Mechanism of selection of side-chain rotamers in α-helices

In this study, a possible mechanism of selection of side-chain rotamers based on the rotamer distributions in known coiled-coil proteins is suggested. According to this mechanism, interhelical hydrophobic, polar, and packing interactions bring alpha-helices closer to each other and this effect squeezes side chains out of the helix-helix interface. As a result, in dimeric coiled coils and long alpha-alpha-hairpins where alpha-helices are packed in a face-to-face manner, most side chains occupying the a-positions have t-rotamers and those in the d-positions g(-)-rotamers. In tetramers, where alpha-helices are packed side-by-side, most side chains in the a-positions adopt g(-)-rotamers and those in the d-positions t-rotamers.     

Packing scheme of α-helices in the histone core of the nucleosome

A nucleosome histone core model is presented which is compatible with experimental data. The model consists of 28 α-helices located as predicted by others^1–4. The histone wheel resembles the one proposed by Klug et al.⁵ Most of the helices are packed nearly parallel to the DNA superhelical axis, forming a bandoleer-like structure. About 10 to 20% of the nucleosomal phosphates may be neutralized by positively charged residues in the α-helices. Disregarding the charge of the NH₂-terminals, this is sifficient for the thermodynamic stability of the nucleosome under physiological conditions. The electrostatic charge on the protein surface is assumed to be relatively fixed due to the participation of the corresponding side chains to the hydrophobically packed helices. Thus, DNA wrapping appears to be determined by the core histones not by the histone NH₂-terminals.     

Signature of n→π* interactions in α-helices

The oxygen of a peptide bond has two lone pairs of electrons. One of these lone pairs is poised to interact with the electron-deficient carbon of the subsequent peptide bond in the chain. Any partial covalency that results from this n→π* interaction should induce pyramidalization of the carbon (C'(i)) toward the oxygen (O(i-1)). We searched for such pyramidalization in 14 peptides that contain both α- and β-amino acid residues and that assume a helical structure. We found that the α-amino acid residues, which adopt the main chain dihedral angles of an α-helix, display dramatic pyramidalization but the β-amino acid residues do not. Thus, we conclude that O(i-1) and C'(i) are linked by a partial covalent bond in α-helices. This finding has important ramifications for the folding and conformational stability of α-helices in isolation and in proteins.     

Energetics and structure of alanine-rich α-helices via adaptive steered molecular dynamics

The energetics and hydrogen bonding profiles of the helix-to-coil transition were found to be an additive property and to increase linearly with chain length, respectively, in alanine-rich α-helical peptides. A model system of polyalanine repeats was used to establish this hypothesis for the energetic trends and hydrogen bonding profiles. Numerical measurements of a synthesized polypeptide Ac-Y(AEAAKA)_kF-NH₂ and a natural α-helical peptide a2N (1–17) provide evidence of the hypothesis’s generality. Adaptive steered molecular dynamics was employed to investigate the mechanical unfolding of all of these alanine-rich polypeptides. We found that the helix-to-coil transition is primarily dependent on the breaking of the intramolecular backbone hydrogen bonds and independent of specific side-chain interactions and chain length. The mechanical unfolding of the α-helical peptides results in a turnover mechanism in which a 3₁₀-helical structure forms during the unfolding, remaining at a near constant population and thereby maintaining additivity in the free energy. The intermediate partially unfolded structures exhibited polyproline II helical structure as previously seen by others. In summary, we found that the average force required to pull alanine-rich α-helical peptides in between the endpoints—namely the native structure and free coil—is nearly independent of the length or the specific primary structure.     

Simulation studies on bacteriorhodopsin α-helices

Bacteriorhodopsin (BR) is a membrane protein which pumps protons through the plasma membrane. Transmembrane BR helical segments are subjected to simulation studies in order to investigate the effect of bilayer environment in various simulation conditions. A bilayer potential is introduced to the system to mimic the lipid membrane. The structures from the simulations are compared with the experimentally determined structures in terms of geometrical properties. Electrostatic contribution to the helix packing is also investigated. The simulation results show that the packing geometry of the transmembrane helices is highly affected by the bilayer potential. The results obtained from the simulations may be used for further simulation studies and analysis in investigating transmembrane helix packing. Received: 5 July 1999 / Revised version: 5 October 1999 / Accepted: 15 October 1999     

Role of the structural context in selection of hydrophobic side-chain rotamers in a- and d-positions of α-helices

It was demonstrated for the first time that the distribution of side-chain rotamers in the a-and d-positions of α-helices of coiled-coil (cc) proteins follows a certain trend, rather then being random. For instance, most side chains adopt t rotamers in the a-positions and g^? rotamers in the d-positions of helical dimers. Vice versa, most side chains adopt g^? rotamers in the a-positions and t rotamers in the d-positions of tetramers. It was concluded that selection of the side-chain rotamers depends on the packing of α-helices and, consequently, depends on the structural context.     

