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...
We have employed pramlintide (prAM) as a surrogate for hAM in CD and NMR studies of the conformational preferences of the N-terminal portion of the structure in media which do not provide long-lived monomeric solutions of hAM due to its rapid conversion to preamyloid beta aggregate states. Direct comparison of hAM and prAM could be made under helix-formation-favoring conditions. On the basis of CD and NMR studies: (i) the Cys(2)-Cys(7) loop conformation has a short-span of helix (Ala(5)-Cys(7)); (ii) the extent to which this helix propagates further into the sequence is medium-dependent; a helix from Ala(5) through Ser(20) (with end fraying from His(18) onward) is observed in aqueous fluoroalcohol media; (iii) in 12+ vol.% HFIP, the amyloidogenic region of hAM forms a second helical domain (Phe(23)-Ser(29)); (iv) the two helical regions of hAM do not have any specific geometric relationship as they are connected by a flexible loop that takes different conformations and (v) although the extreme C-terminus is essential for bioactivity, it is found to be extensively randomized with conformer interconversions occurring at a much faster rate than that is observed in the remainder of the peptide sequence. Two NMR-derived structures of the 1-22 sequence fragment of hAM have been derived. The work also serves to illustrate improved methods for the NMR characterization of helices. A detailed quantitative analysis of the NOE intensities observed in aqueous HFIP revealed alternative conformations in the C-terminal portion of the common amylin helix, a region that is known to be involved in the biorecognition phenomena leading to amyloidogenesis. Even though the SNN sequence appears to be a flexible loop, the chemical shifts (and changes induced upon helix structuring) suggest some interactions between the loop and the amyloidogenic segment of hAM that occur on partial helix formation.
Additional NMR data (local NOE ratios and chemical shifts) for endothelin-1 supporting the existence of a relatively regnlar helix initiated abruptly at Lys 9 (with Asp s as an N-cap) and extending in all cases to Cys 15 (and in a frayed form to Asp 18 in some analogs) is presented. The recent solid-state structure [Janes et al. (1994), Nature Struct. Biol. 1, 311-319], in contrast, places the helix in the extreme C-terminal section of the structure and the Lysg-Tyr ~3 segment is not helical. The X-ray structure does not predict the NOEs or chemical shifts observed for endothelins in aqueous media containing polar organic co-solvents. An analysis of the chemical shift data for reporter groups indicates that the helical conformational preference of endothelins is not significantly altered by the addition of acetonitrile, acetic acid, or ethylene glycol. The validity of the analytic strategy is supported by results for both more rigid and less helical analogs. We conclude that the structure observed in crystals obtained from purely aqueous media is influenced by intermolecular interactions in the solid state and is not a significant contributor to the conformational equilibrium observed for monomeric ET-1.
NMR studies reveal that the conformational equilibrium of the HLDIIW fragment of endothelins, which is the C-terminal extension beyond the disulfide-linked core, is relatively little affected by changes in the structural preference within the bicyclic core. Rather the major determinant appears to be a hydrophobic interaction between the Trp ring and the lie19 sidechain. At subambient temperatures in aqueous medium, this interaction appears to be enhanced in species that have residues 12- 15 present and in a helical conformation.
Many authors have used temperature gradient information in attempts to assess NH exposure in peptides and proteins. We recently established [1] that the common practice of correlating the value with NH sequestration from solvent has zero predictive validity for peptides which have even a modest degree of conformational averaging.
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