The alanine-based peptide Ac-XX(A)7OO-NH2, referred to as XAO (where X, A, and O denote diaminobutyric acid, alanine, and ornithine, respectively), has recently been proposed to possess a well defined polyproline II (P II) conformation at low temperatures. Based on the results of extensive NMR and CD investigations combined with theoretical calculations, reported here, we present evidence that, on the contrary, this peptide does not have any significant amount of organized P II structure but exists in an ensemble of conformations with a distorted bend in the N-and C-terminal regions. The conformational ensemble was obtained by molecular dynamics͞simulated annealing calculations using the AMBER suite of programs with time-averaged distance and dihedralangle restraints obtained from rotating-frame nuclear Overhauser effect (ROE) volumes and vicinal coupling constants 3 JHN⌯␣, respectively. The computed ensemble-averaged radius of gyration R g (7.4 ؎ 1.0) Å is in excellent agreement with that measured by small-angle x-ray scattering (SAXS) whereas, if the XAO peptide were in the P II conformation, Rg would be 11.6 Å. Depending on the pH, peptide concentration, and temperature, the CD spectra of XAO do or do not possess the maximum with positive ellipticity in the 217-nm region, which is characteristic of the P II structure, reflecting a shifting conformational equilibrium rather than an all-or-none transition. The ''P II conformation'' should, therefore, be considered as one of the accessible conformational states of individual amino acid residues in peptides and proteins rather than as a structure of most of the chain in the early stage of folding.CD spectroscopy ͉ molecular dynamics ͉ NMR spectroscopy ͉ polyproline type II structure ͉ unfolded state of proteins T he structures of proteins under denaturing conditions have received considerable attention from experimentalists (1-19) and theoreticians (20-32). The dominating model has been the statistical coil (erroneously called a random coil) developed by Flory (20) and corroborated by Tanford (1). However, recent work (9, 10) suggests that the end-to-end distances in denatured proteins need not conform to statistical-coil polymer-like distributions.Recently, Kallenbach and coworkers (11) carried out CD and NMR studies of an alanine-based peptide Ac-XX(A) 7 OO-NH 2 (where X, A, and O denote diaminobutyric acid, alanine, and ornithine, respectively), hereafter referred to as XAO, and proposed that XAO has a dominant polyproline II (P II ) structure at 2°C and that the content of the ␤-strand is increased by Ϸ10% at 55°C relative to that at 2°C. Based on the temperature dependence of the CD spectra of XAO, they stated that there is a transition from the low-temperature P II conformation to a ␤-structure; in other words, the P II conformation at low temperatures is the state of most residues of the whole peptide rather than the state that pertains to a subset of individual amino acid residues. Subsequently, the XAO peptide has been considered as a model of P II stru...

It has been suggested that the alanine-based peptide with sequence Ac-XX-[A](7)-OO-NH(2), termed XAO where X denotes diaminobutyric acid and O denotes ornithine, exists in a predominantly polyproline-helix (P(II)) conformation in aqueous solution. In our recent work, we demonstrated that this "polyproline conformation" should be regarded as a set of local conformational states rather than as the overall conformation of the molecule. In this work, we present further evidence to support this statement. Differential scanning calorimetry measurements showed only a very small peak in the heat capacity of an aqueous solution of XAO at 57 degrees C, whereas the suggested transition to the P(II) structure should occur at approximately 30 degrees C. We also demonstrate that the temperature dependence of the (3)J(HNHalpha) coupling constants of the alanine residues can be explained qualitatively in terms of Boltzmann averaging over all local conformational states; therefore, this temperature dependence proves that a conformational transition does not occur. Canonical MD simulations with the solvent represented by the generalized Born model, and with time-averaged NMR-derived restraints, demonstrate the presence of an ensemble of structures with a substantial amount of local P(II) conformational states but not with an overall P(II) conformation.