Tetramerization of human guanylate-binding protein 1 is mediated by coiled-coil formation of the C-terminal α-helices

The human guanylate-binding protein 1 (hGBP1) is a large GTP-binding protein belonging to the dynamin family, a common feature of which is nucleotide-dependent assembly to homotypic oligomers. Assembly leads to stimulation of GTPase activity, which, in the case of dynamin, is responsible for scission of vesicles from membranes. By yeast two-hybrid and biochemical experiments we addressed intermolecular interactions between all subdomains of hGBP1 and identified the C-terminal subdomain, α12/13, as a new interaction site for self-assembly. α12/13 represents a stable subdomain of hGBP1, as shown by CD spectroscopy. In addition to contacts between GTPase domains leading to dimer formation, the interaction between two α12/13 subdomains, in the course of GTP hydrolysis, results in tetramer formation of the protein. With the help of CD spectroscopy we showed coiled-coil formation of two α12/13 subdomains and concentration-dependent measurements allow estimating a value for the dissociation constant of 7.3 μM. We suggest GTP hydrolysis-driven release of the α12/13 subdomain, making it available for coiled-coil formation. Furthermore, we can demonstrate the biological relevance of hGBP1 tetramer formation in living cells by chemical cross-link experiments.     

TIM23-mediated insertion of transmembrane α-helices into the mitochondrial inner membrane

While overall hydrophobicity is generally recognized as the main characteristic of transmembrane (TM) α-helices, the only membrane system for which there are detailed quantitative data on how different amino acids contribute to the overall efficiency of membrane insertion is the endoplasmic reticulum (ER) of eukaryotic cells. Here, we provide comparable data for TIM23-mediated membrane protein insertion into the inner mitochondrial membrane of yeast cells. We find that hydrophobicity and the location of polar and aromatic residues are strong determinants of membrane insertion. These results parallel what has been found previously for the ER. However, we see striking differences between the effects elicited by charged residues flanking the TM segments when comparing the mitochondrial inner membrane and the ER, pointing to an unanticipated difference between the two insertion systems.     

Solid-phase synthesis of short α-helices stabilized by the hydrogen bond surrogate approach

Stabilized α-helices and nonpeptidic helix mimetics have emerged as powerful molecular scaffolds for the discovery of protein-protein interaction inhibitors. Protein-protein interactions often involve large contact areas, which are often difficult for small molecules to target with high specificity. The hypothesis behind the design of stabilized helices and helix mimetics is that these medium-sized molecules may pursue their targets with higher specificity because of a larger number of contacts. This protocol describes an optimized synthetic strategy for the preparation of stabilized α-helices that feature a carbon-carbon linkage in place of the characteristic N-terminal main-chain hydrogen bond of canonical helices. Formation of the carbon-carbon bond is enabled by a microwave-assisted ring-closing metathesis reaction between two terminal olefins on the peptide chain. The outlined strategy allows the synthesis and purification of a hydrogen bond surrogate (HBS) α-helix in ～ 1 week.     

Analysis and prediction of the packing of α-helices against a β-sheet in the tertiary structure of globular proteins

The packing of α-helices and β-sheets in six $\text{α}\text{β}$ proteins (e.g. flavodoxin) has been analysed. The results provide the basis for a computer algorithm to predict the tertiary structure of an $\text{α}\text{β}$ protein from its amino acid sequence and actual assignment of secondary structure.The packing of an individual α-helix against a β-sheet generally involves two adjacent ± 4 rows of non-polar residues on the α-helix at the positions i, i + 4, i + 8, i + 1, i + 5, i + 9. The pattern of interacting β-sheet residues results from the twisted nature of the sheet surface and the attendant rotation of the side-chains. At a more detailed level, four of the α-helical residues (i + 1, i + 4, i + 5 and i + 8) form a diamond that surrounds one particular β-sheet residue, generally isoleucine, leucine or valine. In general, the α-helix sits 10 Å above the sheet and lies parallel to the strand direction.The prediction follows a combinational approach. First, a list of possible β-sheet structures (10⁶ to 10¹⁴) is constructed by the generation of all β-sheet topologies and β-strand alignments. This list is reduced by constraints on topology and the location of non-polar residues to mediate the sheet/helix packing, and then rank-ordered on the extent of hydrogen bonding. This algorithm was uniformly applied to 16 $\text{α}\text{β}$ domains in 13 proteins. For every structure, one member of the reduced list was close to the crystal structure; the root-mean-square deviation between equivalenced C^α atoms averaged 5.6 Å for 100 residues. For the $\text{α}\text{β}$ proteins with pure parallel β-sheets, the total number of structures comparable to or better than the native in terms of hydrogen bonds was between 1 and 148. For proteins with mixed β-sheets, the worst case is glyceraldehyde-3-phosphate dehydrogenase, where as many as 3800 structures would have to be sampled. The evolutionary significance of these results as well as the potential use of a combinatorial approach to the protein folding problem are discussed.     

