The electrostatic potential of the alpha helix (electrostatic potential/α-helix/secondary structure/helix dipole)

The active sites of many enzymes are very close to the N-terminus of an α-helix. The helix dipole has been postulated to enhance the binding of anions and speed charge relays in catalysis. We present electrostatic potential maps of α-helices of various lengths using a point charge model. We show that the potential field of the helix can be mimicked by two equal and opposite charges, one at each terminus. The magnitude of these equivalent charges reaches its limiting value of ± 0.2 to 0.3 electron at a helix length of approximately 7–10 residues. We also comment on the relative importance of the helix dipole to that of ionized residues in determining the electrostatics of a protein and discuss what consequences this has for enzymology.     

Role of terminal dipole charges in aggregation of α-helix pair in the voltage gated K+ channel

The voltage sensor domain (VSD) of the potassium ion channel KvAP is comprised of four (S1–S4) α-helix proteins, which are encompassed by several charged residues. Apart from these charges, each peptide α-helix having two inherent equal and opposite terminal dipolar charges behave like a macrodipole. The activity of voltage gated ion channel is electrostatic, where all the charges (charged residues and dipolar terminal charges) interact with each other and with the transmembrane potential. There are evidences that the role of the charged residues dominate the stabilization of the conformation and the gating process of the ion channel, but the role of the terminal dipolar charges are never considered in such analysis. Here, using electrostatic theory, we have studied the role of the dipolar terminal charges in aggregation of the S3b–S4 helix pair of KvAP in the absence of any external field (V = 0). A system attains stability, when its potential energy reaches minimum values. We have shown that the presence of terminal dipole charges (1) change the total potential energy of the charges on S3b–S4, affecting the stabilization of the α-helix pair within the bilayer lipid membrane and (2) the C- and the N-termini of the α-helices favor a different dielectric medium for enhanced stability. Thus, the dipolar terminal charges play a significant role in the aggregation of the two neighboring α-helices.     

Electrostatic interactions between α-helix dipoles in crystals of an uncharged helical undecapeptide

The electrostatic interactions between α-helix dipoles in the crystals of an uncharged helical undecapeptide have been studied in detail. The electrostatic interaction energy between one helix dipole and its 26 nearest neighbors is approximately ?23 kcal mol^?1. A very similar result is obtained when calculating the interactions between one helix dipole and all 988 helix dipoles occurring within a distance of 75 Å. It therefore appears that in these crystals of completely uncharged molecules large, favorable electrostatic interactions occur.     

Correlation between computed conformational properties of cytochrome c peptides and their antigenicity in a T-lymphocyte proliferation assay

By means of conformational energy calculations, we previously showed that the antigenic strength of a series of oligopeptides (derived from the carboxyl terminal sequence of cytochrome c) in a T-lymphocyte proliferation assay depends on their ability to adopt the α-helix conformation. Using experimentally determined statistical weights (within the framework of the Zimm–Bragg theory for the helix–coil transition), here we present a simple free energy analysis of the ability of these peptides to adopt the α-helix conformation in water. The experimental statistical weights have been modified to include the effect of long-range charge–dipole interactions on helix stability. We find that there is a close correlation between the tendency of a peptide to adopt the α-helix conformation and its ability to stimulate antigen-primed T cells. The shortest peptide with a tendency to adopt the α-helix conformation is also the shortest one that exhibits antigenic activity. The rapid and simple method presented here can thus be used to predict relative antigenicities for different peptides derived from cytochrome c.     

Infrared spectra and resonance interactions of amide-I and II vibration of alpha-helix.

Amide-I and II vibrations of α-helix have been treated on the basis of the perturbation theory in a dipole–dipole approximation. The infinite helix and its finite fragments have been considered as models. The calculated infrared spectra for the infinite helix are in good agreement with the spectra of synthetic polypeptides. In the case of finite fragments of the α-helix, the characteristic contours of the amide-I and II bands appear when the nuimber of peptide groups is about 10 and 6, respectively.     

