Alanine-based Peptides Research Articles

Influence of Tyrosine on the Electronic Circular Dichroism of Helical Peptides

Studies of short helical peptides can provide insights into amino acid interactions in proteins and their role in protein folding. A common method of estimating helical structure is electronic circular dichroism (CD) in the far ultraviolet. It is known that aromatic side chains such as tyrosine may influence CD in this region, but this residue has desirable fluorescent properties and is nevertheless often incorporated into peptides. To investigate the relationship between the conformation of tyrosine and its contribution to the CD, we have calculated the CD of some short alanine-based helical peptides from first principles based on molecular dynamics simulations. The calculations are complemented by some analysis of static models. These theoretical studies estimate that a tyrosine residue may contribute up to ′5000 deg cm 2 dmol - 1 to the mean residue ellipticity at 220 nm, with the most typical value being 1000 deg cm 2 dmol - 1 . If uncorrected, this would lead to an underestimate of the helicity of the peptide of 5-20%. For a tyrosine side chain in the trans rotameric state, there is a discernible relationship between its precise orientation and its contribution to CD at 220 nm. Experiments on tyrosine-containing peptides using CD should be interpreted bearing these findings in mind and, preferably, with an independent approach for probing the conformational states of tyrosine.

Direct probing of zwitterion formation in unsolvated peptides.

Molecular beam electric deflection measurements have been used to determine electric susceptibilities for small unsolvated alanine-based peptides. The electric susceptibility provides information about the charge distribution within the peptide and can be used to distinguish between zwitterionic and canonical forms. Measured electric susceptibilities for WAn peptides (n = 1-5) are similar to those for capped Ac-WAn-NH2 peptides (which cannot form zwitterions). Susceptibilities calculated using a simulated tempering-based approach are substantially larger for the zwitterionic form than for the canonical form. The measured susceptibilities are in good agreement with those calculated for the canonical form. For the larger peptides, the lowest potential energy structure found in the simulations is hairpin-like, while the lowest free energy structure found at room temperature is extended. The zwitterionic form is constrained by intramolecular interactions which make it entropically unfavorable.

Noncharged amino acid residues at the solvent-exposed positions in the middle and at the C terminus of the alpha-helix have the same helical propensity.

It was established previously that helical propensities of different amino acid residues in the middle of alpha-helix in peptides and in proteins are very similar. The statistical analysis of the protein helices from the known three-dimensional structures shows no difference in the frequency of noncharged residues in the middle and at the C terminus. Yet, experimental studies show distinctive differences for the helical propensities of noncharged residues in the middle and in the C terminus in model peptides. Is this a general effect, and is it applicable to protein helices or is it specific to the model alanine-based peptides? To answer this question, the effects of substitutions at positions 28 (middle residue) and 32 (C2 position at the C terminus) of the alpha-helix of ubiquitin on the stability of this protein are measured by using differential scanning calorimetry. The two data sets produce similar values for intrinsic helix propensity, leading to a conclusion that noncharged amino acid residues at the solvent-exposed positions in the middle and at the C terminus of the alpha-helix have the same helical propensity. This conclusion is further supported with an excellent correlation between the helix propensity scale obtained for the two positions in ubiquitin with the experimental helix propensity scale established previously and with the statistical distribution of the residues in protein helices.

Electron transfer through a prenucleated bimetalated alanine-based peptide helix.

We have synthesized a 22 residue alanine-based peptide with a tris(bipyridyl)ruthenium(II) amino acid near the middle of the peptide which can act as a photoinducible electron donor. Two histidines spaced i, i + 4 near the C-terminus of the peptide were then cross-linked with a tetraammineruthenium(III) moiety to prenucleate the helix and provide an electron acceptor site. Introduction of the cross-link enhances the average helix content from 67% to 84% at 0 degrees C, as judged by circular dichroism spectroscopy. The temperature dependence of the mean molar residue ellipticity at 222 nm, [THETAV;](222), for the bimetalated peptide was fit to a modified Lifson-Roig helix-coil model to permit extraction of the population of helical conformation at each residue separating the electron donor and acceptor. On average, the residues between the donor and acceptor are 92% helical. Photoinduced electron transfer with a driving force of -1.0 eV and an estimated reorganization energy of 0.82 eV was measured by fluorescence quenching methods in H(2)O and D(2)O, yielding rate constants, k(ET), of 7 +/- 3 x 10(6) s(-)(1) and 5 +/- 1 x 10(6) s(-)(1) at 0 degrees C. Calculation of the electronic coupling matrix element, H(ab), with the Marcus equation yields a value of 0.19 +/- 0.4 cm(-)(1). Analysis in terms of the pathway model for electronic coupling indicates that this magnitude of H(ab) is consistent with the participation of hydrogen bonds in electronic coupling for an isolated alpha-helix.

