The absence of favorable aromatic interactions between beta-sheet peptides

This paper asks whether interactions between phenylalanine (Phe) residues of the non-hydrogen-bonded cross-strand pairs of antiparallel beta-sheets are important and finds that they are not. Peptides 1a-d [o-BuO-C6H4CO-AA1-Orn(i-PrCO-Hao)-Phe-Ile-AA5-NHMe: 1a AA1, AA5 = Phe; 1b AA1, AA5 = Cha (cyclohexylalanine); 1c AA1 = Phe, AA5 = Cha; 1d AA1 = Cha, AA5 = Phe] provide a sensitive system for probing interactions between phenylalanine residues. These peptides form beta-sheet homodimers in organic solvents. When the homodimers of different peptides are mixed, they equilibrate to form heterodimers, as well as homodimers. The position of the equilibrium reflects the propensity of the first (AA1) and fifth (AA5) amino acids to interact within the non-hydrogen-bonded cross-strand pairs of beta-sheets. Mixing peptides 1a-d in all six possible binary combinations provides a measure of the relative propensities of Phe and Cha to pair. Analysis by 1H NMR spectroscopy of the equilibrium constants in CDCl3 solution reveals no significant preference for the formation of Phe-Phe pairs. The equilibria in all six experiments are essentially statistical (K approximately 4), and no (<0.1 kcal/mol) preference is seen for any pairing combination. A survey of Phe-Phe pairs in the Interchain beta-Sheet Database (http://www.igb.uci.edu/servers/icbs/) corroborates that little significant contact occurs between the aromatic rings in the non-hydrogen-bonded cross-strand pairs of antiparallel beta-sheets at the interface between polypeptide chains. Even though contacts between aromatic rings are favorable when they are of suitable geometry, the energetic price of achieving suitable geometries appears to offset the energetic benefits of such contacts in the current model system, as well as in proteins.     

Aromatic-aromatic interactions in crystal structures of helical peptide scaffolds containing projecting phenylalanine residues

Aromatic-aromatic interactions between phenylalanine side chains in peptides have been probed by the structure determination in crystals of three peptides: Boc-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-OMe, I; Boc-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-OMe, II; Boc-Aib-Ala-Phe-Aib-Phe-Ala-Val-Aib-OMe, III. X-ray diffraction studies reveal that all three peptides adopt helical conformations in the solid state with the Phe side chains projecting outward. Interhelix association in the crystals is promoted by Phe-Phe interactions. A total of 15 unique aromatic pairs have been characterized in the three independent crystal structures. In peptides I and II, the aromatic side chains lie on the same face of the helix at i/i + 4 positions resulting in both intrahelix and interhelix aromatic interactions. In peptide III, the Phe side chains are placed on the opposite faces of the helix, resulting in exclusive intermolecular aromatic interactions. The distances between the centroids of aromatic pair ranges from 5.11 to 6.86 A, while the distance of closest approach of ring carbon atoms ranges from 3.27 to 4.59 A. Examples of T-shaped and parallel-displaced arrangements of aromatic pairs are observed, in addition to several examples of inclined arrangements. The results support the view that the interaction potential for a pair of aromatic rings is relatively broad and rugged with several minima of similar energies, separated by small activation barriers.     