Stabilizing effects of 2-methylalanine residues on β-turns and α-helices

¹³C-, ¹H-nmr, CD, and x-ray crystallography revealed β-turns of type III for Boc-Gly-L-Ala-Aib-OMe, Boc-L-Ala-Aib-L-Ala-OMe; the 3₁₀-helix for Boc-Aib-L-Ala-Aib-L-Ala-Aib-OMe; and antiparallel arranged α-helices for Boc-L-Ala-Aib-Ala-Aib-Ala-Glu(OBzl)-Ala-Aib-Ala-Aib-Ala-OMe. An N-terminal rigid α-helical segment is found in the polypeptide antibiotics alamethicin, suzukacillin, and trichotoxin. The α-helix dipole is essential for their voltage-dependent pore formation in lipid bilayer membranes, which is explained by a flip-flop gating mechanism based on dipole–dipole interactions of parallel and antiparallel arranged α-helices within oligomeric structures.     

A competing salt-bridge suppresses helix formation by the isolated C-peptide carboxylate of ribonuclease A

The C-peptide of ribonuclease A (residues 1 to 13) is obtained by cyanogen bromide cleavage at Met13, which converts methionine to a mixture of homoserine lactone (giving C-peptide lactone) and homoserine carboxylate (giving C-peptide carboxylate). The helix-forming properties of C-peptide lactone have been reported. The helix is formed intramolecularly in aqueous solution, is stabilized at low temperatures (0 to 20 °C) and also by a pH-dependent interaction between sidechains. The C-peptide lactone helix is about 1000-fold more stable than expected from “host-guest” data for helix formation in synthetic polypeptides.Here we report the failure of C-peptide carboxylate to form an α-helix in comparable conditions. Formation of a salt-bridge between the α-COO^? group and the imidazolium ring of His12⁺ appears to be responsible for the suppression of helix formation. The presence of the Hse13-COO^? … His12⁺ salt-bridge in C-peptide carboxylate is shown by ¹H nuclear magnetic resonance titration of the amide proton resonances of His12 and Hse13, and is expected from model peptide studies. The most probable reason why C-peptide carboxylate does not form an α-helix is that the Hse13-COO^? … His12⁺ salt-bridge competes successfully with a helix stabilizing salt-bridge (Glu9^? … His12⁺).S-peptide (residues 1 to 20 of ribonuclease A) does form an α-helix with properties similar to those of the C-peptide (lactone) helix, which shows that the lactone ring of C-peptide lactone is not needed for helix formation.These results support the hypothesis that a Glu9^? … His12⁺ salt-bridge stabilizes the C-peptide (lactone) helix, and they show that specific interactions between side-chains can be important in preventing as well as in promoting α-helix formation.     

Residue-specific α-helix propensities from molecular simulation

Formation of α-helices is a fundamental process in protein folding and assembly. By studying helix formation in molecular simulations of a series of alanine-based peptides, we obtain the temperature-dependent α-helix propensities of all 20 naturally occurring residues with two recent additive force fields, Amber ff03w and Amber ff99SB¹. Encouragingly, we find that the overall helix propensity of many residues is captured well by both energy functions, with Amber ff99SB¹ being more accurate. Nonetheless, there are some residues that deviate considerably from experiment, which can be attributed to two aspects of the energy function: i), variations of the charge model used to determine the atomic partial charges, with residues whose backbone charges differ most from alanine tending to have the largest error; ii), side-chain torsion potentials, as illustrated by the effect of modifications to the torsion angles of I, L, D, N. We find that constrained refitting of residue charges for charged residues in Amber ff99SB¹ significantly improves their helix propensity. The resulting parameters should more faithfully reproduce helix propensities in simulations of protein folding and disordered proteins.     