Breaking non-native hydrophobic clusters is the rate-limiting step in the folding of an alanine-based peptide.

The formation mechanism of an alanine-based peptide has been studied by all-atom molecular dynamics simulations with a recently developed all-atom point-charge force field and the Generalize Born continuum solvent model at an effective salt concentration of 0.2M. Thirty-two simulations were conducted. Each simulation was performed for 100 ns. A surprisingly complex folding process was observed. The development of the helical content can be divided into three phases with time constants of 0.06-0.08, 1.4-2.3, and 12-13 ns, respectively. Helices initiate extreme rapidly in the first phase similar to that estimated from explicit solvent simulations. Hydrophobic collapse also takes place in this phase. A folding intermediate state develops in the second phase and is unfolded to allow the peptide to reach the transition state in the third phase. The folding intermediate states are characterized by the two-turn short helices and the transition states are helix-turn-helix motifs-both of which are stabilized by hydrophobic clusters. The equilibrium helical content, calculated by both the main-chain Phi-Psi torsion angles and the main-chain hydrogen bonds, is 64-66%, which is in remarkable agreement with experiments. After corrected for the solvent viscosity effect, an extrapolated folding time of 16-20 ns is obtained that is in qualitative agreement with experiments. Contrary to the prevailing opinion, neither initiation nor growth of the helix is the rate-limiting step. Instead, the rate-limiting step for this peptide is breaking the non-native hydrophobic clusters in order to reach the transition state. The implication to the folding mechanisms of proteins is also discussed.

A simple model for polyproline II structure in unfolded states of alanine-based peptides.

The striking similarity between observed circular dichroism spectra of nonprolyl homopolymers and that of regular left-handed polyproline II (P(II)) helices prompted Tiffany and Krimm to propose in 1968 that unordered peptides and unfolded proteins are built of P(II) segments linked by sharp bends. A large body of experimental evidence, accumulated over the past three decades, provides compelling evidence in support of the original hypothesis of Tiffany and Krimm. Of particular interest are the recent experiments of Shi et al. who find significant P(II) structure in a short unfolded alanine-based peptide. What is the physical basis for P(II) helices in peptide and protein unfolded states? The widely accepted view is that favorable chain-solvent hydrogen bonds lead to a preference for dynamical fluctuations about noncooperative P(II) helices in water. Is this preference simply a consequence of hydrogen bonding or is it a manifestation of a more general trend for unfolded states which are appropriately viewed as chains in a good solvent? The prevalence of closely packed interiors in folded proteins suggests that under conditions that favor folding, water-which is a better solvent for itself than for any polypeptide chain-expels the chain from its midst, thereby maximizing chain packing. Implicit in this view is a complementary idea: under conditions that favor unfolding, chain-solvent interactions are preferred and in a so-called good solvent, chain packing density is minimized. In this work we show that minimization of chain packing density leads to preferred fluctuations for short polyalanyl chains around canonical, noncooperative P(II)-like conformations. Minimization of chain packing is modeled using a purely repulsive soft-core potential between polypeptide atoms. Details of chain-solvent interactions are ignored. Remarkably, the simple model captures the essential physics behind the preference of short unfolded alanine-based peptides for P(II) helices. Our results are based on a detailed analysis of the potential energy landscape which determines the system's structural and thermodynamic preferences. We use the inherent structure formalism of Stillinger and Weber, according to which the energy landscape is partitioned into basins of attraction around local minima. We find that the landscape for the experimentally studied seven-residue alanine-based peptide is dominated by fluctuations about two noncooperative structures: the left-handed polyproline II helix and its symmetry mate.

The initial steps in the hydration of unsolvated peptides: water molecule adsorption on alanine-based helices and globules.

