmembrane alpha-helical segment of a membrane protein with phosphatidylcholine bilayers: differential scanning calorimetric and FTIR spectroscopic studies.

High-sensitivity differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to study the interaction of a synthetic model hydrophobic peptide, Lys2-Gly-Leu24-Lys2-Ala-amide, and members of the homologous series of n-saturated diacylphosphatidylcholines. In the low range of peptide mole fractions, the DSC thermograms exhibited by the lipid/peptide mixtures are resolvable into two components. One of these components is fairly narrow, highly cooperative, and exhibits properties which are similar to but not identical with those of the pure lipid. In addition, the fractional contribution of this component to the total enthalpy change, the peak transition temperature, and cooperativity decrease with an increase in peptide concentration, more or less independently of acyl chain length. The other component is very broad and predominates in the high range of peptide concentration. These two components have been assigned to the chain-melting phase transitions of populations of bulk lipid and peptide-associated lipid, respectively. Moreover, when the mean hydrophobic thickness of the PC bilayer is less than the peptide hydrophobic length, the peptide-associated lipid melts at higher temperatures than does the bulk lipid and vice versa. In addition, the chain-melting enthalpy of the broad endotherm does not decrease to zero even at high peptide concentrations, suggesting that this peptide reduces but do not abolish the cooperative gel/liquid-crystalline phase transition of the lipids with which it is in contact. Our DSC results indicate that the width of the phase transition observed at high peptide concentration is inversely but discontinuously related to hydrocarbon chain length and that gel phase immiscibility occurs when the hydrophobic thickness of the bilayer greatly exceeds the hydrophobic length of the peptide. The FTIR spectroscopic data indicate that the peptide forms a very stable alpha-helix under all of our experimental conditions but that small distortions of its alpha-helical conformation are induced in response to any mismatch between peptide hydrophobic length and bilayer hydrophobic thickness. These results also indicate that the peptide alters the conformational disposition of the acyl chains in contact with it and that the resultant conformational changes in the lipid hydrocarbon chains tend to minimize the extent of mismatch of peptide hydrophobic length and bilayer hydrophobic thickness.     

The interaction between cytoplasmic prion protein and the hydrophobic lipid core of membrane correlates with neurotoxicity

Prion protein (PrP), normally a cell surface protein, has been detected in the cytosol of a subset of neurons. The appearance of PrP in the cytosol could result from either retro-translocation of misfolded PrP from the endoplasmic reticulum (ER) or impaired import of PrP into the ER. Transgenic mice expressing cytoplasmic PrP (cyPrP) developed neurodegeneration in cerebellar granular neurons, although no detectable pathology was observed in other brain regions. In order to understand why granular neurons in the cerebellum were most susceptible to cyPrP-induced degeneration, we investigated the subcellular localization of cyPrP. Interestingly, we found that cyPrP is membrane-bound. In transfected cells, it binds to the ER and plasma/endocytic vesicular membranes. In transgenic mice, it is associated with synaptic and microsomal membranes. Furthermore, the cerebellar neurodegeneration in transgenic mice correlates with the interaction between cyPrP and the hydrophobic lipid core of the membrane but not with either the aggregation status or the dosage of cyPrP. These results suggest that lipid membrane perturbation could be a cellular mechanism for cyPrP-induced neurotoxicity and explain the seemingly conflicting results concerning cyPrP.     

Interaction of a peptide model of a hydrophobic transmembrane alpha-helical segment of a membrane protein with phosphatidylethanolamine bilayers: differential scanning calorimetric and Fourier transform infrared spectroscopic studies.

