2H Relaxation Data Research Articles

Probing Methyl Dynamics from13C Autocorrelated and Cross-Correlated Relaxation

An understanding of side-chain motions in protein is of great interest since side chains often play an important role in protein folding and intermolecular interactions. A novel method for measuring dipole-dipole cross-correlated relaxation in methyl groups of uniformly 13C-labeled proteins without deuteration has been developed by our group. The excellent agreement between dynamic parameters of methyl groups in ubiquitin obtained from the cross-correlated relaxation and 13C spin-lattice relaxation and those derived previously from 2H relaxation data demonstrates the reliability of the method. This method was applied to the study of side-chain dynamics of human intestinal fatty acid binding protein (IFABP) with and without its ligand. Binding of oleic acid to the protein results in decreased mobility of most of the methyl groups in the binding region, whereas no significant change in mobility was observed for methyl groups in the nonbinding region.

Measurement of Methyl 2H Quadrupolar Couplings in Oriented Proteins. How Uniform Is the Quadrupolar Coupling Constant?

2H quadrupolar and one-bond 13C−13C dipolar couplings have been measured at methyl sites in a uniformly 13C and fractionally 2H labeled sample of the N-terminal drk SH3 domain, weakly aligned in a dilute solution of Pf1 phage. An average ratio between the 2H quadrupolar and 13C−13C dipolar couplings of 19.2 ± 0.1 is measured. Assuming rapid rotation about the one-bond 13Cmethyl−13C axis and an angle of 109.5° between the unique principal axis of the electric field gradient tensor and the methyl averaging axis (13Cmethyl−13C bond), an average value of 167 ± 1 kHz is obtained for the quadrupolar coupling constant, e2qQ/h. The profile of 2H quadrupolar vs 13C−13C dipolar couplings suggests that the use of a uniform value for the quadrupolar coupling constant is a good approximation in the analysis of 2H relaxation data measured at methyl sites in proteins.

Backbone and methyl dynamics of the regulatory domain of troponin C: anisotropic rotational diffusion and contribution of conformational entropy to calcium affinity

The N-terminal domain (residues 1 to 90) of chicken skeletal troponin C (NTnC) regulates muscle contraction upon the binding of a calcium ion to each of its two calcium binding loops. In order to characterize the backbone dynamics of NTnC in the apo state (NTnC-apo), we measured and carefully analyzed 15N NMR relaxation parameters T 1, T 2 and NOE at 1H NMR frequencies of 500 and 600 MHz. The overall rotational correlation time of NTnC-apo at 29.6°C is 4.86 (±0.15) ns. The experimental data indicate that the rotational diffusion of NTnC-apo is anisotropic with a diffusion anisotropy, D ∥/ D ⊥, of 1.10. Additionally, the dynamic properties of side-chains having a methyl group were derived from 2H relaxation data of CH 2D groups of a partially deuterated sample. Based on the dynamic characteristics of TnC, two different levels of “fine tuning” of the calcium affinity are presented. Significantly lower backbone order parameters ( S 2), were observed for calcium binding site I relative to site II and the contribution of the bond vector fluctuations to the conformational entropy of sites I and II was calculated. The conformational entropy loss due to calcium binding (ΔΔ S p) differs by 1 kcal/mol between sites I and II. This is consistent with the different dissociation constants previously measured for sites I and II of 16 μM and 1.7 μM, respectively. In addition to the direct role of binding loop dynamics, the side-chain methyl group dynamics play an indirect role through the energetics of the calcium-induced structural change from a closed to an open state. Our results show that the side-chains which will be exposed upon calcium binding have reduced motion in the apo state, suggesting that conformational entropic contributions can be used to offset the free energy cost of exposing hydrophobic groups. It is clear from this work that a complete determination of their dynamic characteristics is necessary in order to fully understand how TnC and other proteins are fine tuned to appropriately carry out their function.

Dimethyl sulfoxide binding to globular proteins: a nuclear magnetic relaxation dispersion study.