The circular dichroism (CD) and Fourier transform infrared (FTIR) methods were applied to the conformational studies of alanine-rich peptide Ac-K-[A]11-KGGY-NH2 (where K is lysine, A is alanine, G is glycine and Y is tyrozyne) in water, methanol (MeOH) and trifluoroethanol (TFE). The analysis of CD-spectra of the peptide in water at different concentrations revealed that the secondary structure content depends on the peptide concentration and pH of the solution. The increase of the peptide concentration causes a decrease of alpha-helix content and, simultaneously, an increase of beta-sheet structure, while the unordered structure is the predominant one. Additional elements are discovered in MeOH and TFE but alpha-helix and beta-turns predominate. Moreover, in these solutions the percentage content of the secondary structure does not depend on the temperature. FTIR measurements, carried out at higher peptide concentration (about one order of magnitude) than these CD measurements mentioned above, revealed that in water solution the solid state beta-sheet, and aggregated structures, dominate. However, in TFE the most abundant are alpha-helix and beta-turns structures. The thioflavine T assay showed the tendency of the studied peptide for aggregate.

We present results of conformational studies by Circular dichroism and NMR spectroscopy, differential scanning calorimetry, and molecular dynamics, of three alanine-based peptides: Ac-KK-(A)(7)-KK-NH(2) (KAK), Ac-OO-(A)(7)-DD-NH(2) (OAD), and Ac-KK-(A)(7)-EE-NH(2) (KAE), where A, K, O, D, and E, denote alanine, lysine, ornithine, aspartic acid, and glutamic acid residues, respectively. For OAD and KAE, canonical MD simulations with time-averaged NMR-derived restraints demonstrate the presence of an ensemble of structures with a variety of conformational states (polyproline II, alpha-helical, alpha', and extended, turn); for KAK the conformational states are predominantly polyproline II and extended. The OAD peptide exhibits a bent shape with its ends close to each other, whereas KAK and KAE are more extended. The bent shape was also observed in our earlier study of the Ac-XX-(A)(7)-OO-NH(2) (XAO) peptide, where X denotes the diaminobutyric acid residue; therefore, the shape seems to depend on the size of the charged side chains at the ends of the alanine sequence and not on their kind. This suggests that the bent shape of the alanine sequence is formed to enable screening of this nonpolar sequence from the solvent by sufficiently short charged side chains. As in our previous study of the XAO peptide, no long polyproline II segments were observed.

We compared the ability of two theoretical methods of pH-dependent conformational calculations to reproduce experimental potentiometric-titration curves of two models of peptides: Ac-K 5 -NHMe in 95% methanol (MeOH)/5% water mixture and Ac-XX(A) 7 OO-NH 2 (XAO) (where X is diaminobutyric acid, A is alanine, and O is ornithine) in water, methanol (MeOH) and dimethylsulfoxide (DMSO), respectively. The titration curve of the former was taken from the literature, and the curve of the latter was determined in this work. The first theoretical method involves a conformational search using the Electrostatically Driven Monte Carlo (EDMC) method with a low-cost energy function (ECEPP/3 plus the SRFOPT surface-solvation model, assumming that all titratable groups are uncharged) and subsequent reevaluation of the free energy at a given pH with the Poisson-Boltzmann equation, considering variable protonation states. In the second procedure, MD simulations are run with the AMBER force field and the Generalized-Born model of electrostatic solvation, and the protonation states are sampled during constant-pH MD runs. In all three solvents, the first pK a of XAO is strongly downshifted compared to the value for the reference compounds (ethyl amine and propyl amine, respectively); the water and methanol curves have one, and the DMSO curve has two jumps characteristic of remarkable differences in the dissociation constants of acidic groups. The predicted titration curves of Ac-K 5 -NHMe are in good agreement with the experimental ones; better agreement is achieved with the MD-based method. The titration curves of XAO in methanol and DMSO, calculated using the MD-based approach, trace the shape of the experimental curves, reproducing the pH jump, while those calculated with the EDMC-based approach, and the titration curve in water calculated using the MD-based approach, have smooth shapes characteristic of the titration of weak multifunctional acids with small differences between the dissociation constants. Nevertheless, quantitative agreement between theoretically predicted and experimental titration curves is not achieved in all three solvents even with the MD-based approach which is manifested by a smaller pH range of the calculated titration curves with respect to the experimental curves. The poorer agreement obtained for water than for the non-aqueous solvents suggests a significant role of specific solvation in water, which cannot be accounted for by the meanfield solvation models.