The helical alanine controversy: an (Ala)6 insertion dramatically increases helicity

Employing chemical shift melts and hydrogen/deuterium exchange NMR techniques, we have determined the stabilization of the Trp-cage miniprotein due to multiple alanine insertions within the N-terminal alpha-helix. Alanine is shown to be uniquely helix-stabilizing and this stabilization is reflected in the global fold stability of the Trp-cage. The associated free energy change per alanine can be utilized to calculate the alanine propagation value. From the Lifson-Roig formulation, the calculated value (wAla = 1.6) is comparable to those obtained for short, solubilized, alanine-rich helices and is much larger than the values obtained by prior host-guest techniques or in N-terminally templated helices and peptides bearing long contiguous strings of alanines with no capping or solubilizing units present.     

Peptide pinwheels

Electrospraying a mixture of Ac-(GA)7K and Ac-A(GA)7K (Ac = acetyl, G = glycine, A = alanine, and K = lysine) peptides produces strong signals for unsolvated dimers and trimers. The conformations of these multimers have been examined with use of ion mobility measurements in conjunction with molecular dynamics simulations. The results suggest that the trimers adopt a pinwheel arrangement of helices with the C-termini tethered together by the protonated lysine side chain from one peptide interacting with the C-terminus of a neighboring helix. This arrangement leads to a cooperative electrostatic stabilization of all the helices through the interaction of the combined charge with the helix dipoles. The dimer adopts a related V-shaped arrangement of helices which is also cooperatively stabilized.     

Folding and unfolding of helix-turn-helix motifs in the gas phase

Ion mobility measurements and molecular dynamic simulations have been performed for a series of peptides designed to have helix-turn-helix motifs. For peptides with two helical sections linked by a short loop region: AcA(14)KG(3)A(14)K+2H(+), AcA(14)KG(5)A(14)K+2H(+), AcA(14)KG(7)A(14)K+2H(+), and AcA(14)KSar(3)A(14)K+2H(+) (Ac = acetyl, A = alanine, G = glycine, Sar = sarcosine and K = lysine); a coiled-coil geometry with two anti-parallel helices is the lowest energy conformation. The helices uncouple and the coiled-coil unfolds as the temperature is raised. Equilibrium constants determined as a function of temperature yield enthalpy and entropy changes for the unfolding of the coiled-coil. The enthalpy and entropy changes depend on the length and nature of the loop region. For a peptide with three helical sections: protonated AcA(14)KG(3)A(14)KG(3)A(14)K; a coiled-coil bundle with three helices side-by-side is substantially less stable than a geometry with two helices in an antiparallel coiled-coil and the third helix collinear with one of the other two.     

Proton transfer-induced conformational changes and melting in designed peptides in the gas phase

The conformations of protonated RA15K, RA20K and RA15H (R = arginine, A = alanine, K = lysine, and H = histidine) have been examined in the gas phase as a function of temperature. These peptides were designed so that intramolecular proton transfer will trigger conformational changes between a helix (proton sequestered at the C-terminus) and globule (proton sequestered at the N-terminus). Kinetically controlled structural transitions occur below 400 K (from helix to globule for RA15H, and from globule to helix for RA15K and RA20K). As the temperature is raised, the compact globule found at room temperature expands, accesses more configurations, and becomes entropically favored. At around 500 K, the RA15K and RA20K helices undergo a melting transition. The transition is broad, as expected for a phase transition in a finite system, and becomes narrower as the peptide size increases. In the helical conformation, the two basic residues are well separated; as a result, the proton transfer necessary to drive the melting transition probably involves a mobile proton. For doubly protonated RA15K, a dumbbell-like conformation (resulting from repulsion between the two protonated basic residues) is found at high temperature.     

Solvent dependence of PII conformation in model alanine peptides

Alanine residues in two model peptides, the pentapeptide AcGGAGGNH(2) and the 11mer AcO(2)A(7)O(2)NH(2), have been reported to have substantial PII conformation in water. The PII structure in both peptides is sensitive to solvent. In the presence of the organic solvent TFE, the conformation of the pentamer changes from PII to internally H-bonded gamma or beta turns, while the chain with seven alanines forms alpha helix. The PII structure in the 11mer is more stable than that in the shorter peptide as the TFE concentration increases. For the pentamer, a comparison of short-chain aliphatic alcohols to water shows that the PII content decreases in the order water > methanol > ethanol > 2-propanol, linearly according to empirical scales of solvent polarity. Thus, depending on the extent of local solvation as folding progresses, the peptide backbone as modeled by alanine oligomers shifts from PII to internally H-bonded (gamma or beta turn) conformations and to alpha helix in longer segments. On the other hand, the PII content of AcO(2)A(7)O(2)NH(2) increases significantly in the presence of guanidine, as does that of oligoproline peptides, while detergent sodium dodecyl sulfate (SDS) favors alpha helix in this peptide. The shorter peptide does not show a parallel increase in PII with guanidine.     