Equilibrium constants for the adsorption of the first water molecule onto a variety of unsolvated alanine-based peptides have been measured and Delta H degrees and DeltaS degrees have been determined. The studies were designed to examine the effects of conformation, charge, and composition on the propensity for peptides to bind water. In general, water adsorption occurs significantly more readily on the globular peptides than on helical ones: several of the singly charged helical peptides were not observed to adsorb a water molecule even at -50 degrees C. These results place a limit on the free energy change for interaction between a water molecule and the helical peptide group. Molecular dynamics simulations reproduce most of the main features of the results. The ability to establish a network of hydrogen bonds to several different hydrogen-bonding partners emerges as a critical factor for strong binding of the water molecule. Whether the charge site is involved in water adsorption depends on how well it is shielded. Peptides containing a protonated histidine bind water much more strongly that those containing a protonated lysine because the delocalized charge on histidine is difficult to shield. The entropy change for adsorption of the first water molecule is correlated with the enthalpy change.

Osmolyte effects on helix formation in peptides and the stability of coiled-coils.

The ability of several naturally occurring substances known as osmolytes to induce helix formation in an alanine-based peptide have been investigated. As predicted by the osmophobic effect hypothesis, the osmolytes studies here do induce helix formation. Trimethylamine-N-oxide (TMAO) is the best structure-inducing osmolytes investigated here, but it is not as effective in promoting helix formation as the common cosolvent trifluoroethanol (TFE). We also provide a semiquantitative study of the ability of TMAO to induce helix formation and urea, which acts as a helix (and protein) denaturant. We find that on a molar basis, these agents are exactly counteractive as structure inducing and unfolding agents. Finally, we extend the investigations to the effects of urea and TMAO on the stability of a dimeric coiled-coil peptide and find identical results. Together these results support the tenets of the osmophobic hypothesis and highlight the importance of the polypeptide backbone in protein folding and stability.

The Activation Energy for Insertion of Transmembrane α-Helices is Dependent on Membrane Composition

The physical mechanisms that govern the folding and assembly of integral membrane proteins are poorly understood. It appears that certain properties of the lipid bilayer affect membrane protein folding in vitro, either by modulating helix insertion or packing. In order to begin to understand the origin of this effect, we investigate the effect of lipid forces on the insertion of a transmembrane α-helix using a water-soluble, alanine-based peptide, KKAAAIAAAAAIAAWAAIAAAKKKK-amide. This peptide binds to preformed 1,2-dioleoyl- l-α-phosphatidylcholine (DOPC) vesicles at neutral pH, but spontaneous transmembrane helix insertion directly from the aqueous phase only occurs at high pH when the Lys residues are de-protonated. These results suggest that the translocation of charge is a major determinant of the activation energy for insertion. Time-resolved measurements of the insertion process at high pH indicate biphasic kinetics with time constants of ca 30 and 430 seconds. The slower phase seems to correlate with formation of a predominantly transmembrane α-helical conformation, as determined from the transfer of the tryptophan residue to the hydrocarbon region of the membrane. Temperature-dependent measurements showed that insertion can proceed only above a certain threshold temperature and that the Arrhenius activation energy is of the order of 90 kJ mol −1. The kinetics, threshold temperature and the activation energy change with the mole fraction of 1,2-dioleoyl- l-α-phosphatidylethanolamine (DOPE) introduced into the DOPC membrane. The activation energy increases with increasing DOPE content, which could reflect the fact that this lipid drives the bilayer towards a non-bilayer transition and increases the lateral pressure in the lipid chain region. This suggests that folding events involving the insertion of helical segments across the bilayer can be controlled by lipid forces.

Structural transitions in biomolecules - a numerical comparison of two approaches for the study of phase transitions in small systems

We compare two recently proposed methods for the characterization of phase transitions in small systems. The usefulness of these techniques is evaluated for the case of structural transition in alanine-based peptides.

Origin of the different strengths of the (i,i+4) and (i,i+3) leucine pair interactions in helices

Pairs of leucine side chains, spaced either (i,i+3) or (i,i+4), are known to stabilize alanine-based peptide helices, Experiments with new peptide sequences confirm that the (i,i+4) pair interaction is markedly stronger than the (i,i+3) pair interaction. This result is not expected from reported Monte Carlo simulations, which predict that the (i,i+3) interaction is slightly stronger. The interaction strength can be predicted from recently reported measurements of buried non-polar surface area, obtained from structures in the Protein Data Bank: the agreement is reasonable for the (i,i+3) LL interaction but underestimates the (i,i+4) LL interaction. Solvation of peptide groups in the helix backbone may contribute to the different strengths of the two LL pair interactions because different χ 1 leucine rotamers are used and the (i,i+3) pair shields two peptide groups whereas the (i,i+4) pair shields only one. A rough estimate of the backbone solvation effect, based on the difference between the helix propensities of leucine and alanine, agrees with the size of the difference between the (i,i+3) and (i,i+4) leucine pair interactions.