The 2H magnetic relaxation dispersion (NMRD) technique was used to characterize interactions of dimethyl sulfoxide (DMSO) with globular proteins. A difference NMRD experiment involving the N-acetylglucosamine trisaccharide inhibitor, demonstrated that the DMSO 2H NMRD profile in lysozyme solution is due to a single DMSO molecule bound in the active cleft, with a molecular order parameter of 0.47 +/- 0.05 and a residence time in the range 10 ns to 5 ms. With the aid of transverse 2H relaxation data, the upper bound of the residence time was further reduced to 100 microns. A 1H shift titration experiment was also performed, yielding a binding constant of 2.3 +/- 0.3 M-1 at 27 degrees C. In contrast to lysozyme, no DMSO dispersion was observed for bovine pancreatic trypsin inhibitor (BPTI), indicating that a stable DMSO-protein complex requires a cleft of appropriate geometry in addition to hydrogen-bond and hydrophobic interactions.

Hydrogen Exchange and Protein Hydration: The Deuteron Spin Relaxation Dispersions of Bovine Pancreatic Trypsin Inhibitor and Ubiquitin

Water deuteron ( 2H) spin relaxation was used to study hydrogen exchange hydration, and protein dynamics in aqueous soltuions of the globular proteins bovine pancreatic trypsin inhibitor (BPTI) and ubiquitin. The frequency dispersion of the longitudinal 2H relaxation rate was measured in the pD range 2 to 11 at 27°C. In contrast to the previously reported water 17O relaxation dispersion from the same samples, the 2H dispersion depends strongly on pD. This pD dependence is due to labile protein deuterons in acidic side-chains and surface peptide groups, which exchange rapidly with water deuterons. The pD dependence of the 2H relaxation in BPTI solutions could be quantitatively accounted for in terms of known p Kvalues and hydrogen exchange rate constants. For ubiquitin, labile protein deuterons contribute importantly to the 2H relaxation dispersion even in the neutral pD range. The 2H relaxation data also provided information about the orientational order and internal motion of OD and ND bonds in side-chains and surface peptides. A comparison of the water contribution to the 2H dispersion with the 17O dispersion indicates that one of the four internal water molecules of BPTI, presumably the deeply buried water molecule W122, exchanges more slowly (10 −6to 10 −4second) than the other three (10 −8to 10 −6second).

2H and 13C nuclear magnetic relaxation studies of the cubic liquid-crystalline phase I1 in the sodium octanoate–octane–water system

An extensive n.m.r. relaxation study of the cubic I1 phase formed in the sodium octanoate–octane–water system is presented. 13C relaxation data are presented at three magnetic field strengths and 2H relaxation data at twelve magnetic field strengths (from 0.3 to 8.5 T). All the relaxation data depend on the magnetic field strength. The data are analysed with the ‘two-step’ model of relaxation, and information concerning both the dynamic (correlation times) and static (order parameters) properties of the hydrocarbon chains in the surfactant aggregates are obtained. Comparisons are made with results from sodium octanoate–water in other phase situations. As regards the structure of the surfactant aggregates in the I1 phase, the relaxation data support the structure of hemisphere-capped rods suggested by Fontell et al.(K. Fontell, K. V. Fox and E. Hansson, Mol. Cryst. Liq. Cryst., 1985, 1, 9.) In particular, it is shown that the complete set of 2H relaxation data can be rationalized with the two-step model, if the axial ratio of the hemisphere-capped rods is ca. 1.3 to 1.

Internal rotation of methyl groups in NN-dimethylamides studied by nuclear magnetic resonance methods

The diffusion constants for anisotropic reorientation and internal rotation rates of methyl groups in DMF have been recalculated using longitudinal relaxation times for 14N, 17O, 2H, 13C, and quadrupolar parameters available in the literature. Within the framework of an isotropic reorientation model the rates of internal rotation of CH2D groups in NN-di([2H]methyl)-formamide, -benzamide, and -2-methoxybenzamide have been determined on the basis of 14N and 2H relaxation data. It seems that generally in NN-dimethylamides internal rotation of the E-methyl group is more hindered than that of the Z-methyl group.