The energy landscape of unsolvated peptides: helix formation and cold denaturation in Ac-A4G7A4 + H+

Ion mobility measurements and molecular dynamics simulations were performed for unsolvated A4G7A4 + H+ and Ac-A4G7A4 + H+ (Ac = acetyl, A = alanine, G = glycine) peptides. As expected, A4G7A4 + H+ adopts a globular conformation (a compact, random-looking, three-dimensional structure) over the entire temperature range examined (100-410 K). Ac-A4G7A4 + H+ on the other hand is designed to have a flat energy landscape with a marginally stable helical state. This peptide shows at least four different conformations at low temperatures (<230 K). The two conformations with the largest cross sections are attributed to - and partial -helices, while the one with the smallest cross section is globular. The other main conformation may be partially helical. Ac-A4G7A4 + H+ becomes predominantly globular at intermediate temperatures and then becomes more helical as the temperature is raised further. This unexpected behavior may be due to the helix having a higher vibrational entropy than the globular state, as predicted by some recent calculations (Ma, B.; Tsai, C.-J.; Nussinov, R. Biophys. J. 2000, 79, 2739-2753).     

Energetic characterization of short helical polyalanine peptides in water: analysis of 13C=O chemical shift data

Measured at 2 degrees C in water, NMR chemical shifts of (13)C=O labeled central alanine residues of peptides W-Lys(5)-(t)L(3)-Ala(n)-(t)L(3)-Lys(5)NH(2), n = 9, 11, 13, 15, 19 and W-Lys(5)-(t)L(3)-a-Ala(n)-A-Inp-(t)L(2)-Lys(5)NH(2) (a = D-Ala; (t)L = tert-leucine; Inp = 4-carboxypiperidine) are used to assign jt(L) and ct(L), the N- and C-terminal (t)L capping parameters and length-dependent values for w(Ala)(n), the alanine helical propensity for Ala(n) peptides. These parameters allow Lifson-Roig characterization of the stabilities of Ala(n)() helices in water. To facilitate chemical shift characterization, different (13)C/(12)C ratios are incorporated into specific Ala sites to code up to six residue sites per peptide. Large left/right chemical shift anisotropies are intrinsic to helical polyalanines, and a correcting L-R-based model is introduced. Capping parameters jt(L) = ct(L) lie in the range of 0.3 to 0.5; the (t)L residues are thus moderately helix-destabilizing. For helical conformations of lengths shorter than eight residues, assigned values for w(Ala) approach 1.0 but increase monotonically with length to a value of 1.59 for w(Ala)(19).     

Helix formation inside a nanotube: possible influence of backbone-water hydrogen bonding by the confining surface through modulation of water activity

Recent molecular dynamics simulations of Sorin and Pande [J. Am. Chem. Soc. 128, 6316 (2006)] in explicit solvent found that helix formation of an alanine peptide is disfavored inside a nanotube relative to that in bulk solution. Here, we present a theory to quantitatively rationalize their simulation results. The basic idea is that the nonpolar inner surface of the nanotube creates a depletion layer and raises the activity of the confined water. The raised water activity, in turn, stabilizes the coil state through hydrogen bonding with the backbone amides and carbonyls. We account for the influence of water activity on helix formation within the Lifson-Roig theory. With physically reasonable parameters, the dependence of the helical content on the diameter of the nanotube obtained in the simulations is well reproduced.     

Helix-turn-helix motifs in unsolvated peptides

The conformations of unsolvated Ac-A14KG3A14K + 2H+ (Ac = acetyl, A = alanine, K = lysine, G = glycine) have been examined by ion mobility measurements and molecular dynamics simulations. This peptide was designed as a model helix-turn-helix motif. It was found to adopt three distinct geometries which were assigned to an extended helical conformation which is only stable at low temperatures (<230 K), a relatively high energy but metastable structure with exchanged lysines, and a coiled-coil. The coiled coil (which consists of an antiparallel arrangement of two helical alanine sections linked by a flexible glycine loop) is the dominant conformation. For temperatures >350 K, the experimental results indicate the helices uncouple and the loop randomizes. From equilibrium constants determined for this helix coupling right arrow over left arrow uncoupling transition, we found DeltaH degrees = -45 kJ mol-1 and DeltaS degrees = 114 J K-1 mol-1. -DeltaH degrees is essentially the enthalpy change for docking the two helices together while DeltaS degrees is essentially the entropy change for freeing up the glycine loop.     