Minimalist protein design: a beta-hairpin peptide that binds ssDNA

A 28-residue beta-hairpin dimer (WKWK)2 with two Trp and two Lys residues on one face of each beta-sheet was shown to form a complex with single-stranded oligonucleotides at low micromolar concentrations. Each beta-hairpin of the dimer contains a cross-strand Trp-Trp pair in a diagonal orientation which has previously been shown to create a cleft for the intercalation of aromatic guests such as adenine (J. Am. Chem. Soc. 2003, 125, 9580). The beta-hairpin dimer binds 5-residue ssDNA sequences 5'-AAAAA-3', 5'-TTTTT-3', and 5'-CCCCC-3' in water with dissociation constants in the range of 12-30 muM. A weak energetic preference for binding to sequence 5'-AAAAA-3' was observed, which is believed to result from stronger stacking interactions between Trp and the adenine base. The interaction of 5'-AAAAA-3' with the Lys and Trp residues of the peptide was evident by NMR, and a 1:1 association was demonstrated. The recognition of an 11-residue ssDNA sequence occurred with a dissociation constant of 3 muM under near-physiological ionic strength and pH, demonstrating that the beta-hairpin dimer binds ssDNA as strongly as many naturally occurring proteins. The salt dependence of the interaction of the 11-residue oligonucleotide with the peptide dimer indicates that Trp-nucleobase stacking interactions contribute about -4 kcal/mol to recognition, which is much greater than the contribution of nonionic interactions in unstructured peptides containing Trp. Moreover, recognition of the ssDNA demonstrated reduced salt dependence relative to the corresponding duplex, resulting in selectivity for ssDNA under high salt conditions. Peptide (WKWK)2 is a relevant mimic of OB-fold (oligonucleotide/oligosaccharide-binding) proteins which bind ssDNA on the surface of a beta-sheet.     

Effect of halogenation on edge-face aromatic interactions in a beta-hairpin peptide: enhanced affinity with iodo-substituents

[reaction: see text] In a model beta-hairpin peptide, we have found that the favorable interaction of cross-strand aromatic rings can be enhanced by up to 1 kcal mol(-1) with halogen substituents. It appears that the polarizability of the halogen atoms accounts for the increase in stability and that there is a direct interaction between the N-terminal phenylalanine and the halogen atom. Thermal denaturation studies indicate that the interaction is enthalpically driven with an associated entropic cost. These findings have relevance to areas of molecular recognition and drug design.     

Experimental measurement of carbohydrate-aromatic stacking in water by using a dangling-ended DNA model system

Protein-carbohydrate recognition is of fundamental importance for a large number of biological processes; carbohydrate-aromatic stacking is a widespread, but poorly understood, structural motif in this recognition. We describe, for the first time, the measurement of carbohydrate-aromatic interactions from their contribution to the stability of a dangling-ended DNA model system. We observe clear differences in the energetics of the interactions of several monosaccharides with a benzene moiety depending on the number of hydroxy groups, the stereochemistry, and the presence of a methyl group in the pyranose ring. A fucose-benzene pair is the most stabilizing of the studied series (-0.4 Kcal mol(-1)) and this interaction can be placed in the same range as other more studied interactions with aromatic residues of proteins, such as Phe-Phe, Phe-Met, or Phe-His. The noncovalent forces involved seem to be dispersion forces and nonconventional hydrogen bonds, whereas hydrophobic effects do not seem to drive the interaction.     

Expected and unexpected results from combined beta-hairpin design elements

A model beta-hairpin dodecapeptide [EFGWVpGKWTIK] was designed by including a favorable D-ProGly Type II' beta-turn sequence and a Trp-zip interaction, while also incorporating a beta-strand unfavorable glycine residue in the N-terminal strand. This peptide is highly folded and monomeric in aqueous solution as determined by combined analysis with circular dichroism and 1H NMR spectroscopy. A peptide representing the folded conformation of the model beta-hairpin [cyclic(EFGWVpGKWTIKpG)] and a linear peptide representing the unfolded conformation [EFGWVPGKWTIK] yield unexpected relative deviations between the CD and 1H NMR spectroscopic results that are attributed to variations in the packing interactions of the aromatic side chains. Mutational analysis of the model beta-hairpin indicates that the Trp-zip interaction favors folding and stability relative to an alternate hydrophobic cluster between Trp and Tyr residues [EFGYVpGKWTIK]. The significance of select diagonal interactions in the model beta-hairpin was tested by rearranging the cross-strand hydrophobic interactions to provide a folded peptide [EWFGIpGKTYWK] displaying evidence of an unusual backbone conformation at the hydrophobic cluster. This unusual conformation does not appear to be a result of the glycine residue in the beta-strand, as replacement with a serine results in a peptide [EWFSIpGKTYWK] with a similar and seemingly characteristic CD spectrum. However, an alternate arrangement of hydrophobic residues with a Trp-zip interaction in a similar position to the parent beta-hairpin [EGFWVpGKWITK] results in a folded beta-hairpin conformation. The differences between side chain packing of these peptides precludes meaningful thermodynamic analysis and illustrates the caution necessary when interpreting beta-hairpin folding thermodynamics that are driven, at least in part, by aromatic cross strand interactions.     

