Extracting Information from the Temperature Gradients of Polypeptide NH Chemical Shifts. 1. The Importance of Conformational Averaging

Abstract

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...

Structure of phosphorylated UBL domain and insights into PINK1-orchestrated parkin activation

“…Amide temperature coefficients (∆δ NH /∆T) for pUBL also showed large decreases near the S65 phosphorylation site compared with UBL, including Q63 and Q64 in the α2-L structural element ( Fig. S4) indicative of amide hydrogen bonding (28,29). In contrast, the amide of pSer65 showed increased temperature dependency, reflecting the loss of hydrogen bonds with F4 in β1, shortening the terminal β4 strand.…”

A New Level of Conotoxin Diversity, a Non-native Disulfide Bond Connectivity in α-Conotoxin AuIB Reduces Structural Definition but Increases Biological Activity

“…In the bottom graph a change in temperature coefficient from that for random coil of less than Ϫ3 ppb/K indicates residues that may be solvent-protected. The variation in amide proton chemical shifts with temperature provides a measure of exposure to solvent, and hence potentially yields information on hydrogen bonding (47,48). With the exception of Phe-9 all residues from Ser-4 to Cys-15 of the native isomer of AuIB have temperature coefficients less negative than Ϫ4 parts per billion (ppb)/K, a standard measure of amide proton protection from solvent.…”

Temperature-dependent Kinetics Study for Hydrogen Exchange of Type I Antifreeze Protein from Winter Flounder

“…Similarly, the /T values of the S4 amide protons in A17L-and A20L-AFPs are 0.018 ± 0.001 and 0.017 ± 0.001 ppm/°C, respectively. These /T values are greater than the average /T value (0.0275 ppm/°C) of hydrogen bonded amide protons based on statistical analysis of proteins and peptides, 12 indicating that the S4 amide protons of all three of the AFPs are not exchange-protected by hydrogen bonding interaction. However, the N16/N27 amide protons in wt-AFP have /T values of approximately 0.010 ppm/°C at  5 °C.…”