High-sensitivity differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to study the interaction of a synthetic alpha-helical hydrophobic transmembrane peptide, Acetyl-Lys2-Gly-Leu24-Lys2-Ala-Amide, and members of a homologous series of n-saturated diacylphosphatidylethanolamines (PEs). In the lower range of peptide mol fractions, the DSC endotherms exhibited by the lipid/peptide mixtures consist of two components. The temperature and cooperativity of the sharper, higher-temperature component are very similar to those of pure PE bilayers and are almost unaffected by variations in the peptide/lipid ratio. However, the fractional contribution of this component to the total enthalpy change decreases with increases in peptide concentration, and this component completely disappears at higher peptide mol fractions. The other component, which is less cooperative and occurs at a lower temperature, predominates at higher peptide concentrations. These two components of the DSC endotherm can be attributed to the chain-melting phase transitions of peptide-nonassociated and peptide-associated PE molecules, respectively. Although the temperature at which the peptide-associated PE molecules melt is progressively decreased by increases in peptide concentration, the magnitude of this shift is independent of the length of the PE hydrocarbon chain. In addition, the width of the phase transition observed at higher peptide concentrations is also relatively insensitive to PE hydrocarbon chain length, except that peptide gel-phase immiscibility occurs in very short- or very long-chain PE bilayers. Moreover, the enthalpy of the chain-melting transition of the peptide-associated PE does not decrease to 0 even at high peptide concentrations, suggesting that this peptide does not abolish the cooperative gel/liquid-crystalline phase transition of the lipids with which it is in contact. The FTIR spectroscopic data indicate that the peptide remains in a predominantly alpha-helical conformation, but that the peptide alpha-helix is subject to small distortions coincident with the changes in hydrophobic thickness that accompany the chain-melting phase transition of the PE bilayer. These data also indicate that the peptide significantly disorders the hydrocarbon chains of adjacent PE molecules in both the gel and liquid-crystalline states relatively independently of lipid hydrocarbon chain length. The relative independence of many aspects of PE-peptide interactions on the hydrophobic thickness of the host bilayer observed in the present study is in marked contrast to the results of our previous study of peptide-phosphatidylcholine (PC) model membranes (Zhang et al. (1992) Biochemistry 31:11579-11588), where strong hydrocarbon chain length-dependent effects were observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Kinetic and conformational properties of a novel T-cell antigen receptor transmembrane peptide in model membranes

Core peptide (CP; GLRILLLKV) is a 9-amino acid peptide derived from the transmembrane sequence of the T-cell antigen receptor (TCR) alpha-subunit. CP inhibits T-cell activation both in vitro and in vivo by disruption of the TCR at the membrane level. To elucidate CP interactions with lipids, surface plasmon resonance (SPR) and circular dichroism (CD) were used to examine CP binding and secondary structure in the presence of either the anionic dimyristoyl-L-alpha-phosphatidyl-DL-glycerol (DMPG), or the zwitterionic dimyristoyl-L-alpha-phoshatidyl choline (DMPC).Using lipid monolayers and bilayers, SPR experiments demonstrated that irreversible peptide-lipid binding required the hydrophobic interior provided by a membrane bilayer. The importance of electrostatic interactions between CP and phospholipids was highlighted on lipid monolayers as CP bound reversibly to anionic DMPG monolayers, with no detectable binding observed on neutral DMPC monolayers.CD revealed a dose-dependent conformational change of CP from a dominantly random coil structure to that of beta-structure as the concentration of lipid increased relative to CP. This occurred only in the presence of the anionic DMPG at a lipid : peptide molar ratio of 1.6:1 as no conformational change was observed when the zwitterionic DMPC was tested up to a lipid : peptide ratio of 8.4 : 1.     

Binding of a de novo designed peptide to specific glycosaminoglycans

The binding of glycosaminoglycans to a synthetic peptide (SKAQKAQAKQAKQAQKAQKAQAKQAKQW-CONH(2)), consisting of a hybrid consensus heparin binding sequence, is studied using circular dichroism, fluorescence anisotropy and nuclear magnetic resonance techniques. The results unveil certain novel features, most importantly, the peptide binds preferentially to iduronic acid containing glycosaminoglycans and the dissociation constant for the peptide-heparin complex was found to be 30 nM. Interestingly, higher order intermolecular association(s)/aggregation was not observed, especially at saturating concentrations of the ligand. The helical structure of the peptide backbone, induced upon binding to a particular glycosaminoglycan is directly related to their binding affinity. In our opinion, studies on such unconventional hybrid peptide sequences containing low density basic amino acid residues would lead to the design of sequence specific glycosaminoglycan binding peptides.     