ω-Helices in Proteins

A modification of the α-helix, termed the ω-helix, has four residues in one turn of a helix. We searched the ω-helix in proteins by the HELFIT program which determines the helical parameters—pitch, residues per turn, radius, and handedness—and p = rmsd/(N ? 1)^1/2 estimating helical regularity, where “rmsd” is the root mean square deviation from the best fit helix and “N” is helix length. A total of 1,496 regular α-helices 6–9 residues long with p ≤ 0.10 Å were identified from 866 protein chains. The statistical analysis provides a strong evidence that the frequency distribution of helices versus n indicates the bimodality of typical α-helix and ω-helix. Sixty-two right handed ω-helices identified (7.2% of proteins) show non-planarity of the peptide groups. There is amino acid preference of Asp and Cys. These observations and analyses insist that the ω-helices occur really in proteins.     

The Role of Arrestin α-Helix I in Receptor Binding

Arrestins rapidly bind phosphorylated activated forms of their cognate G protein-coupled receptors, thereby preventing G protein coupling and often switching signaling to other pathways. Amphipathic α-helix I (residues 100-111) has been implicated in receptor binding, but the mechanism of its action has not been determined yet. Here we show that several mutations in the helix itself and in adjacent hydrophobic residues in the body of the N-domain reduce arrestin1 binding to light-activated phosphorylated rhodopsin (P-Rh^?). On the background of phosphorylation-independent mutants that bind with high affinity to both P-Rh^? and light-activated unphosphorylated rhodopsin, these mutations reduce the stability of the arrestin complex with P-Rh^?, but not with light-activated unphosphorylated rhodopsin. Using site-directed spin labeling, we found that the local structure around α-helix I changes upon binding to rhodopsin. However, the intramolecular distances between α-helix I and adjacent β-strand I (or the rest of the N-domain), measured using double electron-electron resonance, do not change, ruling out relocation of the helix due to receptor binding. Collectively, these data demonstrate that α-helix I plays an indirect role in receptor binding, likely keeping β-strand I, which carries several phosphate-binding residues, in a position favorable for its interaction with receptor-attached phosphates.     

The contribution of helical potential to the in vitro receptor binding activity of a neuropeptide Y N-terminal deletion fragment

In its dimeric form neuropeptide Y (NPY) folds into a compact structure in which the antiparallel oriented proline and α-helices apparently associate to form a primitive hydrophobic core. To investigate the contribution of helical stability to the receptor binding activity of NPY and its N-terminal deletion fragments, we synthesized and studied the solution conformational properties and in vitro activities of NPY, N^α-acetyl-NPY_2–36, NPY_15–36, N^α-propinonly-NPY_15–36, and N^α-succinyl-NPY_15–36 is significantly less helical than both NPY and N^α-acetyl-NPY_2–36, and this decreased helical potential is attributed of the absence of the intramolecular stabilizing interaction afforded by the proline helix in the latter analogues. However, in accord with the helix dipole model, the helical potential of NPY_15–36 is significantly increased by N-terminal succinlyation, whereas propionylation has no effect. In addition to an increase in helical potential, N^α-succinyl-NPY_15–36 is 2.5 and 4.6 times more active than NPY_15–36 and N^α-propionly-NPY_15–36, respectively and is equipotent with N^α-acteyl-NPY_2–36 in displacing 1mM[³H]-NPY from specific binding sites in rat brain membranes. The demonstration of positive correlation between % α-helix content and in vitro binding activity suggests that the helical potential of N-terminal NPY deletion fragments contributes to their in vitro activity in the rat brain, and that a second role of the proline helix might be to stabilize the receptor-active conformation of the NPY α-helix. © 1993 John Wiley & Sons, Inc.     