Effect of phosphorylation on alpha-helix stability as a function of position.

We have investigated the effect of placing phosphoserine at the N-cap, N1, N2, N3, and interior position in alanine-based alpha-helical peptides. Helix contents of each peptide were measured by CD spectroscopy and titrations performed to determine pK(a) values. Data were analyzed with modified Lifson-Roig theory to determine helix-coil parameters (n, n(1), n(2), n(3), and w) and free energy changes for phosphoserine at each helical position. Results are given for a -1 and -2 phosphoserine charge state. Results show that phosphoserine stabilizes at the N-terminal positions by as much as 2.3 kcal.mol(-1), while destabilizes in the helix interior by 1.2 kcal.mol(-1), relative to serine. The rank order of free energies relative to serine at each position is N2 > N3 > N1 > N-cap > interior. Moreover, -2 phosphoserine is the most preferred residue known at each of these N-terminal positions. Experimental pK(a) values for the -1 to -2 phosphoserine transition are in the order N2 < N-cap < N1 < N3 < interior. This order agrees well with electrostatics calculations carried out with phosphoserine at the N-terminal positions and interior positions. Combining these with calculations at the C3, C2, C1, and C-cap positions gives results for phosphoserine along the length of the helix. We see a transition from phosphoserine stabilization at the N-terminus to destabilization at the C-terminus and can explain this in terms of the balance of protein solvation, favorable interactions, and dehydration. These results give insight into the phosphorylatable control of biological systems through positive or negative changes in stability.

A ruthenium(II) tris(bipyridyl) amino acid: synthesis and direct incorporation into an alpha-helical peptide by solid-phase synthesis.

The synthesis of a ruthenium(II) tris(bipyridyl) amino acid is described along with its incorporation into a helix-forming peptide using standard solid-phase peptide synthesis methods. The physical properties of the metalloamino acid separately and in the context of the peptide are reported. The electrochemical potential, absorption spectrum, emission and excitation spectra, and emission lifetime data for the ruthenium(II) tris(bipyridyl) amino acid are similar to those of ruthenium(II) tris(bipyridine), indicating that the amino acid functionality introduced on one of the bipyridine rings does not strongly perturb the properties of the metal complex. The ruthenium(II) tris(bipyridyl) amino acid is amenable to direct incorporation into a synthetic peptide using solid-phase methods and BOC/benzyl chemistry. The 22 amino acid alanine-based peptide produced is 67% helical at 0 degrees C in aqueous buffer, as measured by circular dichroism spectroscopy. The 2,2,2-trifluorethanol helix induction curve and the temperature dependence of the helicity are typical for alanine-based peptides. The Lifson-Roig helix propagation parameter, w, derived from circular dichroism and NMR-monitored kinetic hydrogen-deuterium exchange is 0.5 +/- 0.1 at 0 degrees C, indicating a moderate helix propensity for this metalloamino acid.

Thermodynamics of interactions of urea and guanidinium salts with protein surface: relationship between solute effects on protein processes and changes in water-accessible surface area.