A structural study of model peptides derived from HIV-1 integrase central domain

The HIV-1 integrase (IN) catalyzes the integration of viral DNA in the human genome. In vitro the enzyme displays an equilibrium of monomers, dimers, tetramers and larger oligomers. However, its functional oligomeric form in vivo is not known. We report a study of the auto-associative properties of three peptides denoted K156, E156 and E159. These derive from the alpha4 helix of the IN catalytic core. The alpha4 helix is an amphipatic helix exposed at the surface of the protein and could be involved in the oligomerization process through its hydrophobic face. The peptides were obtained from the replacement of several amino acid residues by more helicogenic ones in the alpha4 helix peptide. K156 carries the basic residues Lys156 and Lys159, which have been shown important for the binding of IN to viral DNA. In E156 and E159 they are replaced with the acidic residue Glu. A fourth peptide K(E)156 obtained from the replacement of hydrophobic residues with Glu in K156 in order to abolish the auto-associative properties is used as a negative control. The capacity shown by peptides for alpha-helical formation is demonstrated by circular dichroism (CD) analysis performed in aqueous solution and in aqueous trifluoroethanol (TFE) mixtures. Both electrospray ionization mass spectrometry (ESI-MS) and glutaraldehyde chemical cross-linking show that peptides adopt different solvent-dependent equilibriums of monomers, dimers, trimers and tetramers. Oligomerization of peptides in aqueous solution is related to their ability to form helical structures. Addition of a small amount of TFE (<10%) stimulates helix stabilization and the interhelical hydrophobic contacts. Higher amounts of TFE alter the hydrophobic contacts and disrupt the oligomeric species. In addition to hydrophobic interactions, the patterns indicate that the biologically important Lys156 and Lys159 residues also participate in helix association. K(E)156 despite its ability to adopt a helical structure is unable to associate into oligomers, demonstrating the importance of hydrophobic contacts for oligomerization. Thus, the designed peptides provide us information on the functional properties of the alpha4 IN that seems to hold a dual role in DNA recognition and protein oligomerization.     

Helix unfolding in unsolvated peptides

The conformations of unsolvated Ac-K(AGG)(5)+H(+) and Ac-(AGG)(5)K+H(+) peptides (Ac = acetyl, A = alanine, G = glycine, and K = lysine) have been examined by ion mobility measurements over a wide temperature range (150-410 K). The Ac-K(AGG)(5)+H(+) peptide remains a globule (a compact, roughly spherical structure) over the entire temperature range, while both an alpha-helix and a globule are found for Ac-(AGG)(5)K+H(+) at low temperature. As the temperature is raised the alpha-helix unfolds. Rate constants for loss of the helix (on a millisecond time scale) have been determined as a function of temperature and yield an Arrhenius activation energy and preexponential factor of 38.2 +/- 1.0 kJ mol(-1) and 6.5 +/- 3.7 x 10(9) s(-1), respectively. The alpha-helix apparently does not unfold directly into the globule, but first converts into a long-lived intermediate which survives to a significantly higher temperature before converting. According to molecular dynamics simulations, there is a partially untwisted helical conformation that has both a low energy and a well-defined geometry. This special structure lies between the helix and globule and may be the long-lived intermediate.     

Molecular Dynamics Simulations of a β-Hairpin Fragment of Protein G by Means of Atom-Bond Electronegativity Equalization Method Fused into Molecular Mechanics (ABEEMσπ/MM)

There are some controversial opinions about the origin of folding β‐hairpin stability in aqueous solution. In this study, the structural and dynamic behavior of a 16‐residue β‐hairpin from B1 domain of protein G has been investigated at 280, 300, 350 and 450 K using molecular dynamics (MD) simulations by means of Atom‐Bond Electronegativity Equalization Method Fused into Molecular Mechanics i.e., ABEEMδπ/MM and the explicit ABEEM‐7P water solvent model. In addition, a 300 K simulation of one mutant having the aromatic residues substituted with alanines has been performed. The hydrophobic surface area, hydrophilic surface area and some structural properties have been used to measure the role of the hydrophobic interactions. It is found that the aromatic residues substituted with alanines have shown an evident destabilization of the structure and unfolding started after 1.5 ns. It is also found that the number of the main chain hydrogen bonds have different distributions through three different simulations. All above demonstrate that the hydrophobic interactions and the main chain hydrogen bonds play an important role in the stability of the folding structure of β‐hairpin in solution. Furthermore, through the structural analyses of the β‐hairpin structures from four temperature simulations and the comparison with other MD simulations of β‐hairpin peptides, the new ABEEMδπ force field can reproduce the structural data in good agreement with the experimental data.     

