Truncation and mutation of a poorly folded 39-residue peptide has produced 20-residue constructs that are >95% folded in water at physiological pH. These constructs optimize a novel fold, designated as the 'Trp-cage' motif, and are significantly more stable than any other miniprotein reported to date. Folding is cooperative and hydrophobically driven by the encapsulation of a Trp side chain in a sheath of Pro rings. As the smallest protein-like construct, Trp-cage miniproteins should provide a testing ground for both experimental studies and computational simulations of protein folding and unfolding pathways. Pro Trp interactions may be a particularly effective strategy for the a priori design of self-folding peptides.

The Trp-cage, as the smallest miniprotein, remains the subject of numerous computational and experimental studies of protein folding dynamics and pathways. The original Trp-cage (NLYIQWLKDGGPSSGRPPPS, Tm = 42 degrees C) can be significantly stabilized by mutations; melting points as high as 64 degrees C are reported. In helical portions of the structure, each allowed replacement of Leu, Ile, Lys or Ser residues by Ala results in a 1.5 (+/-0.35) kJ/mol fold stabilization. No changes in structure or fluxionality of the core results upon stabilization. Contrary to the initial hypothesis, specific Pro/Trp interactions are not essential for core formation. The entropic advantage of Pro versus Ala (DeltaDeltaS(U) = 11 +/- 2 J/mol K) was measured at the solvent-exposed P17 site. Pro-Ala mutations at two of the three prolines (P12 and P18) that encage the indole ring result in less fold destabilization (2.3-3.4 kJ/mol). However, a P19A mutation reduces fold stability by 16 kJ/mol reflecting a favorable Y3/P19 interaction as well as Trp burial. The Y3/P19 hydrophobic staple interaction defines the folding motif as an 18-residue unit. Other stabilizing features that have been identified include a solvent-exposed Arg/Asp salt bridge (3.4-6 kJ/mol) and a buried H-bonded Ser side chain ( approximately 10 kJ/mol).

A detailed analysis of backbone amide NH chemical shift temperature gradients (∆δ/∆T values) for proteins and highly cross-linked peptides reveals that hydrogen-bonded exchange-protected NHs are characterized by ∆δ/∆T values of -2.0 ( 1.4 ppb/°C while exposed NHs typically display gradients of -6.0 f -8.5 ppb/°C; however, numerous exceptions to these generalizations occur. For partially folded peptides (rather than proteins), exceptions are more common than concordance with this rule; ∆δ/∆T values ranging from -28 to +12 ppb/°C have been observed. In the case of the peptide systems for which exchange protection data is available, the common practice of assuming that a ∆δ/∆T value less negative than -4 ppb/°C indicates that the NH is sequestered from solvent is shown to have zero predictive validity. The analysis of the data for partially folded peptides, protein fragments, and other peptides which are expected to display minimal structuring reveals a significant correlation between ∆δ/∆T and the deviation of δ NH from the random coil reference shift. The analysis was facilitated by plotting NH chemical shift deviations (NH-CSD) Versus the ∆δ/∆T values. Using such plots, slow-exchanging hydrogen-bonded sites in proteins can be determined with much higher confidence than using the value of the gradient alone. For peptides, the occurrence of large shift deviations and abnormal gradients are diagnostic for partial structuring at lower temperatures which becomes increasingly randomized on warming. A good correlation coefficient (R g 0.75) for NH-CSD and ∆δ/∆T values indicates that essentially all of the NH shift deviation from reference values is due to the concerted formation of a single structured state on cooling. Correlation coefficients greater than 0.95 were observed for both helix and -hairpin forming peptides. The slope of the correlation plot (parts per thousand/°C) is a measure of the decrease in the population of the structured state upon warming. A detailed model which rationalizes the effects of conformational equilibria upon NH shifts is presented. A positive ∆Cp for unfolding is required to rationalize the linearity of δ NH with temperature that is routinely observed for partially structured peptides. This analysis suggests that ordered states of short peptides achieve significant populations in water only when the hydrophobic effect favors the structured state. This conclusion is pertinent to the current questions concerning the temporal sequence of secondary Versus tertiary structure formation during protein folding. Further, it is suggested that the use of NMR parameters (scalar and dipolar couplings) to derive the structural preferences of protein fragments which might serve a "seeding" role in the folding pathway is justified only when the CSD/gradient plot displays both a correlation coefficient greater than 0.70 and significant NH-CSD values (|CSD| > 0.3).With the development of 2D NMR methods, peptide/protein structure elucidation has been dominated by methods based on NOE-derived distance const...

Exendin-4, a 39 amino acid peptide originally isolated from the oral secretions of the lizard Heloderma suspectum, has been shown to share certain activities with glucagon-like-peptide-1 (GLP-1), a 30 amino acid peptide. We have determined the structuring preferences of exendin-4 and GLP-1 by NMR in both the solution and dodecylphosphocholine (DPC) micelle-associated states. Based on both chemical shift deviations and the pattern of intermediate range NOEs, both peptides display significant helicity from residue 7 to residue 28 with greater fraying at the N-terminus. Thornton and Gorenstein [(1994) Biochemistry 33, 3532-3539] reported that the presence of a flexible, helix-destabilizing, glycine at residue 16 in GLP-1 was an important feature for membrane and receptor binding. Exendin-4 has a helix-favoring glutamate as residue 16. In the micelle-associated state, NMR data indicate that GLP-1 is less helical than exendin-4 due to the presence of Gly16; chemical shift deviations along the peptide sequence suggest that Gly16 serves as an N-cap for a second, more persistent, helix. In 30 vol-% trifluoroethanol (TFE), a single continuous helix is evident in a significant fraction of the GLP-1 conformers present. Exendin-4 has a more regular and less fluxional helix in both media and displays stable tertiary structure in the solution state. In the micelle-bound state of exendin-4, a single helix (residues 11-27) is observed with residues 31-39 completely disordered and undergoing rapid segmental motion. In aqueous fluoroalcohol or aqueous glycol, the Leu21-Pro38 span of exendin-4 forms a compact tertiary fold (the Trp-cage) which shields the side chain of Trp25 from solvent exposure and produces ring current shifts as large as 3 ppm. This tertiary structure is partially populated in water and fully populated in aqueous TFE. The Leu21-Pro38 segment of exendin-4 may be the smallest protein-like folding unit observed to date. When the Trp-cage forms, fraying of the exendin-4 helix occurs exclusively from the N-terminus; backbone NHs for the C-terminal residues of the helix display H/D exchange protection factors as large as 10(5) at 9 degrees C. In contrast, no tertiary structure is evident when exendin-4 binds to DPC micelles. An energetically favorable insertion of the tryptophan ring into the DPC micelle is suggested as the basis for this change. With the exception of exendin-4 in media containing fluoro alcohol cosolvents, NMR structure ensembles generated from the NOE data do not fully reflect the conformational averaging present in these systems. Secondary structure definition from chemical shift deviations may be the most appropriate treatment for peptides that lack tertiary structure.