To interpret effects of urea and guanidinium (GuH(+)) salts on processes that involve large changes in protein water-accessible surface area (ASA), and to predict these effects from structural information, a thermodynamic characterization of the interactions of these solutes with different types of protein surface is required. In the present work we quantify the interactions of urea, GuHCl, GuHSCN, and, for comparison, KCl with native bovine serum albumin (BSA) surface, using vapor pressure osmometry (VPO) to obtain preferential interaction coefficients (Gamma(mu3)) as functions of nondenaturing concentrations of these solutes (0-1 molal). From analysis of Gamma(mu3) using the local-bulk domain model, we obtain concentration-independent partition coefficients K(nat)(P) that characterize the accumulation of these solutes near native protein (BSA) surface: K(nat)(P,urea)= 1.10 +/- 0.04, K(nat)(P,SCN(-)) = 2.4 +/- 0.2, K(nat)(P,GuH(+)) = 1.60 +/- 0.08, relative to K(nat)(P,K(+)) identical with 1 and K(nat)(P,Cl(-)) = 1.0 +/- 0.08. The relative magnitudes of K(nat)(P) are consistent with the relative effectiveness of these solutes as perturbants of protein processes. From a comparison of partition coefficients for these solutes and native surface (K(nat)(P)) with those determined by us previously for unfolded protein and alanine-based peptide surface K(unf)(P), we dissect K(P) into contributions from polar peptide backbone and other types of protein surface. For globular protein-urea interactions, we find K(nat)(P,urea) = K(unf)(P,urea). We propose that this equality arises because polar peptide backbone is the same fraction (0.13) of total ASA for both classes of surface. The analysis presented here quantifies and provides a physical basis for understanding Hofmeister effects of salt ions and the effects of uncharged solutes on protein processes in terms of K(P) and the change in protein ASA.

Time-resolved two-dimensional vibrational spectroscopy of a short α-helix in water

Nonlinear two-dimensional (2D) vibrational spectroscopy has been used to investigate the amide I band of an alanine-based 21-residue α-helical peptide in aqueous solution. Whereas the linear absorption spectrum consists of a single, broad amide I band, the 2D vibrational spectrum clearly reveals that this band is composed of two amide I transitions, which are assigned to the A and E1 modes. The A–E1 frequency splitting is found to be approximately 10 cm−1. We find that the amide I band is inhomogeneously broadened due to conformational disorder of the helix. The 2D line shapes can be well described using distributions of the dihedral angles (φ,ψ) around their average values with a width of 20°, confirming previous molecular-dynamics studies. Time-resolved 2D measurements show that the conformation fluctuates on a time scale of picoseconds.

Structure, stability and folding of the α-helix

Pauling first described the alpha-helix nearly 50 years ago, yet new features of its structure continue to be discovered, using peptide model systems, site-directed mutagenesis, advances in theory, the expansion of the Protein Data Bank and new experimental techniques. Helical peptides in solution form a vast number of structures, including fully helical, fully coiled and partly helical. To interpret peptide results quantitatively it is essential to use a helix/coil model that includes the stabilities of all these conformations. Our models now include terms for helix interiors, capping, side-chain interactions, N-termini and 3(10)-helices. The first three amino acids in a helix (N1, N2 and N3) and the preceding N-cap are unique, as their amide NH groups do not participate in backbone hydrogen bonding. We surveyed their structures in proteins and measured their amino acid preferences. The results are predominantly rationalized by hydrogen bonding to the free NH groups. Stabilizing side-chain-side-chain energies, including hydrophobic interactions, hydrogen bonding and polar/non-polar interactions, were measured accurately in helical peptides. Helices in proteins show a preference for having approximately an integral number of turns so that their N- and C-caps lie on the same side. There are also strong periodic trends in the likelihood of terminating a helix with a Schellman or alpha L C-cap motif. The kinetics of alpha-helix folding have been studied with stopped-flow deep ultraviolet circular dichroism using synchrotron radiation as the light source; this gives a far superior signal-to-noise ratio than a conventional instrument. We find that poly(Glu), poly(Lys) and alanine-based peptides fold in milliseconds, with longer peptides showing a transient overshoot in helix content.

Design and characterization of asparagine- and lysine-containing alanine-based helical peptides that bind selectively to A.T base pairs of oligonucleotides immobilized on a 27 mhz quartz crystal microbalance.

We have systematically designed and synthesized six kinds of 16-17 mer alanine-based peptides containing four to six lysine (K) and one to four asparagine (N) residues to achieve the selective binding to A.T base pairs of DNA duplexes. The position and number of K and N residues were changed in the helical structure according to common features of the DNA-binding proteins, in which K and N residues are expected to interact electrostatically with phosphate groups and to interact with A.T base pairs by hydrogen bonding, respectively. The time courses of binding of these peptides to dA(30).dT(30) and dG(30).dC(30) duplexes immobilized on a 27 MHz quartz crystal microbalance (QCM) were studied in 10 mM phosphate buffer (pH 7.5) and 40 mM NaCl at 10 degrees C. The maximum binding amounts (Deltam(max)) on a nanogram scale and binding constants (K(a)) could be obtained from the frequency decrease (mass increase) of the oligonucleotide-immobilized QCM. The conformation changes of the peptides upon binding to DNAs were monitored by circular dichroism (CD) spectroscopy. The four properly arranged N residues in the six-cationic K peptide, K6N4(d), resulted in a 5-fold higher affinity for A.T base pairs (K(a) = 5.9 x 10(5) M(-1)) than for G.C base pairs (K(a) = 1.2 x 10(5) M(-1)), and alpha-helices were clearly promoted by the binding to A.T base pairs from CD spectral changes.