Water molecule adsorption on short alanine peptides: how short is the shortest gas-phase alanine-based helix?

Water adsorption measurements have been performed under equilibrium conditions for unsolvated Ac-A(n)K+H(+) and Ac-KA(n)+H(+) peptides with n = 4 - 10. Previous work on larger alanine peptides has shown that two dominant conformations (helices and globules) are present for these peptides and that water adsorbs much more strongly to the globules than to the helices. All the Ac-KA(n)+H(+) peptides studied here (which are expected to be globular) adsorb water strongly, and so do the Ac-A(n)K+H(+) peptides with n < 8. However, for Ac-A(n)K+H(+) with n = 8-10 there is a substantial drop in the propensity to adsorb water. This result suggests that Ac-A(8)K+H(+) is the smallest Ac-A(n)K+H(+) peptide to have a significant helical content in the gas phase. Water adsorption measurements for Ac-V(n)K+H(+) and Ac-L(n)K+H(+) with n = 5-10 suggest that the helix emerges at n = 8 for these peptides as well.     

The energy landscape of unsolvated peptides: the role of context in the stability of alanine/glycine helices

Ion mobility measurements have been used to examine the conformations present for unsolvated Ac-(AG)(7)A+H(+) and (AG)(7)A+H(+) peptides (Ac = acetyl, A = alanine, and G = glycine) over a broad temperature range (100-410 K). The results are compared to those recently reported for Ac-A(4)G(7)A(4)+H(+) and A(4)G(7)A(4)+H(+), which have the same compositions but different sequences. Ac-(AG)(7)A+H(+) shows less conformational diversity than Ac-A(4)G(7)A(4)+H(+); it is much less helical than Ac-A(4)G(7)A(4)+H(+) at the upper end of the temperature range studied, and at low temperatures, one of the two Ac-A(4)G(7)A(4)+H(+) features assigned to helical conformations is missing for Ac-(AG)(7)A+H(+). Molecular dynamics simulations suggest that the different conformational preferences are not due to differences in the stabilities of the helical states, but differences in the nonhelical states: it appears that Ac-(AG)(7)A+H(+) is more flexible and able to adopt lower energy globular conformations (compact random looking three-dimensional structures) than Ac-A(4)G(7)A(4)+H(+). The helix to globule transition that occurs for Ac-(AG)(7)A+H(+) at around 250-350 K is not a direct (two-state) process, but a creeping transition that takes place through at least one and probably several intermediates.     

Pi-helix preference in unsolvated peptides

Ion mobility measurements have been used to examine helix formations in the gas phase for a series of alanine/glycine-based peptides that incorporate a glutamic acid (E) and lysine (K) at various positions along the backbone. Incorporation of an EK pair lowers the percent helix for all positions (presumably because hydrogen bonding between the backbone and the E and K side chains stabilize the nonhelical globular conformations). The largest percent helix is found when the EK pair is in an i,i+5 arrangement, which suggests that the preferred helical conformation for these peptides is a pi-helix. This conclusion is supported by comparison of cross sections deduced from the ion-mobility measurements to average cross sections calculated for conformations obtained from molecular dynamics simulations. The glutamic acid and lysine may form an ion pair that is stabilized by interactions with the helix macro-dipole.     

Entropic stabilization of isolated beta-sheets

Temperature-dependent electric deflection measurements have been performed for a series of unsolvated alanine-based peptides (Ac-WA(n)-NH(2), where Ac = acetyl, W = tryptophan, A = alanine, and n = 3, 5, 10, 13, and 15). The measurements are interpreted using Monte Carlo simulations performed with a parallel tempering algorithm. Despite alanine's high helix propensity in solution, the results suggest that unsolvated Ac-WA(n)-NH(2) peptides with n > 10 adopt beta-sheet conformations at room temperature. Previous studies have shown that protonated alanine-based peptides adopt helical or globular conformations in the gas phase, depending on the location of the charge. Thus, the charge more than anything else controls the structure.     