Hydrogen bonding and dipolar interactions between quinolines and organic solvents. Nuclear magnetic resonance and ultraviolet-visible spectroscopic studies

Solvatochromic studies on quinoline (Q), 3-cyanoquinoline (CNQ), 3-bromoquinoline (BrQ) and 8-hydroxyquinoline (OHQ) in pure solvents and alcohol-cyclohexane mixtures have been performed. The results are compared with Proton Nuclear Magnetic Resonance, 1H NMR. studies and AMI calculations. Taft and Kamlet's solvatochromic comparison method was used to disclose solvent effects in pure solvents. These studies shows that the hydrogen bond acceptor ability of the Q ring is diminished and its polarity is increased by the presence of the cyano group in CNQ and the bromo group in BrQ. In OHQ, intramolecular hydrogen bonding has been observed. This interaction is weakened by the interaction with protic solvents. The studies in binary mixtures, alcohol-cyclohexane, show solute-solvent interactions, which compete with solvent self-association in the preferential solvation phenomena. Alcohols with strong ability to self-associate have less preference toward solvation of these compounds. The association constants for solute-ethanol systems were determined by 1H NMR. The results show that the solvent hydrogen bond donor ability is the main factor involved in the interaction with these solutes at the aza aromatic site.     

Stacking and T-shape competition in aromatic-aromatic amino acid interactions

The potential of mean force of interacting aromatic amino acids is calculated using molecular dynamics simulations. The free energy surface is determined in order to study stacking and T-shape competition for phenylalanine-phenylalanine (Phe-Phe), phenylalanine-tyrosine (Phe-Tyr), and tyrosine-tyrosine (Tyr-Tyr) complexes in vacuo, water, carbon tetrachloride, and methanol. Stacked structures are favored in all solvents with the exception of the Tyr-Tyr complex in carbon tetrachloride, where T-shaped structures are also important. The effect of anchoring the two alpha-carbons (C(alpha)) at selected distances is investigated. We find that short and large C(alpha)-C(alpha) distances favor stacked and T-shaped structures, respectively. We analyze a set of 2396 protein structures resolved experimentally. Comparison of theoretical free energies for the complexes to the experimental analogue shows that Tyr-Tyr interaction occurs mainly at the protein surface, while Phe-Tyr and Phe-Phe interactions are more frequent in the hydrophobic protein core. This is confirmed by the Voronoi polyhedron analysis on the database protein structures. As found from the free energy calculation, analysis of the protein database has shown that proximal and distal interacting aromatic residues are predominantly stacked and T-shaped, respectively.     

Spontaneous self-assembly of aromatic cyclic dipeptide into fibre bundles with high thermal stability and propensity for gelation

Cyclic dipeptides (CDPs) are the simplest members of cyclic peptides exhibiting exceptional rigidity and stability. A detailed study on the spontaneous self-assembly of aromatic CDP (Phe-Phe) into fibre bundles with 1–2 μm thick and several millimetres long is described. These fibre bundles exhibit the structural hierarchy that is found in natural fibres such as spider silk and collagen. The solubility studies in various solvents provided more insights into the existence and nature of fibre bundles. Fibre bundles transform to gel in chloroform at critical concentration of added trifluoroacetic acid. Thermogravimetric analysis data indicated high thermal stability with multiple transitions attributed to the existence of structural hierarchy in fibre bundles. NMR studies revealed that aromatic π–π interactions along with intermolecular (N–H…O) hydrogen-bonded molecular chains are mainly responsible for the formation as well as observed high stability of fibre bundles. This detailed study of structural hierarchy, solubility, gelation and thermal stability demonstrates the robustness of aromatic CDP (Phe-Phe) to form fibre bundles.     

