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.

Simple analytical functions consisting of electrostatic, polarization, Lennard-Jones or modified Lennard-Jones, and cavity terms are proposed to express the potentials of mean force analytically for spherical particles interacting in water. The cavity term was expressed either through the molecular-surface area of the solute or by using the Gaussian-overlap model of hydrophobic hydration developed in paper 1 of this series. The analytical expressions were fitted to the potentials of mean force of a methane homodimer, heterodimers composed of a methane molecule, and an ammonium cation or a chloride anion, respectively, and dimers consisting of two chloride anions, two ammonium cations, or a chloride ion and an ammonium cation. The potentials of mean force for these dimers were determined by umbrella-sampling molecular dynamics simulations with the AMBER 7.0 force field with TIP3P water either in our earlier work or in this work. For all systems, the analytical formulas fitted the potentials of mean force very well. However, using the molecular-surface area to express the cavity term provided a good fit only when the nonbonded interactions were expressed by an all-repulsive modified Lennard-Jones potential but also resulted in non-physical values of some of the parameters. Conversely, the use of our new Gaussian-overlap-based expression for the cavity term provided a good fit to the potentials of mean force (PMFs) with Lennard-Jones nonbonded potential, and the values of all parameters were physically reasonable.