EXTRACTING INFORMATION FROM THE TEMPERATURE-GRADIENTS OF POLYPEPTIDE NH CHEMICAL-SHIFTS .1. THE IMPORTANCE OF CONFORMATIONAL AVERAGING

A detailed analysis of backbone amide NH chemical shift temperature gr adients (Delta delta/Delta T values) for proteins and highly cross-lin ked peptides reveals that hydrogen-bonded exchange-protected NHs are c haracterized by Delta delta/Delta T values of -2.0 +/- 1.4 ppb/degrees C while exposed NHs typically display gradients of -6.0 --> -8.5 ppb/ degrees C; however, numerous exceptions to these generalizations occur . For partially folded peptides (rather than proteins), exceptions are more common than concordance with this rule; Delta delta/Delta T valu es ranging from -28 to +12 ppb/degrees C have been observed. In the ca se of the peptide systems for which exchange protection data is availa ble, the common practice of assuming that a Delta delta/Delta T value less negative than -4 ppb/degrees C indicates that the NH is sequester ed from solvent is shown to have zero predictive validity. The analysi s of the data for partially folded peptides, protein fragments, and ot her peptides which are expected to display minimal structuring reveals a significant correlation between Delta delta/Delta T and the deviati on of delta(NH) from the random coil reference shift. The analysis was facilitated by plotting NH chemical shift deviations (NH-CSD) versus the Delta delta/Delta T values. Using such plots, slow-exchanging hydr ogen-bonded sites in proteins can be determined with much higher confi dence than using the value of the gradient alone. For peptides, the oc currence of large shift deviations and abnormal gradients are diagnost ic for partial structuring at lower temperatures which becomes increas ingly randomized on warming. A good correlation coefficient (R greater than or equal to 0.75) for NH-CSD and Delta delta/Delta T values indi cates that essentially all of the NH shift deviation from reference va lues 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 beta-hairpin forming peptides. The slope of the correl ation plot (parts per thousand/degrees C) is a measure of the decrease in the population of the structured state upon warming. A detailed mo del which rationalizes the effects of conformational equilibria upon N H shifts is presented. A positive Delta Cp for unfolding is required t o rationalize the linearity of delta(NH) With temperature that is rout inely observed for partially structured peptides. This analysis sugges ts that ordered states of short peptides achieve significant populatio ns in water only when the hydrophobic effect favors the structured sta te. This conclusion is pertinent to the current questions concerning t he temporal sequence of secondary versus tertiary structure formation during protein folding. Further, it is suggested that the use of NMR p arameters (scalar and dipolar couplings) to derive the structural pref erences of protein fragments which might serve a ''seeding'' role in t he folding pathway is justified only when the CSD/gradient plot displa ys both a correlation coefficient greater than 0.70 and significant NH -CSD values (\CSD\ > 0.3).