Molecular conformation and packing of peptide beta hairpins in the solid state: structures of two synthetic octapeptides containing 1-aminocycloalkane-1-carboxylic acid residues at the i+2 position of the beta turn

Peptide beta-hairpin formation is facilitated by centrally positioned D-Pro-Xxx segments. The synthetic peptides Boc-Leu-Phe-Val-D-Pro-Ac(6)c-Leu-Phe-Val-OMe (1) and Boc-Leu-Phe-Val-D-Pro-Ac(8)c-Leu-Phe-Val-OMe (2) were synthesized in order to explore the role of bulky 1-aminocycloalkane-1-carboxylic acid residues (Ac(n)c, where n is the number of carbon atoms in the ring), at the i+2 position of the nucleating beta turn in peptide beta hairpins. Peptides 1 and 2 crystallize in the monoclinic space group P2(1) with two molecules in the asymmetric unit. The crystal structures of 1 and 2 provide conformational parameters for four peptide hairpin molecules. In all cases, the central segments adopts a type II' beta-turn conformation, and three of the four possible cross-strand hydrogen bonds are observed. Fraying of the hairpins at the termini is accompanied by the observation of NHpi interaction between the Leu(1)NH group and Phe(7) aromatic group. Cross strand stabilizing interactions between the facing residues Phe(2) and Phe(7) are suggested by the observed orientation of aromatic rings. Anomalous far-UV CD spectra observed in solution suggest that close proximity of the Phe rings is maintained even in isolated molecules. In both peptides 1 and 2, the asymmetric unit consists of approximately orthogonal hairpins, precluding the formation of a planar beta-sheet arrangement in the solid state. Solvent molecules, one dioxane and one water in 1, three water molecules in 2, mediate peptide association. A comparison of molecular conformation and packing motifs in available beta-hairpin structures permits delineation of common features. The crystal structures of beta-hairpin peptides provide a means of visualizing different modes of beta-sheet packing, which may be relevant in developing models for aggregates of polypeptides implicated in disease situations.     

Influence of the dynamic positions of cations on the structure of the DNA minor groove: sequence-dependent effects

Different models for minor groove structures predict that the conformation is essentially fixed by sequence and has an influence on local ion distribution or alternatively that temporal positions of ions around the minor groove can affect the structure if they neutralize cross-strand phosphate charges. Our previous studies show that the minor groove in an AATT dodecamer responds to local sodium ion positions and is narrow when ions neutralize cross-strand phosphate-phosphate charges [J. Am. Chem. Soc. 2000, 122, 10513-10520]. Previous results from a number of laboratories have shown that G-tracts often have a wider minor groove than A-tracts, but they do not indicate whether this is due to reduced flexibility or differences in ion interactions. We have undertaken a molecular dynamics study of a d(TATAGGCCTATA) duplex to answer this question. The results show that the G-tract has the same amplitude of minor groove fluctuations as the A-tract sequence but that it has fewer ion interactions that neutralize cross-strand phosphate charges. These results demonstrate that differences in time-average groove width between A- and G-tracts are due to differences in ion interactions at the minor groove. When ions neutralize the cross-strand phosphates, the minor groove is narrow. When there are no neutralizing ion interactions, the minor groove is wide. The population of structures with no ion interactions is larger with the GGCC than with the AATT duplex, and GGCC has a wider time-average minor groove in agreement with experiment.     