Dependence on the dielectric model and pH in a synthetic helical peptide studied by Monte Carlo simulated annealing

Monte Carlo simulated annealing is applied to the tertiary structure prediction of a 17-residue synthetic peptide, which is known by experiment to exhibit high helical content at low pH. Two dielectric models are considered: sigmoidal distance-dependent dielectric function and a constant dielectric function (? = 2). Starting from completely random initial conformations, our simulations for both dielectric models at low pH gave many helical conformations. The obtained low-energy conformations are compared with the nuclear Overhauser effect spectroscopy cross-peak data for both main chain and side chains, and it is shown that the results for the sigmoidal dielectric function are in remarkable agreement with the experimental data. The results predict the existence of two disjoint helices around residues 5–9 and 11–16, while nmr experiments imply significant α-helix content between residues 5 and 14. Simulations with high pH, on the other hand, hardly gave a helical conformation, which is also in accord with the experiment. These findings indicate that when side chains are charged, electrostatic interactions due to these charges play a major role in the helix stability. Our results are compared with the previous 500 ps molecular dynamics simulations of the same peptide. It is argued that simulated annealing is superior to molecular dynamics in two respects: (1) direct folding of α-helix from completely random initial conformations is possible for the former, whereas only unfolding of an α-helix can be studied by the latter; (2) while both methods predict high helix content for low pH, the results for high pH agree with experiment (low helix content) only for the former method. © 1994 John Wiley & Sons, Inc.     

Thermodynamic model of secondary structure for α-helical peptides and proteins

A thermodynamic model describing formation of α-helices by peptides and proteins in the absence of specific tertiary interactions has been developed. The model combines free energy terms defining α-helix stability in aqueous solution and terms describing immersion of every helix or fragment of coil into a micelle or a nonpolar droplet created by the rest of protein to calculate averaged or lowest energy partitioning of the peptide chain into helical and coil fragments. The α-helix energy in water was calculated with parameters derived from peptide substitution and protein engineering data and using estimates of nonpolar contact areas between side chains. The energy of nonspecific hydrophobic interactions was estimated considering each α-helix or fragment of coil as freely floating in the spherical micelle or droplet, and using water/cyclohexane (for micelles) or adjustable (for proteins) side-chain transfer energies. The model was verified for 96 and 36 peptides studied by ¹H-nmr spectroscopy in aqueous solution and in the presence of micelles, respectively ([set I] and [set 2]) and for 30 mostly α-helical globular proteins ([set 3]). For peptides, the experimental helix locations were identified from the published medium-range nuclear Overhauser effects detected by ¹H-nmr spectroscopy. For sets 1, 2, and 3, respectively, 93, 100, and 97% of helices were identified with average errors in calculation of helix boundaries of 1.3, 2.0, and 4.1 residues per helix and an average percentage of correctly calculated helix—coil states of 93, 89, and 81%, respectively. Analysis of adjustable parameters of the model (the entropy and enthalpy of the helix—coil transition, the transfer energy of the helix backbone, and parameters of the bound coil), determined by minimization of the average helix boundary deviation for each set of peptides or proteins, demonstrates that, unlike micelles, the interior of the effective protein droplet has solubility characteristics different from that for cyclohexane, does not bind fragments of coil, and lacks interfacial area. © 1997 John Wiley & Sons, Inc. Biopoly 42: 239–269, 1997     

Positional dependence of the effects of negatively charged Glu side chains on the stability of two-stranded α-helical coiled-coils

The effects on protein stability of negatively charged Glu side chains at different positions along the length of the α-helix were investigated in the two-stranded α-helical coiled-coil. A native coiled-coil has been designed which consists of two identical 35 residue polypeptide chains with a heptad repeat Q_gV_aG_bA_cL_dQ_eK_f and a Cys residue at position 2 to allow the formation of an interchain 2-2′ disulphide bridge. This coiled-coil contains no intra- or interchain electrostatic interactions and served as a control for peptides in which Glu was substituted for Gln in the e or g heptad positions. The effect of the substitutions on stability was determined by urea denaturation at 20°C with the degree of unfolding monitored by circular dichroism spectroscopy. A Glu substituted for Gln near the N-terminus in each chain of the coiled-coil stabilizes the coiled-coil at pH 7, consistent with the charge–helix dipole interaction model. This stability increase is modulated by pH change and the addition of salt (KCl or guanidine hydrochloride), confirming the electrostatic nature of the effect. In contrast, Glu substitution in the middle of the helix destabilizes the coiled-coil because of the lower helical propensity and hydrophobicity of Glu compared with Gln at pH 7. Taking the intrinsic differences into account, the apparent charge–helix dipole interaction at the N-terminus is approximately 0.35 kcal/mol per Glu substitution. A Glu substitution at the C-terminus destabilizes the coiled-coil more than in the middle owing to the combined effects of intrinsic destabilization and unfavourable charge–helix dipole interaction with the negative pole of the helix dipole. The estimated destabilizing charge–helix dipole interaction of 0.08 kcal/mol is smaller than the stabilizing interaction at the N-terminus. The presence of a 2-2′disulphide bridge appears to have little influence on the magnitude of the charge–helix dipole interactions at either end of the coiled-coil. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational constraints of amino acid side chains in alpha-helices

