There is growing appreciation of the functional relevance of unfolded proteins in biology. However, unfolded states of proteins have proven inaccessible to the usual techniques for high-resolution structural and energetic characterization. Unfolded states are still generally conceived of as statistical coils, based on the pioneering work of Flory [(1969 T he process by which a protein acquires its native structure is among the most complex reactions known, and challenges remain in defining the nature of the transition state(s), the structure and role of intermediates, and the properties of the starting ensemble of states (1-4). According to Flory (5) and Tanford (6), unfolded proteins can be represented as statistical random coils, in which a given residue has no strong preference for any specific conformation. Confirming earlier conclusions by Tiffany and Krimm (7-9), recent evidence from a variety of spectroscopic probes (10-22), theoretical studies (23-34), and coil library surveys (35-43) consistently point to a major role for the polyproline II (PPII, ⌽ ϭ Ϫ75°, ⌿ ϭ ϩ145°) conformation in oligo-Ala (for review, see ref. 3 and related articles in the same volume), oligo-Lys, and oligo-Glu peptides (44). We have reported that in a seven-Ala peptide model PPII converts to a ␤-like structure with increasing temperature (13). These findings raise several important questions regarding the structure of unfolded proteins: Although alanine is arguably a reasonable model for the unperturbed peptide backbone, is PPII also present in unfolded peptide chains composed of nonalanine nonproline residues? Is there an intrinsic PPII propensity for each individual side chain? If PPII is in equilibrium with ␤-structure, is there a correlation between scales of PPII propensity and analogous ␤-sheet scales? To what extent is PPII sequence and context dependent?Here, we address these questions by analyzing a series of end-blocked host pentapeptides AcGGXGGNH 2 , where X denotes 19 natural amino acids except glycine. Members of the series are found to differ in their extent of PPII conformation as determined by NMR and CD spectroscopy. Our results lead to the following conclusions: PPII is present as a dominant conformation in the majority of AcGGXGGNH 2 peptides. Different side chains show distinct propensities to adopt PPII in these unfolded molecules. Importantly, we find an inverse correlation between the determined PPII scale and the ␤-sheet-forming propensities derived from a zinc-finger model system (45) when 18 aa (except Gly and Pro) are divided into two groups: one, the nonpolar ␤-branched and bulky aromatic residues (VIWFY) and the other all of the remaining side chains. Finally, we find a correlation between our PPII scale in AcGGXGGNH 2 and a PPII scale derived from alternative model peptides such as AcPPPXPPPGYNH 2 (46). Still there are indications that the PPII scale is likely to be sequence and context dependent (47). Materials and MethodsPeptides Synthesis and Purification. Peptides were assembled on Rink Amide res...

The finding that short alanine peptides possess a high fraction of polyproline II (PII) structure (⌽ ‫؍‬ ؊75°, ⌿ ‫؍‬ ؉145°) at low temperature has broad implications for unfolded states of proteins. An important question concerns whether or not this structure is locally determined or cooperative. We have monitored the conformation of alanine in a series of model peptides AcGG(A)n-GGNH2 (n ‫؍‬ 1-3) over a temperature range from ؊10°C to ؉80°C. Use of 15 N-labeled alanine substitutions makes it possible to measure 3 J␣N coupling constants accurately over the full temperature range. Based on a 1D next-neighbor model, the cooperative parameter of PII nucleation is evaluated from the coupling constant data. The finding that is close to unity (1 ؎ 0.2) indicates a noncooperative role for alanine in PII structure formation, consistent with statistical surveys of the Protein Data Bank that suggest that most PII structure occurs in isolated residues. Lack of cooperativity in these models implies that hydration effects that influence PII conformation in water are highly localized. Using a nuclear Overhauser effect ratio strategy to define the alanine ⌿ angle, we estimate that, at 40°C, the time-averaged alanine conformation (⌽ ‫؍‬ ؊80°, ⌿ ‫؍‬ ؉170°) deviates from canonical PII structure, indicating that PII melts at high temperature. Thus, the high-temperature state of short alanine peptides seems to be an unfolded ensemble with higher distribution in the extended ␤ structure basin, but not a coil. Rather than the statistical coil expected from analysis of the dimensions of unfolded proteins in solvent such as guanidine hydrochloride (3), unfolded proteins seem to be locally ordered, with substantial amounts of polyproline II (PII) structure. PII is a left-handed 3 1 helical structure occupied by collagen and peptides containing proline with dihedral angles ⌽ ϭ Ϫ75°and ⌿ ϭ ϩ145°. Unfolded structures of peptides in native folding conditions can be studied by using chains too short to nucleate normal ␣-helix or ␤-strand structure; these chains have the advantage of being amenable to theoretical as well as experimental analysis. A wealth of spectroscopic and theoretical evidence indicates that oligomers of two or three alanines, the simplest models for the peptide backbone, have predominantly PII conformation in water (4-9). Similarly, the single alanine residue in the pentapeptide AcGGAGGNH 2 (GGAGG) is predominantly PII in water at 20°C (10). The glycines flanking alanine in this pentapeptide have the maximal possible conformational freedom for peptide bonds. This and related model peptides have been used to represent unfolded backbone protein structure for many years (11-13). The solvent trifluoroethanol (TFE) has been widely used as an ␣-helix-stabilizing agent (14,15). When the solvent is changed from water to neat TFE, PII structure disappears, judging from the CD spectrum (16). NMR analysis of the alanine dihedral angles of GGAGG in TFE shows that an internally H-bonded C7eq turn conformation is favored over PII or ␣ ...