The interaction of carbohydrates and amino acids with aromatic systems studied by density functional and semi-empirical molecular orbital calculations with dispersion corrections

Density functional theory (DFT-D) and semi-empirical (PM3-D) methods having an added dispersion correction have been used to study stabilising carbohydrate-aromatic and amino acid-aromatic interactions. The interaction energy for three simple sugars in different conformations with benzene, all give interaction energies close to 5 kcal mol(-1). Our original parameterization of PM3 (PM3-D) seriously overestimates this value, and has prompted a reparametrization which includes a modified core-core interaction term. With two additional parameters, the carbohydrate complexes, as well as the S22 data set, are well reproduced. The new PM3 scheme (PM3-D*) is found to describe the peptide bond-aromatic ring interactions accurately and, together with the DFT-D method, it is used to investigate the interaction of six amino acids with pyrene. Whilst the peptide backbone can adopt both stacked and T-shaped structures in the complexes with similar interaction energies, there is a preference for the unsaturated ring to adopt a stacked structure. Thus, peptides in which the latter interactions are maximised are likely to be the most effective for the functionalisation of carbon nanotubes.     

Self-association of the anionic form of the DNA-binding anticancer drug mithramycin

The aqueous-phase self-association of mithramycin (MTR), an aureolic acid anticancer antibiotic, has been studied using different spectroscopic techniques such as absorption, fluorescence, circular dichroism, and 1H nuclear magnetic resonance spectroscopy. Results from these studies indicate self-association of the anionic antibiotic at pH 8.0 over a concentration range from micromolar to millimolar. These results could be ascribed to the following steps of self-association: M + M left arrow over right arrow M2, M2 + M left arrow over right arrow M3, and M3 + M left arrow over right arrow M4, where M, M2, M3, and M4 represent the monomer, dimer, trimer, and tetramer of mithramycin, respectively. Dynamic light scattering and isothermal titration calorimetry studies also support aggregation. In contrast, an insignificant extent of self-association is found for the neutral drug (at pH 3.5) and the [(MTR)2Mg2+] complex (at pH 8.0). Analysis of 2D NMR spectra of 1 mM MTR suggests that the sugar moieties play a role in the self-association process. Self-association of the drug might occur either via hydrophobic interaction of the sugar residues among themselves or water-mediated hydrogen bond formation between sugar residue(s). On the other hand, absence of a significant upfield shift of the aromatic protons from 100 microM to 1 mM MTR suggests against the possibility of stacking interactions between the aromatic rings as a stabilizing force for the formation of the dimer and higher oligomers.     

Model chemistry calculations of thiophene dimer interactions: origin of pi-stacking

The intermolecular interaction energies of thiophene dimers have been calculated by using an aromatic intermolecular interaction (AIMI) model (a model chemistry for the evaluation of intermolecular interactions between aromatic molecules). The CCSD(T) interaction energy at the basis set limit has been estimated from the MP2 interaction energy near the basis set limit and the CCSD(T) correction term obtained by using a medium-size basis set. The calculated interaction energies of the parallel and perpendicular thiophene dimers are -1.71 and -3.12 kcal/mol, respectively. The substantial attractive interaction in the thiophene dimer, even where the molecules are well separated, shows that the major source of attraction is not short-range interactions such as charge transfer but rather long-range interactions such as electrostatic and dispersion. The inclusion of electron correlation increases the attraction significantly. The dispersion interaction is found to be the major source of attraction in the thiophene dimer. The calculated total interaction energy of the thiophene dimer is highly orientation dependent. Although electrostatic interaction is substantially weaker than dispersion interaction, it is highly orientation dependent, and therefore electrostatic interaction play an important role in the orientation dependence of the total interaction energy. The large attractive interaction in the perpendicular dimer is the cause of the preference for the herringbone structure in the crystals of nonsubstituted oligothiophenes (alpha-terthienyls), and the steric repulsion between the beta-substituents is the cause of the pi-stacked structure in the crystals of some beta-substituted oligothiophenes.     