The conformational freedom of amino acid side chains is strongly reduced when the side chains occur on an α-helix. A quantitative evaluation of this freedom has been carried out by means of conformational energy computations for all naturally occurring amino acids and for α-aminobutyric acid when they are placed in the middle of a right-handed poly(L-alanine) α-helix. One of the three possible rotameric states for rotation around the C^α ? C^β bond (viz. g⁺) is excluded completely on the helix because of steric hindrance, and the relative populations of the other two rotamers (t and g^?) are altered because of steric interactions and the reduction of hydrogen-bonding possibilities. The computed tendencies of the changes in distributions of rotamers, on going from an ensemble of all backbone conformations to the α-helix, agree with the observed tendencies in proteins. Minimum-energy side-chain conformations in an α-helix have been tabulated for use in conformational energy computations on polypeptides.     

The alpha helix of the intermediate region in hGBP-1 acts as a coupler for enhanced GMP formation

The interferon-gamma inducible large GTPase human guanylate binding protein-1 (hGBP-1) plays a key role in anti-pathogenic and anti-proliferative functions. This protein hydrolyzes GTP to both GDP and GMP (predominant product) through sequential phosphate cleavages, which makes it functionally distinct from other GTPases. Previous study on truncated variants of hGBP-1 suggested that the α-helix present in the intermediate region is essential for dimerization and thus for GMP formation. However, the role of this helix in the full-length protein in GMP formation is not clearly understood. Here, we present that substitution of the helix with a Gly-rich flexible (GGS)₃ sequence in the full-length hGBP-1 (termed as linker protein) showed a drastic decrease in GMP formation. Unlike wild-type, the linker protein is not capable of undergoing substrate-induced dimerization and thereby transition state-induced tetramerization, suggesting the importance of the helix in oligomerization. Furthermore, we examined the effect of interactions between this helix and the α2-helix of the globular domain in GMP formation through mutational studies. The L118G mutation in the α2-helix showed a significantly reduced GMP formation. These results indicate that the interactions of the α-helix with the α2-helix are essential for enhanced GMP production. We propose that these interactions help in the oligomerization-assisted proper positioning of the catalytic machinery for efficient second phosphate cleavage. These findings thus provide a better understanding into the regulation of GMP formation in a large GTPase hGBP-1.     

The structure of a thermally stable 3-phosphoglycerate kinase and a comparison with its mesophilic equivalent

The structure of the phosphoglycerate kinase (PGK) from Bacillus stearothermophilus, a moderate thermophile, has been determined and compared with that of its mesophilic equivalent from yeast. The Bacillus enzyme structure was solved by molecular replacement and improved using constrained rigid-body, molecular dynamics and conventional refinement procedures. The refinement residual, calculated using all the measured data between 8 and 1.65 Å, is 0.18(1). The stereo chemical deviations of the final model from ideality are 0.01 Å for both bonds and planes. The mid-point temperatures of the Bacillus and yeast enzymes are 67 and 53°C, respectively. Differential scanning calorimetry indicates that the energy difference (ΔΔG) between the mesophilic and thermophilic enzymes is of the order of 5 kcal mol^?1 at room temperature. The structure comparison indicates that the features most likely to be responsible for the increased thermal stability of the Bacillus enzyme are the increased internal hydrophobicity, additional ion pairs, and better α-helix stability resulting from the removal of helix destablising residues and extra helix–dipole/helix side chain ionic interactions. © 1993 Wiley-Liss, Inc.     