Helix Folding of an Alanine-Based Peptide in Explicit Water

Computer simulations using full atomic representations for both the peptide and water molecules were performed to study the folding of a 16-residue alanine-based helical peptide in aqueous solution. Using a recently developed self-guided molecular dynamics (SGMD) method, which was shown to improve the conformational searching efficiency significantly as compared to conventional MD simulation method, reversible folding (folding, unfolding and refolding) of a 16-residue alanine-based synthetic peptide in explicit water at 274 K was successfully accomplished. Consistent with experimental results, the helix was found to be the major secondary structural element in aqueous solution, and among different helix forms, the α-helix is the dominant form. Conformational analysis of our simulation results showed that turns and 310-helices play an essential role in the folding of α-helix. Interestingly, our results showed that the propagation of a helix segment is more frequent at the C-end than at the N-end. In most helix conformations, the backbone carbonyl groups of the peptide prefer to simultaneously form intramolecular hydrogen bonds with the backbone amide groups of the peptide and intermolecular hydrogen bonds with water molecules, indicating water accessibility to the backbone carbonyl groups is crucial for helix formation in water. Therefore, the helical propensities of amino acids may be related to the water accessibility of their backbone groups in helical conformation. Water molecules also function as hydrogen bonding bridges linking helical residue pairs (i, i + n, with n = 3, 4, 5), suggesting a role of water bridges in helix folding.

Molecular Dynamics Simulations of Three-Strand β-Sheet Folding

Traditionally, the empirical force field had great difficulties in simulating β-sheet folding. In the current study, we tested molecular dynamics simulations of β-sheet folding using a solvent-referenced potential. Three available β-sheet-forming synthetic peptides, TWIQNGSTKWYQNGSTKIYT, RGWSVQNGKYTNNGKTTEGR, and VFITSDPGKTYTEVDPGOKILQ, were simulated at their experimental temperatures. From extended initial conformations, all three peptides folded into β-sheet conformations. The calculated ratios of the β-structure from the 100 ns simulations were 26.5%, 17.8%, and 28.5%, respectively, for the three peptides. From different initial conformations, folding into β-sheets was also observed. With the same energy functions, the alanine-based peptide folded into helical conformations, demonstrating the sequence dependence of folding. During simulations, the β-sheet folding is usually initiated by the fast formation of turns. The three-strand compact structures with favorable inter-strand side-chain interactions occur prior to backbone hydrogen bonding. The conversion of the compact structure to β-sheet is slow, and the peptide spends most of the time in these two states. The attractive side-chain interaction is mainly due to the solvent effect, especially the hydrophobic interactions. Without this solvent effect, β-sheet did not form in the simulations. For the first two sequences, the simulations suggest that the experimentally observed structure may include an ensemble of β-sheet structures. For the DP-containing peptide, one β-sheet structure with type II‘ β-turns is much more stable than other structures.

A role of the Trp–His interaction in the conformational switch between α-helix and β-sheet in short alanine-based peptides

The interaction between aromatic residues (Trp, Tyr, and Phe) and a histidine residue (His) is often present in proteins and plays an important role in determining the conformation of peptides and the folding of globular proteins. The role of the Trp–His interaction in the conformation of peptides and the folding of globular proteins has been investigated for a series of alanine-based peptides having a pair of Trp–His in different geometrical spacing and positions. A conformational switch between the α-helix and β-sheet due to the Trp–His interaction was found with the result that the pairs of Trp–His with (i, i + 4) and (i, i + 2) spacing at the C terminus led to α-helix and β-sheet conformations, respectively. The possible factors contributing to the positional effect of the Trp–His interaction are also discussed in the paper. The role of the Trp–His interaction in the conformational switch between the α-helix and β-sheet in peptides is important in the evolution of new protein folding by accumulations of simple mutations in peptides and proteins.