Hydrophobic core malleability of a de novo designed three-helix bundle protein

De novo protein design provides a tool for testing the principles that stabilize the structures of proteins. Recently, we described the design and structure determination of alpha(3)D, a three-helix bundle protein with a well-packed hydrophobic core. Here, we test the malleability and adaptability of this protein's structure by mutating a small, Ala residue (A60) in its core to larger, hydrophobic side-chains, Leu and Ile. Such changes introduce strain into the structures of natural proteins, and therefore generally destabilize the native state. By contrast, these mutations were slightly stabilizing ( approximately 1.5 kcal mol(-1)) to the tertiary structure of alpha(3)D. The value of DeltaC(p) for unfolding of these mutants was not greatly affected relative to wild-type, indicating that the change in solvent accessibility for unfolding was similar. However, two-dimensional heteronuclear single quantum coherence spectra indicate that the protein adjusts to the introduction of steric bulk in different ways. A60L-alpha(3)D showed serious erosion in the dispersion of both the amide backbone as well as the side-chain methyl chemical shifts. By contrast, A60I-alpha(3)D showed excellent dispersion of the backbone resonances, and selective changes in dispersion of the aliphatic side-chains proximal to the site of mutation. Together, these data suggest that alpha(3)D, although folded into a unique three-dimensional structure, is nevertheless more malleable and flexible than most natural, native proteins.     

Effects of detergent alkyl chain length and chemical structure on the properties of a micelle-bound bacterial membrane targeting peptide

The effects of phospholipid or detergent chain length on the structure and translational diffusion coefficient of the membrane-targeting peptide corresponding to the N-terminal amphipathic sequence of Escherichia coli enzyme IIA(Glc) were investigated by nuclear magnetic resonance (NMR) spectroscopy. Three anionic phospholipids (dihexanoyl phosphatidylglycerol, dioctanoyl phosphatidylglycerol, and didecanoyl phosphatidylglycerol) and four lipid-mimicking anionic detergents (sodium hexanesulfonate, 2,2-dimethyl-silapentane-5-sulfonate, sodium nonanesulfonate, and sodium dodecylsulfate) were evaluated. In all cases, the cationic peptide adopts an amphipathic helical structure. While the chain length of the two-chain phospholipids has a negligible effect on the peptide conformation, the effect of chain length of those single-chain detergents on the helix length is more pronounced. The diffusion coefficients of the peptide/micelle complexes were found to correlate with the chain lengths of both the lipid and the detergent groups. Taken together, short-chain anionic phospholipids are proposed to be useful membrane-mimetic models for the structural elucidation of membrane-binding peptides such as cationic antimicrobial peptides. DSS does not form micelles by itself according to the diffusion coefficient data, but it does associate with this cationic peptide. Consequently, both DSS and its analog may be chosen as NMR chemical shift reference compounds depending on the nature of the biomolecules under investigation.     

Control of the transmembrane orientation and interhelical interactions within membranes by hydrophobic helix length

We examined the effect of the length of the hydrophobic core of Lys-flanked poly(Leu) peptides on their behavior when inserted into model membranes. Peptide structure and membrane location were assessed by the fluorescence emission lambdamax of a Trp residue in the center of the peptide sequence, the quenching of Trp fluorescence by nitroxide-labeled lipids (parallax analysis), and circular dichroism. Peptides in which the hydrophobic core varied in length from 11 to 23 residues were found to be largely alpha-helical when inserted into the bilayer. In dioleoylphosphatidylcholine (diC18:1PC) bilayers, a peptide with a 19-residue hydrophobic core exhibited highly blue-shifted fluorescence, an indication of Trp location in a nonpolar environment, and quenching localized the Trp to the bilayer center, an indication of transmembrane structure. A peptide with an 11-residue hydrophobic core exhibited emission that was red-shifted, suggesting a more polar Trp environment, and quenching showed the Trp was significantly displaced from the bilayer center, indicating that this peptide formed a nontransmembranous structure. A peptide with a 23-residue hydrophobic core gave somewhat red-shifted fluorescence, but quenching demonstrated the Trp was still close to the bilayer center, consistent with a transmembrane structure. Analogous behavior was observed when the behavior of individual peptides was examined in model membranes with various bilayer widths. Other experiments demonstrated that in diC18:1PC bilayers the dilution of the membrane concentration of the peptide with a 23-residue hydrophobic core resulted in a blue shift of fluorescence, suggesting the red-shifted fluorescence at higher peptide concentrations was due to helix oligomerization. The intermolecular self-quenching of rhodamine observed when the peptide was rhodamine-labeled, and the concentration dependence of self-quenching, supported this conclusion. These studies indicate that the mismatch between helix length and bilayer width can control membrane location, orientation, and helix-helix interactions, and thus may mismatch control both membrane protein folding and the interactions between membrane proteins.     