The peptide chain of tyrosyl tRNA synthetase: No evidence for a super-secondary structure of four α-helices

A detailed backbone model has been built for 274 residues of tyrosyl tRNA synthetase, based on an X-ray diffraction study. This includes eight helical sections and a six-stranded pleated sheet. The four helices near the carboxyl terminal end are not arranged like the helices of TMV disk protein and hemerythrin, and the structure gives no support to the idea that four antiparallel helices form a common structural unit in proteins.     

Submolecular-scale imaging of α-helices and C-terminal domains of tubulins by frequency modulation atomic force microscopy in liquid

In this study, we directly imaged subnanometer-scale structures of tubulins by performing frequency modulation atomic force microscopy (FM-AFM) in liquid. Individual α-helices at the surface of a tubulin protofilament were imaged as periodic corrugations with a spacing of 0.53 nm, which corresponds to the common pitch of an α-helix backbone (0.54 nm). The identification of individual α-helices allowed us to determine the orientation of the deposited tubulin protofilament. As a result, C-terminal domains of tubulins were identified as protrusions with a height of 0.4 nm from the surface of the tubulin. The imaging mechanism for the observed subnanometer-scale contrasts is discussed in relation to the possible structures of the C-terminal domains. Because the C-terminal domains are chemically modified to regulate the interactions between tubulins and other biomolecules (e.g., motor proteins and microtubule-associated proteins), detailed structural information on individual C-terminal domains is valuable for understanding such regulation mechanisms. The results obtained in this study demonstrate that FM-AFM is capable of visualizing the structural variation of tubulins with subnanometer resolution. This is an important first step toward using FM-AFM to analyze the functions of tubulins.     

Statistical mechanical treatment of α-helices and extended structures in proteins with inclusion of short- and medium-range interactions

A statistical mechanical model of protein conformation with medium-range interactions between theith and (i+k)^th residues (k<-4) is presented. Two two-state models, an α-helix-coil and an extended-structure-coil model, are formulated using the same form of the partition function, but the two models are applied independently to predict the locations of α-helical, extended, and coil segments; in the relatively few cases (<2%) where the predictions from the two models are in conflict, the prediction is scored as an incorrect one. Two independent sets of statistical weights (one set for each model) are derived to describe the interactions between the 20 amino acid residues for each range of interactionk; they are evaluated by minimizing an objective function so that the probability profiles for the α-helix or extended structure, respectively, in proteins computed from these statistical weights correlate optimally with the experimentally observed native conformations of these proteins. Examination of the resulting statistical weights shows that those for the interactions between hydrophobic residues and between a hydrophobic and a hydrophilic residue have reasonable magnitudes compared to what would be expected from the spatial arrangements of the side chains in the α-helix and the extended structure, and that those for the α-helix-coil model correlate well with experimentally determined values of the Zimm-Bragg parameterss and σ of the helix-coil transition theory. From the point of view of a method to predict the conformational states (i.e., α-helix, extended structure, and coil) of each residue, the statistical weights (as inall empirical prediction schemes) depend very much on the proteins used for the data base, since the presently available set of proteins of known structure is still too small for very high predictability; as a result, the correctness of the prediction is not very good for proteins not included in the data base. However, the correctness of the prediction, at least for the 37 proteins utilized as the data base in this study, is 91% and 87% for the α-helix-coil and the extended-structure-coil models, respectively; further, 79% of all the residues are predicted correctly when both the α-helix-coil and extended-structure-coil models are applied independently.     

Temperature-dependent structural changes in intrinsically disordered proteins: Formation of α-helices or loss of polyproline II?

