Conformation of Valine Side Chains in Ribonuclease T1 Determined by NMR Studies of Homonuclear and Heteronuclear 3J Coupling Constants

Abstract

A conformational analysis of the valine side chains of ribonuclease T1 (RNase T1) was performed using NMR spectroscopy, in particular homonuclear (1H, 1H and 13C, 13C) and heteronuclear (1H, 15N and 1H, 13C) vicinal spin-spin coupling constants as obtained from E.COSY-type NMR experiments. The coupling constants related to the chi 1 dihedral angle in valine, 3JH alpha H beta, 3JNH beta, 3JC'H beta, 3JH alpha C gamma 1, 3JH alpha C gamma 2, 3JC'C gamma 1, and 3JC'C gamma 2, were evaluated in a quantitative manner. The analysis of 3J data allowed for the stereospecific assignment of the valine methyl resonances. On the basis of various models for motional averaging of coupling constants, a fit of the torsion angles chi 1 to a set of the experimental 3J coupling constants (3JH alpha H beta, 3JNH beta, 3JC'H beta) was carried out. The resulting side-chain conformations were examined with respect to NOE distance informations. Single rotameric states emerged for Val16, Val67, Val79, and Val101, while conformational equilibria between staggered rotamers were found for Val33 and Val78. Using a different model approach, Val52 and Val89 are also likely to exhibit unimodal chi 1 angle distributions. The analysis was found to depend critically on the set of Karplus parameters used. Except for Val52 and Val78, the predominant rotamers obtained from 3J coupling informations agree with the conformation in the crystal structure of ribonuclease T1 (Martinez-Oyanedel et al., 1991).

Mechanistic Analysis of a Suicide Inactivator of Histone Demethylase LSD1

“…Although there was some contamination observed in this mass spectrum with unreacted and dealkylated 1*, the latter being unlabeled should be invisible in the characterization of the 1*-flavin adduct. We used a combination of HSQC (34) and HCCH-COSY (35,36) experiments to identify the labeled threecarbon fragment predicted for 3 along with the protons on these carbons. As shown in panels A and B of Figure 4, the R-and γ-carbons cluster in the expected chemical shift range of 160-170 ppm.…”

Refinement of Protein Tertiary Structure by Using Spin-Spin Coupling Constants from Nuclear Magnetic Resonance Measurements

“…A variety of angular-mobility models can be applied to cases in which a single fixed torsion does not explain the observed data satisfactorily. Analyses commonly assume the  1 torsion either to dwell preferentially in energetically favourable staggered states (Pachler, 1963(Pachler, , 1964Hansen et al, 1975) or to librate about a mean value according to a Gaussian probability profile (Jardetzky, 1980;Karimi-Nejad et al, 1994;Brüschweiler & Case, 1994).…”

A Method to Explore Protein Side Chain Conformational Variability Using Experimental Data

“…Hence, the time average depends on both the coupling type, for instance, 3 J NHb or 3 J HaHb , which determines the parameters of the Karplus relation, and the values of q. J-coupling values are generally not restrained quantitatively to experimental values in MD simulations. Instead, they are interpreted using methods that can be loosely classed into three categories: 1) A single corresponding angle value is sought; [16,32,33] this has the advantage of requiring the least experimental data, as there is only one adjustable parameter, [33] meaning that in some cases the ambiguities of the Karplus relation ship may be resolved completely with the measurement of just two coupling values. On the other hand, with large quantities of data, it may be impossible to locate a single angle value that fits all of it simultaneously.…”

“…[42,43,29] 2) A Gaussian distribution is fitted. [23,29,33] This represents an improvement relative to (1) as thermal motion around an average angle value is accounted for. At least two measurements are required to fit the two parameters of this model.…”

“…At least two measurements are required to fit the two parameters of this model. [23,29,33] A Gaussian description has proved successful in many cases. [29,33] It cannot, however, account for situations where more than one value of the dihedral angle is significantly populated, that is, when the dihedral angle distribution possesses more than one maximum value.…”