Yeast autophagosomes: de novo formation of a membrane structure

Autophagy - the degradation of organelles and cytoplasmic material - occurs through dynamic rearrangements of cellular membrane structures. Following the induction of autophagy, newly formed autophagosomes transfer cytosolic materials to the lysosome or vacuole for degradation. The autophagosome is an organelle destined for degradation, suggesting that the membrane structure is formed de novo many times. The autophagosome is formed through the nucleation, assembly and elongation of membrane structures. The concerted action of several Apg/Aut/Cvt proteins around a characteristic subcellular structure (the preautophagosomal structure) is the key to understanding this novel type of membrane-formation process.     

Correlation between hydrophobic properties and efficiency of carrier-mediated membrane transduction and apoptosis of a p53 C-terminal peptide

Two membrane transporters, the 17 amino acid (aa) oligopeptide penetratin derived from the homeodomain of Antennapedia (Ant) and an analogue of the basic domain of TAT (aa 47-57) (TAT-a) from HIV-1, were tested as carriers for a p53 C-terminal peptide (aa 361-382) into human breast cancer cells. The studies were performed to determine whether the membrane-transduction efficiency of membrane carriers: Ant, TAT or TAT analogue (TAT-a) correlated with peptide hydrophobic features. Peptide-sequence analysis clearly demonstrated that the Ant sequence and p53 peptide sequence (p53p) together created a peptide with enhanced hydrophobic characteristics; while the TAT or TAT analogue (TAT-a) and p53p sequence together created a peptide with significantly less hydrophobic qualities. The degree of hydrophobic moment and helical wheel plots for these peptides correlated directly with their ability to transduce the p53 peptide. Western blot analysis revealed that Ant was able to transduce p53 C-terminal peptide into human breast cancer cells as a highly efficient membrane transporter. Compared to Ant, TAT-a fused to the C-terminus of p53 peptide (p53p-TAT-a) was a less efficient carrier into these cells under the conditions of our study. Additionally, N-terminal linked TAT-a to p53p (TAT-a-p53p) showed even lower efficiency as a transporter than p53-TAT-a. Apoptosis assays showed that the p53 peptide, fused at its C-terminus to Ant (p53p-Ant), induced a higher percentage of apoptotic cells in human breast cancer cell lines expressing mutant or wild-type p53 as compared to p53 peptide fused at its C-terminus to the TAT-a sequence (p53p-TAT-a) or when fused at the N-terminus to TAT-a (TAT-a-p53p). These data suggested a direct correlation between hydrophobic characteristics and efficiency as a transporter. Sequence study, using hydrophobic moment and helical wheel analyses, may be useful predictive tools for choosing the best carrier for a peptide.     

Yeast mitochondrial inner membrane 30 kd hydrophobic protein: properties and interaction with phospholipids

A 30 kd hydrophobic protein is extracted from yeast mitochondrial inner membrane. It is present in wild yeast strains but absent in mitochondrial DNA lacking mutants. The isoelectric point of the protein and its solubility in various organic solvents are determined. The fluorescence of a tryptophan residue near the surface of the 30 kd protein dissolved in butanol-1, can be quenched by phospholipids containing unsaturated fatty acids. Results are in accordance with the 30 kd protein being an integral protein of the yeast mitochondrial inner membrane.     

StarD7 behaves as a fusogenic protein in model and cell membrane bilayers

StarD7 is a surface active protein, structurally related with the START lipid transport family. So, the present work was aimed at elucidating a potential mechanism of action for StarD7 that could be related to its interaction with a lipid-membrane interface. We applied an assay based on the fluorescence de-quenching of BD-HPC-labeled DMPC-DMPS 4:1 mol/mol SUVs (donor liposomes) induced by the dilution with non-labeled DMPC-DMPS 4:1 mol/mol LUVs (acceptor liposomes). Recombinant StarD7 accelerated the dilution of BD-HPC in a concentration-dependent manner. This result could have been explained by either a bilayer fusion or monomeric transport of the labeled lipid between donor and acceptor liposomes. Further experiments (fluorescence energy transfer between DPH-HPC/BD-HPC, liposome size distribution analysis by dynamic light scattering, and the multinuclear giant cell formation induced by recombinant StarD7) strongly indicated that bilayer fusion was the mechanism responsible for the StarD7-induced lipid dilution. The efficiency of lipid dilution was dependent on StarD7 electrostatic interactions with the lipid-water interface, as shown by the pH- and salt-induced modulation. Moreover, this process was favored by phosphatidylethanolamine which is known to stabilize non-lamellar phases considered as intermediary in the fusion process. Altogether these findings allow postulate StarD7 as a fusogenic protein.