Stabilization of beta-hairpin peptides by salt bridges: role of preorganization in the energetic contribution of weak interactions

A model beta-hairpin peptide has been used to investigate the context-dependent contribution of cross-strand Lys-Glu interactions to hairpin stability. We have mutated two Ser-Lys interstrand pairs to Glu-Lys salt bridges, one close to the type I' Asn-Gly turn sequence (Ser6 --> Glu), and one close to the N- and C-termini (Ser15 --> Glu). Each individual interaction contributes approximately 1.2-1.3 kJ mol(-1) to stability; however, introducing the two salt bridges simultaneously produces a much larger overall contribution (-3.6 kJ mol(-1)) consistent with an important role for preorganization and cooperativity in determining the energetics of weak interactions. We compare and contrast CD and NMR data on the highly folded hairpin with the two Glu-Lys pairs to shed light on the nature of the folded state in water. We show that large cosolvent-induced changes in the CD spectrum, in contrast with the modest effects observed on Halpha chemical shifts, support a hydrophobically collapsed entropy-driven conformation in water whose stability is modulated by long-range Coulombic interactions from the Glu-Lys interactions. Cosolvent stabilizes the structure enthalpically, as is evident from CD melting profiles.     

Supramolecular self-organization in PEO-modified C60 fullerene/water solutions: influence of polymer molecular weight and nanoparticle concentration

Utilizing a first-principles-based coarse-grained implicit solvent model, we have investigated the self-association of C(60) fullerenes that have been symmetrically modified with six grafted poly(ethylene oxide) (PEO) chains in aqueous solution. Despite the highly symmetric nature of the pair interactions between PEO-grafted fullerenes, their supramolecular assemblies are highly anisotropic and resemble the linear clusters formed in Stockmayer fluids. The dipole-like interaction between these symmetrically modified fullerenes results from the shielding of the C(60) fullerenes by PEO, favoring the addition of more PEO-grafted fullerenes to the linear clusters at the relatively unprotected ends. At low nanoparticle concentrations, self-association is dominated by the formation of stable dimers and trimers resulting from fullerene-fullerene contact and favorable PEO-fullerene interactions. With increasing nanoparticle concentration, larger clusters become increasingly probable. The molecular weight of the PEO tethers can be treated as a temperature-like analogue, with a reduction in average cluster size with increasing chain length due to increased steric repulsion, which is qualitatively similar to effects observed in Stockmayer fluids with increasing temperature. The role of PEO in supramolecular self-organization in PEO-modified C(60) fullerene/water solutions is complex, contributing not only to steric stabilization but also to favorable energetic interactions, nanoparticle shielding, and depletion-driven aggregation.     

NMR analysis of aromatic interactions in designed peptide beta-hairpins

Designed octapeptide beta-hairpins containing a central (D)Pro-Gly segment have been used as a scaffold to place the aromatic residues Tyr and Trp at various positions on the antiparallel beta-strands. Using a set of five peptide hairpins, aromatic interactions have been probed across antiparallel beta-sheets, in the non-hydrogen bonding position (Ac-L-Y-V-(D)P-G-L-Y/W-V-OMe: peptides 1 and 2), diagonally across the strands (Boc-Y/W-L-V-(D)P-G-W-L-V-OMe: peptides 3 and 6), and along the strands at positions i and i + 2 (Boc-L-L-V-(D)P-G-Y-L-W-OMe: peptide 4). Two peptides served as controls (Boc-L-L-V-(D)P-G-Y-W-V-OMe: peptide 5; Boc-L-Y-V-(D)P-G-L-L-V-OMe: peptide 7) for aromatic interactions. All studies have been carried out using solution NMR methods in CDCl(3) + 10% DMSO-d(6) and have been additionally examined in CD(3)OH for peptides 1 and 2. Inter-ring proton-proton nuclear Overhauser effects (NOEs) and upfield shifted aromatic proton resonances have provided firm evidence for specific aromatic interactions. Calculated NMR structures for peptides 1 and 2, containing aromatic pairs at facing non-hydrogen bonded positions, revealed that T-shaped arrangements of the interacting pairs of rings are favored, with ring current effects leading to extremely upfield chemical shifts and temperature dependences for specific aromatic protons. Anomalous far-UV CD spectra appeared to be a characteristic feature in peptides where the two aromatic residues are spatially proximal. The observation of the close approach of aromatic rings in organic solvents suggests that interactions of an electrostatic nature may be favored. This situation may be compared to the case of aqueous solutions, where clustering of aromatic residues is driven by solvophobic (hydrophobic) forces.     