Electron capture induced dissociation of doubly protonated pentapeptides: dependence on molecular structure and charge separation

We have studied electron capture induced dissociation of a set of doubly protonated pentapeptides, all composed of one lysine (K) and either four glycine (G) or four alanine (A) residues, as a function of the sequence of these building blocks. Thereby the separation of the two charges, sequestered on the N-terminal amino group and the lysine side chain, is varied. The characteristic cleavage of N-C(α) bonds is observed for all peptides over the whole backbone length, with the charge carrying fragments always containing K. The resulting fragmentation patterns are very similar if G is replaced by A. In the case of [XKXXX+2H](2+) (X=A or G), a distinct feature is observed in the distribution of backbone cleavage fragments and the probability for ammonia loss is drastically reduced. This may be due to an isomer with an amide oxygen as protonation site giving rise to the observed increase in breakage at a specific site in the molecule. For the other peptides, a correlation with the distance between amide oxygen and the charge at the lysine side chain has been found. This may be an indication that it is only the contribution from this site to the charge stabilization of the amide π(*) orbitals which determines relative fragment intensities. For comparison, complexes with two crown ether molecules have been studied as well. The crown ether provides a shielding of the charge and prevents the peptide from folding and internal hydrogen bonding, which leads to a more uniform fragmentation behavior.     

Helix Nucleation by the Smallest Known α‐Helix in Water

Cyclic pentapeptides (e.g. Ac‐(cyclo‐1,5)‐[KAXAD]‐NH₂; X=Ala, 1 ; Arg, 2 ) in water adopt one α‐helical turn defined by three hydrogen bonds. NMR structure analysis reveals a slight distortion from α‐helicity at the C‐terminal aspartate caused by torsional restraints imposed by the K(i)–D(i+4) lactam bridge. To investigate this effect on helix nucleation, the more water‐soluble 2 was appended to N‐, C‐, or both termini of a palindromic peptide ARAARAARA (≤5 % helicity), resulting in 67, 92, or 100 % relative α‐helicity, as calculated from CD spectra. From the C‐terminus of peptides, 2 can nucleate at least six α‐helical turns. From the N‐terminus, imperfect alignment of the Asp5 backbone amide in 2 reduces helix nucleation, but is corrected by a second unit of 2 separated by 0–9 residues from the first. These cyclic peptides are extremely versatile helix nucleators that can be placed anywhere in 5–25 residue peptides, which correspond to most helix lengths in protein–protein interactions.     

A new scheme for determining the intramolecular seven-membered ring N-H...O=C hydrogen-bonding energies of glycine and alanine peptides

In this paper a new scheme was proposed to calculate the intramolecular hydrogen-bonding energies in peptides and was applied to calculate the intramolecular seven-membered ring N-H...O=C hydrogen-bonding energies of the glycine and alanine peptides. The density-functional theory B3LYP6-31G(d) and B3LYP6-311G(d,p) methods and the second-order Moller-Plesset perturbation theory MP26-31G(d) method were used to calculate the optimal geometries and frequencies of glycine and alanine peptides and related structures. MP26-311++G(d,p), MP26-311++G(3df,2p), and MP2/aug-cc-pVTZ methods were then used to evaluate the single-point energies. It was found that the B3LYP6-31G(d), MP26-31G(d), and B3LYP6-311G(d,p) methods yield almost similar structural parameters for the conformers of the glycine and alanine dipeptides. MP2/aug-cc-pVTZ predicts that the intramolecular seven-membered ring N-H...O=C hydrogen-bonding strength has a value of 5.54 kcal/mol in glycine dipeptide and 5.73 and 5.19 kcal/mol in alanine dipeptides, while the steric repulsive interactions of the seven-membered ring conformers are 4.13 kcal/mol in glycine dipeptide and 6.62 and 3.71 kcal/mol in alanine dipeptides. It was also found that MP26-311++G(3df,2p) gives as accurate intramolecular N-H...O=C hydrogen-bonding energies and steric repulsive interactions as the much more costly MP2/aug-cc-pVTZ does.     

The gas-phase structure of alanine

The jet-cooled rotational spectrum of neutral alanine has been studied using laser-ablation molecular-beam Fourier transform microwave spectroscopy (LA-MB-FTMW). The spectra of the two most stable forms were observed in the frequency range 6-18 GHz for the parent, (15)N alanine, three single (13)C species, and four single D species. The (14)N nuclear quadrupole coupling hyperfine structures have been resolved, and their comparison with those calculated theoretically confirms unambiguously the conformer assignments. The independent structures of both conformers have been determined experimentally for the first time using r(s) and r(0) procedures. In both cases, the amino acid backbone is nonplanar. For the most stable conformer I, the COOH group adopts a cis configuration and an asymmetric bifurcated hydrogen bond is formed between the amino group and carbonyl oxygen (r(N-H(a)...O=C) = 2.70(2) A and r(N-H(b)...O=C) = 2.88(2) A). For conformer IIa, the COOH group adopts a trans configuration and is stabilized by a O-H...N hydrogen bond (r(O-H...N) = 1.96(2) A). The relative conformer abundances in the supersonic expansion have also been investigated.