Hydrophobic organization of α-helix membrane bundle in bacteriorhodopsin

The hydrophobic organization of the intramembraneα-helical bundle in bacteriorhodopsin (BRh) was assessed based on a new approach to characterization of spatial hydrophobic properties of transmembrane (TM)α-helical peptides. The method employs two independent techniques: Monte Carlo simulations of nonpolar solvent around TM peptides and analysis of molecular hydrophobicity potential on their surfaces. The results obtained by the two methods agree with each other and permit precise hydrophobicity mapping of TM peptides. Superimposition of such data on the experimentally derived spatial model of the membrane moiety together with 2D maps of hydrophobic hydrophilic contacts provide considerable insight into the hydrophobic organization of BRh. The helix bundle is stabilized to a large extent by hydrophobic interactions between helices—neighbors in the sequence of BRh, by long-range interactions in helix pairs C-E, C-F, and C-G, and by nonpolar contracts between retinal and helices C, D, E, F. Unlike globular proteins, no polar contacts between residues distantly separated in the sequence of BRh were found in the bundle. One of the most striking results of this study is the finding that the hydrophobic organization of BRh is significantly different from those in bacterial photoreaction centers. Thus, TMα-helices in BRh expose their most nonpolar sides to the bilayer as well as to the neighboring helices and to the interior of the bundle. Some of them contact lipids with their relatively hydrophilic surfaces. No correlation was found between disposition of the most hydrophobic and the most variable sides of the TM helices.     

Evaluation of hydrophobicity versus chaperonelike activity of bovine alphaA- and alphaB-crystallin

Calf lens A-crystallin isolated by reversed-phase HPLC demonstrates a slightly more hydrophobic profile than B-crystallin. Fluorescent probes in addition to bis-ANS, like cis-parinaric acid (PA) and pyrene, show higher quantum yields or Ham ratios when bound to A-crystallin than to B-crystallin at room temperature. Bis-ANS binding to both A- and B-crystallin decreases with increase in temperature. At room temperature, the chaperone-like activity of A-crystallin is lower than that of B-crystallin whereas at higher temperatures, A-crystallin shows significantly higher protection against aggregation of substrate proteins compared to B-crystallin. Therefore, calf lens A-crystallin is more hydrophobic than B-crystallin and chaperone-like activity of -crystallin subunits is not quantitatively related to their hydrophobicity.     

Temperature coefficients of amide proton NMR resonance frequencies in trifluoroethanol: A monitor of intramolecular hydrogen bonds in helical peptides?

Summary 2D ¹H NMR spectroscopy of two -helical peptides which differ in their amphipathicity has been used to investigate the relationships between amide-proton chemical shifts, amide-proton exchange rates, temperature, and trifluoroethanol (TFE) concentration. In 50% TFE, in which the peptides are maximally helical, the amide-proton chemical shift and temperature coefficient patterns are very similar to each other in each peptide. Temperature coefficients from –10 to –6 ppb/K, usually indicative of the lack of intramolecular hydrogen bonds, were observed even for hydrophobic amino acids in the center of the -helices. However, slow hydrogen isotope exchange for residues from 4 to 16 in both 18-mer helices indicates intact intramolecular hydrogen bonds over most of the length of these peptides. Based on these anomalous observations, we suggest that the pattern of amide-proton shifts in -helices in H₂O/TFE solvents is dominated by bifurcated intermolecular hydrogen-bond formation between the backbone carbonyl groups and TFE. The amide-proton chemical shift changes with increasing temperature may be interpreted by a disruption of intermolecular hydrogen bonds between carbonyl groups and the TFE in TFE/water rather than by the length of intramolecular hydrogen bonds in -helices. Supplementary Material is available upon request, comprising seven pages with listings of experimental details and the NMR shift data for the two peptides.     

Synthesis of antiparallel 4α-helix bundle TASP by chemoselective ligation

Summary The use of chemoselective ligation methods and orthogonal protection techniques allows access to Template-Assembled Synthetic Protein (TASP) molecules exhibiting a large variety of packing topologies. This is demonstrated for the synthesis of an antiparallel 4-helical bundle TASP by condensing amphiphilic peptide blocks, containing aldehyde functions at the C- or N-terminus, to a selectively addressable topological template via oxime bond formation. The resulting antiparallel 4-helix TASP is obtained in high yield and shows a template-induced helical conformation.     

A Nonstatistical Approach to the Prediction of Regular Structures in Proteins by the Example of α Helices

A new approach to the analysis of regular structures in proteins that is based on the method of molecular mechanics is proposed. The method uses only the information about the amino acid sequence. The -helical conformation was simulated using the ICM program of molecular mechanics. Energy profiles of the sequences in the -helical conformation, spanning the entire polypeptide chain, were plotted for eight proteins from the Protein Data Bank. The regions of each profile that exhibit energy minima were found to correspond to the -helical regions of the real spatial structure of the protein. Twenty-four out of 25 helices were distinctly pronounced, which indicates a rather high accuracy of the prediction. The energy profiles also help reveal the short regions that correspond to 3/10-helices and the turns that include local -helical conformations. Unlike the known statistical methods of prediction, this method makes it possible to establish the physical principles of the formation of -helical conformations.     