Comparison of C-H...pi and hydrophobic interactions in a beta-hairpin peptide: impact on stability and specificity

We have examined the impact of C-H...pi and hydrophobic interactions in the diagonal position of a beta-hairpin peptide through comparison of the interaction of Phe, Trp, or Cha (cyclohexylalanine) with Lys or Nle (norleucine). NMR studies, including NOESY and chemical shift perturbation studies, of the Lys side chain indicates that Lys interacts in a specific geometry with Phe or Trp through the polarized C epsilon. In contrast, Nle does not interact in a specific manner with the diagonal aromatic residue. Thermal denaturation provides additional support that Lys and Nle interact in fundamentally different manners. Folding of the peptide with a diagonal Trp...Lys interaction was found to be enthalpically driven, whereas the peptide with a diagonal Trp...Nle interaction displayed cold denaturation, as did the control peptide with a diagonal Cha...Nle interaction, indicating different driving forces for interaction of Lys and Nle with Trp. These findings have significant implications for specificity in protein folding and de novo protein design.     

Context-dependence of the contribution of disulfide bonds to beta-hairpin stability

Incorporation of disulfide bonds to stabilize protein and peptide structures is not always a successful strategy. To advance current knowledge on the contribution of disulfide bonds to beta-hairpin stability, a previously reported beta-hairpin-forming peptide was taken as a template to design a series of Cys-containing peptides. The conformational behavior of these peptides in their oxidized, disulfide-cyclized peptides, and reduced, linear peptides, was investigated on the basis of NMR parameters: NOEs, and 1H and 13C chemical shifts. We found that the effect of disulfide bonds on beta-hairpin stability depends on its location within the beta-hairpin structure, being very small or even destabilizing when connecting two hydrogen-bonded facing residues. When the disulfide bond is linking non-hydrogen-bonded facing residues, we estimated that its contribution to the free-energy change of beta-hairpin folding is approximately -1.0 kcal mol(-1). This value is larger than those reported for most beta-hairpin-stabilizing cross-strand side-chain-side-chain interactions, except for some aromatic-aromatic interactions, in particular the Trp-Trp one, and the cation-pi interaction between Trp and the non-natural methylated Arg/Lys. As disulfide bonds are frequently used to stabilize peptide conformations, our conclusions can be useful for peptide, peptidomimetic, and protein design, and may even extend to other chemical cross-links.     

Proton-coupled electron transfer in a biomimetic peptide as a model of enzyme regulatory mechanisms

Proton-coupled electron-transfer reactions are central to enzymatic mechanism in many proteins. In several enzymes, essential electron-transfer reactions involve oxidation and reduction of tyrosine side chains. For these redox-active tyrosines, proton transfer couples with electron transfer, because the phenolic pKA of the tyrosine is altered by changes in the tyrosine redox state. To develop an experimentally tractable peptide system in which the effect of proton and electron coupling can be investigated, we have designed a novel amino acid sequence that contains one tyrosine residue. The tyrosine can be oxidized by ultraviolet photolysis or electrochemical methods and has a potential cross-strand interaction with a histidine residue. NMR spectroscopy shows that the peptide forms a beta-hairpin with several interstrand dipolar contacts between the histidine and tyrosine side chains. The effect of the cross-strand interaction was probed by electron paramagnetic resonance and electrochemistry. The data are consistent with an increase in histidine pKA when the tyrosine is oxidized; the effect of this thermodynamic coupling is to increase tyrosyl radical yield at low pH. The coupling mechanism is attributed to an interstrand pi-cation interaction, which stabilizes the tyrosyl radical. A similar interaction between histidine and tyrosine in enzymes provides a regulatory mechanism for enzymatic electron-transfer reactions.