Kinetic steps for α-helix formation

The kinetics of α-helix formation in polyalanine and polyglycine eicosamers (20-mers) were examined using torsional-coordinate molecular dynamics (MD). Of one hundred fifty-five MD experiments on extended (Ala)₂₀ carried out for 0.5 ns each, 129 (83%) formed a persistent α-helix. In contrast, the extended state of (Gly)₂₀ only formed a right-handed α-helix in two of the 20 MD experiments (10%), and these helices were not as long or as persistent as those of polyalanine. These simulations show helix formation to be a competition between the rates of (a) forming local hydrogen bonds (i.e. hydrogen bonds between any residue i and its i + 2, i + 3, i + 4, or i + 5th neighbor) and (b) forming nonlocal hydrogen bonds (HBs) between residues widely separated in sequence. Local HBs grow rapidly into an α-helix; but nonlocal HBs usually retard helix formation by “trapping” the polymer in irregular, “balled-up” structures. Most trajectories formed some nonlocal HBs, sometimes as many as eight. But, for (Ala)₂₀, most of these eventually rearranged to form local HBs that lead to α-helices. A simple kinetic model describes the rate of converting nonlocal HBs into α-helices. Torsional-coordinate MD speeds folding by eliminating bond and angle degrees of freedom and reducing dynamical friction. Thus, the observed 210 ps half-life for helix formation is likely to be a lower bound on the real rate. However, we believe the sequential steps observed here mirror those of real systems. Proteins 33:343–357, 1998. © 1998 Wiley-Liss, Inc.     

Theoretical π-π* circular dichroic spectra of helical poly(glycine) and poly(L-alanine) as functions of backbone torsion angles

Circular dichroic spectra and oscillator strengths of the π-π transition near 190 nm are calculated for helical (Gly)₆ and (Ala)₆ at 30° intervals of the backbone torsion angles (?,ψ) over the range -180° ≤ ? ≤ -60°, ?60° ≤ ψ ≤ 180°, using the partially dispersive normal mode treatment of the dipole interaction model. Polarizabilities of atoms and the NC′O group are those determined semiempirically in previous studies. Calculations for (Ala)₆ at (?,ψ) angles corresponding to the α-helix, the poly(Pro) II helix, a collagen single helix, a poly-(MeAla) helix, and single β-helices are found to agree well with most of the available experimental data.     

Modeling of Arylamide Helix Mimetics in the p53 Peptide Binding Site of hDM2 Suggests Parallel and Anti-Parallel Conformations Are Both Stable

The design of novel α-helix mimetic inhibitors of protein-protein interactions is of interest to pharmaceuticals and chemical genetics researchers as these inhibitors provide a chemical scaffold presenting side chains in the same geometry as an α-helix. This conformational arrangement allows the design of high affinity inhibitors mimicking known peptide sequences binding specific protein substrates. We show that GAFF and AutoDock potentials do not properly capture the conformational preferences of α-helix mimetics based on arylamide oligomers and identify alternate parameters matching solution NMR data and suitable for molecular dynamics simulation of arylamide compounds. Results from both docking and molecular dynamics simulations are consistent with the arylamides binding in the p53 peptide binding pocket. Simulations of arylamides in the p53 binding pocket of hDM2 are consistent with binding, exhibiting similar structural dynamics in the pocket as simulations of known hDM2 binders Nutlin-2 and a benzodiazepinedione compound. Arylamide conformations converge towards the same region of the binding pocket on the 20 ns time scale, and most, though not all dihedrals in the binding pocket are well sampled on this timescale. We show that there are two putative classes of binding modes for arylamide compounds supported equally by the modeling evidence. In the first, the arylamide compound lies parallel to the observed p53 helix. In the second class, not previously identified or proposed, the arylamide compound lies anti-parallel to the p53 helix.