A high-resolution 1H NMR approach for structure determination of membrane peptides and proteins in non-deuterated detergent: Application to mastoparan X solubilized in n-octylglucoside

Summary Application of ¹H 2D NMR methods to solubilized membrane proteins and peptides has up to now required the use of selectively deuterated detergents. The unavailability of any of the common biochemical detergents in deuterated form has therefore limited to some extent the scope of this approach. Here a ¹H NMR method is described which allows structure determination of membrane peptides and small membrane proteins by ¹H 2D NMR in any type of non-deuterated detergent. The approach is based on regioselective excitation of protein resonances with DANTE-Z or spin-pinging pulse trains. It is shown that regioselective excitation of the amide-aromatic region of solubilized membrane proteins and peptides leads to an almost complete suppression of the two orders of magnitude higher contribution of the protonated detergent to the ¹H NMR spectrum. Consistently TOCSY, COSY and NOESY sequences incorporating such regioselective excitation in the F2 dimension yield protein ¹H 2D NMR spectra of quality comparable to those obtained in deuterated detergents. Regioselective TOCSY and NOESY spectra display all through-bond and through-space correlations within amide-aromatic protons and between these protons and aliphatic and -protons. Regioselective COSY spectra provide scalar coupling constants between amide and -protons. Application of the method to the membrane-active peptide mastoparan X, solubilized in n-octylglucoside, yields complete sequence-specific assignments and extensive secondary structure-related spatial proximities and coupling constants. It is shown that mastoparan adopts an -helical conformation when bound to nonionic detergent micelles. The present method is expected to increase the applicability of ¹H solution NMR methods to membrane proteins and peptides.Abbreviations 2D NMR two-dimensional NMR - COSY correlated spectroscopy - DANTE delays alternating nutations for tailored excitation - NOESY nuclear Overhauser enhancement spectroscopy - TOCSY total correlation spectroscopy     

The α-helix dipole in membranes: a new gating mechanism for ion channels

Electric dipoles placed side by side attract each other if antiparallel and repel each other if parallel. The hydrophobic -helical sections of proteins that span membranes are known to possess large electric dipole moments. The first part of the paper consists of a calculation of the interaction energies between such helices including screening effects. Interaction energies remain comparable with a typical thermal energy of KT up to separations of order 20 Å. In addition it is shown that, due solely to its dipole moment, an -helix which completely spans the membrane has an energy up to 5 KT lower than one which terminates within the membrane width. The second part of the paper describes the electrical interaction of the charge structure of a membrane channel and the protein helices that surround the pore. The gating charge transfer that is measured when a voltage sensitive ion channel switches, means that the dipole moment of the ion channel changes. This in turn results in a change in the radial forces that act between the pore and the -helices that surround it. A change in these radial forces which tend to open or to close the pore constitutes an electrically silent gating mechanism that must necessarily act subsequent to the gating charge transfer. The gating mechanism could consist of the radial translation of the neighbouring proteins or in their axial rotation under the influence of the torque that would act on a pair of approximately equidistant but oppositely directed -helices. An attempt to calculate the interaction energy of a typical pore and a single -helix spanning the membrane results in an energy of many times KT.     

Local helix content in an alanine-rich peptide as determined by the complete set of 3JHNα coupling constants

Summary Alanine-rich peptides serve as models for exploring the factors that control helix structure in peptides and proteins. Scalar CH-NH couplings (³J_HN) are an extremely useful measure of local helix content; however, the large alanine content in these peptides leads to significant signal overlap in the CH region of ¹H 2D NMR spectra. Quantitative determination of all possible ³J_HN values is, therefore, very challenging. Szyperski and co-workers [(1992) J. Magn. Reson., 99, 552–560] have recently developed a method for determining ³J_HN from NOESY spectra. Because ³J_HN may be determined from 2D peaks outside of the CH region, there is a much greater likelihood of identifying resolved resonances and measuring the associated coupling constants. It is demonstrated here that ³J_HN can be obtained for every residue in the helical peptide Ac-(AAAAK)₃A-NH₂. The resulting ³J_HN profile clearly identifies a helical structure in the middle of the peptide and further suggests that the respective helix termini unfold via distinct pathways.Abbreviations ³J_HN three-bond CH-NH scalar coupling constant - NOE nuclear Overhauser enhancement - NOESY two-dimensional nuclear Overhauser spectroscopy - COSY two-dimensional correlated spectroscopy - DQF-COSY two-dimensional double-quantum-filtered correlated spectroscopy - TOCSY two-dimensional total correlation spectroscopy To whom correspondence should be addressed.Deceased March 5, 1996.     

Entropy Change for Free Polypeptide Chain upon Hydrogen Bonding

Formation probabilities of different hydrogen bonds between carbonyl oxygen and amide hydrogen were determined by Monte Carlo simulations using a computer model in the space of sterically allowable conformations of alanine and glycine oligopeptides, and the corresponding entropy losses for the peptide backbone, TS, were calculated. The model was studied at different criteria of steric interactions. Comparison with the data of other authors showed the values of TSto be mainly determined by overall extent and type of the state space and to be only slightly dependent on its energy profile. Both short-range and long-range steric interactions were shown to prevent hydrogen bonding, especially in alanine peptides. In the model studied, the initiation of (R)-helices is associated with TS= 8–10 kT, and prior formation of a 3/10-turn or one three-center H-bond does not appreciably decrease this entropy barrier. Elongation of the (R)-helix by one residue leads to TS= 3.0–3.7 kT, the helices begin to stabilize after at least three sequential H-bonds are formed. The difference in the probability of insertion of Ala and Gly into the helix is lower than it follows from comparison of their mobility. The results could be explained assuming that factors different from helical H-bonds take part in the stabilization of the helices. One may suppose upon modeling of folding that even three sequential H-bonds are unable to fix the structure of a flexible peptide loop, while the elongation of (R)-helices in the supersecondary helix-loop-helix structure is favorable as long as the loop conformation remains nearly optimal.