Motional dynamics of residues in a β‐hairpin peptide measured by ¹³C‐NMR relaxation

Abstract

Structurally characterizing partially folded peptides is problematic given the nature of their transient conformational states. I3C-NMR relaxation data can provide information on the geometry of bond rotations, motional restrictions, and correlated bond rotations of the backbone and side chains and, therefore, is one approach that is useful to assess the presence of folded structure within a conformational ensemble. A peptide 12mer, RIGITVNG7KTYGRI2, has been shown to partially fold in a relatively stable /3-hairpin conformation centered at NG. Here, five residues, G2, V5, G7, Y10, G11, were selectively 13C-enriched, and 13C-NMR relaxation experiments were performed to obtain auto-and cross-correlation motional order parameters, correlation times, bond rotation angular variances, and bond rotational correlation coeffkients. Our results indicate that, of the three glycines, G7 within the hairpin @turn displays the most correlated #J(t),$(t) rotations with its axis of rotation bisecting the angle defined by the H-C-H bonds. These positively correlated bond rotations give rise to "twisting" type motions of the HCH group. V5 and Y10 #J,$ bond rotations are also positively correlated, with their C&,H groups undergoing similar "twisting" type motions. Motions of nearterminal residues G2 and G11 are less restricted and less correlated and are best described as wobbling-in-a-cone. V5 and Y10 side-chain motions, aside from being highly restricted, were found to be correlated with #J,$ bond rotations. At 303 K, where the hairpin is considered "unfolded," the peptide exists in a transient, collapsed state because backbone and side-chain motions of V5, G7, and Y10 remain relatively restricted, unlike their counterparts in GXG-based tripeptides. These results provide unique information toward understanding conformational variability in the unfolded state of proteins, which is necessary to solve the protein folding problem.Keywords: I3C-NMR; cross-correlation; motional dynamics; peptides; relaxation Short linear peptides most often exist in solution as a distribution of transient, rapidly interconverting conformations. Structurally characterizing such conformational ensembles has proved problematic. Conventional approaches to NMR structural analysis are limited because few, if any, long-range, conformationally informative 'H-'H NOES are usually observed and J-coupling constants and chemical shifts render only average structures. On the other hand, I3C-NMR relaxation measurements provide information concerning motional restrictions and preferred axes of bond rotations at both backbone and side-chain positions (Daragan et al., 1997). Such relaxation studies offer a unique approach to discerning the limits of conformational space accessible to a given residue within the peptide conformational ensemble and to elucidating correlated

Angular variances for internal bond rotations of side chains in GXG-based tripeptides derived from13C-NMR relaxation measurements: Implications to protein folding

“…(referred to as E ROT in that paper 12 ) for either methyl group of 2.2 kcal/mol. In GVG, E j values for valine methyl group rotations are considerably larger (4.7 kcal/ mol for C ␥1 and 3.4 kcal/mol for C ␥2 ), suggesting increased side-chain/ backbone and/or side-chain/solvent interactions for valine methyls in this tripeptide.…”

“…Since one methyl group is always closer to the carbonyl oxygen of the backbone, this energetic difference most probably results from interactions of these methyl groups with backbone atoms. 12 In a partially folded, ␤-hairpin peptide, 12 valine (V5) methyl i values at 303 K are equal to 28 ps for C ␥1 and 23 ps for C ␥2 with a rotational activation energy E j a Correlation times CH , for motions of methyl group CH bonds given in picoseconds. Activation energies E CH , for these motions are given in kcal/mol.…”

“…Even methyl group rotational correlation times are sensitive to local environments, conformation, and accessibility to solvent. 8 -10 Nuclear magnetic resonance relaxation measurements have recently provided evidence from different proteins and peptides [11][12][13][14][15][16] that the "unfolded state" is in fact not fully unfolded with all residues being solvent exposed, but rather it is an ensemble of highly transiently collapsed or partially collapsed species that may include populations of partially folded states. In this respect, it is crucial to establish a baseline or point of reference for internal motions and motional restrictions (or angular variances) for individual residues in a fully, unfolded state (distribution).…”

“…Rotations about specific side-chain bonds, for example, are often more sensitive to changes in protein conformation and folding than are backbone bond rotations. [1][2][3][4][5][6][7][8][9][10][11][12] In folded proteins and peptides, backbone bond rotations that occur primarily via rotational fluctuations within potential wells are often easier to model, whereas side chains can generally probe greater conformational space and also undergo jumps among various rotomer states. From studies of the temperature dependence of nmrderived motional parameters, it is apparent that in partially folded peptides, 11,12 amplitudes of internal rotations about backbone , bonds vary little with temperature, whereas in folded structures, correlations between various i (t) and k (t) bond rotations can be substantial and can change dramatically with temperature.…”

Internal motional amplitudes and correlated bond rotations in an α‐helical peptide derived from 13C and 15N NMR relaxation

“…Using molecular dynamics simulations on model peptides in a-helix and b-sheet structures, it was observed that the magnitude and sign of c fc are dependent upon f, c angles within a structural element: in an a-helix, c fc is negative and in a b-sheet, c fc is positive. As initial verification of the model, c fc was found to be positive in two partially-folded b-sheet0hairpin-forming peptides, indicating that C a H internal motions primarily occur along the sheet as "twisting" type motions~Daragan et al Ramirez-Alvarado et al, 1998!. In the present study, 13 C and 15 N NMR relaxation experiments were performed on the hydrophobic staple-helix peptide in water and in water0TFE solution from 5 to 30 8C. Five hydrophobic residues, F2, A5, L7, A8, and A10, were uniformly 13 C0 15 Nenriched, and proton-coupled and -decoupled NMR relaxation measurements were acquired at four Larmor frequencies~62.5, 125, 150, and 200 MHz for 13 C!.…”