Structural characterization of intrinsically disordered proteins (IDPs) is mandatory for deciphering their potential unique physical and biological properties. A large number of circular dichroism (CD) studies have demonstrated that a structural change takes place in IDPs with increasing temperature, which most likely reflects formation of transient α-helices or loss of polyproline II (PPII) content. Using three IDPs, ACTR, NHE1, and Spd1, we show that the temperature-induced structural change is common among IDPs and is accompanied by a contraction of the conformational ensemble. This phenomenon was explored at residue resolution by multidimensional NMR spectroscopy. Intrinsic chemical shift referencing allowed us to identify regions of transiently formed helices and their temperature-dependent changes in helicity. All helical regions were found to lose rather than gain helical structures with increasing temperature, and accordingly these were not responsible for the change in the CD spectra. In contrast, the nonhelical regions exhibited a general temperature-dependent structural change that was independent of long-range interactions. The temperature-dependent CD spectroscopic signature of IDPs that has been amply documented can be rationalized to represent redistribution of the statistical coil involving a general loss of PPII conformations.     

Thoeretical study of the packing of α-helices of poly(l-alanine) into transmembrane bundles. Possible significance for ion-transfer

Energy optimizations are carried out on packages of Nα-helices of poly(l-alanine) from N = 3−7, starting from an initial arrangement of the helices at the vertices of various polygonal prisms, in view of the possible formation of channel-making bundles in membranes. The results show: that, for each N, a number of stable packages exist; that the presence of one pair (and even two) of adjacent parallel helices in a package is not incompatible with its stability, due to the overcompensation of its unfavorable electrostatic energy by the sum of the corresponding favorable terms for the antiparallel pairs; and that some packages provide ready-made pores in their interior. The energy profile computed for Na⁺ inside one of the pores (resulting from five helices) shows a favorable energy all the way through, in spite of the methyl groups protruding into the channel. Similarly one water molecule interacts favorably with this pore throughout.     

The structure of the ends of α-helices in globular proteins: effect of additional hydrogen bonds and implications for helix formation

We prepared a set of about 2000 α-helices from a relational database of high-resolution three-dimensional structures of globular proteins, and identified additional main chain i ← i+3 hydrogen bonds at the ends of the helices (i.e., where the hydrogen bonding potential is not fulfilled by canonical i ← i+4 hydrogen bonds). About one-third of α-helices have such additional hydrogen bonds at the N-terminus, and more than half do so at the C-terminus. Although many of these additional hydrogen bonds at the C-terminus are associated with Schellman loops, the majority are not. We compared the dihedral angles at the termini of α-helices having or lacking the additional hydrogen bonds. Significant differences were found, especially at the C-terminus, where the dihedral angles at positions C2 and C1 in the absence of additional hydrogen bonds deviate substantially from those occurring within the α-helix. Using a novel approach we show how the structure of the C-terminus of the α-helix can emerge from that of constituent overlapping α-turns and β-turns, which individually show a variation in dihedral angles at different positions. We have also considered the direction of propagation of the α-helix using this approach. If one assumes that helices start as a single α-turn and grow by successive addition of further α-turns, the paths for growth in the N → C and C → N directions differ in a way that suggests that extension in the C → N direction is favored.     

Theoretical study of the packing of α-helices into possible transmembrane bundles. Sequences including alanines,leucines and serines

Energy optimizations are carried out on packages of two and five α-helices containing leucines on their faces of contact and made otherwise of alanines. The effect of these bulky side-chains on the optimal arrangements is analysed and compared to the results previously obtained for pure poly(l-alanine) packages; the essential pairing properties are conserved (near antiparallelism, preponderous role of the non-bonded interactions, possibility of existence of parallel pairs); five α-helices made of 8 alanines and 6 leucines (three on each interface) can pack in different stable P5L bundles including various holes, according to the tilt and relative sliding of the helices. Substitution of serines to the alanines lying on the inner wall affects very little the interhelix packing. The seryl side chains adapt their conformation at best to their surroundings. The P5L packages can be used to represent individual subunits arranged in ‘superbundles’ around a central pit in a channel-forming protein.     

Molecular dynamics exposes α-helices in myelin basic protein

Molecular dynamics simulations of models of unmodified and deiminated MBP (myelin basic protein) have been performed on solvated structures with added counterions, for 10 ns using AMBER (assisted model building with energy refinement). The protein structures became extended, and a considerable number of -helical segments formed spontaneously. The degree of molecular extension was greater in the deiminated species, and the -helices were more transient. These structural disruptions may be operative in vivo during multiple sclerosis.Figure A model of the C1 isoform of myelin basic protein showing major -helical segments that were stable over the last 1 ns of a molecular dynamics simulation. The -helices are colored red, the -strands are colored yellow, the -turns are colored blue, and random coils are colored green