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Semi-automatic Stereospecific Resonance and NOESY Assignment of Proteins Based on the Tertiary Structure – the Program nmr2st
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DETERMINATION OF H-ALPHA,H-BETA AND H-BETA,C' COUPLING-CONSTANTS IN C-13 LABELED PROTEINS
No full textA sensitive method to assign H(beta) protons stereospecifically as well as to determine rotamer populations about chi1 in two 3D experiments is presented. The SOFT-HCCH-COSY experiment allowed us to measure the 3J(H(beta),C') couplings, using constant time evolution of C(alpha) in t2 and C(aliphatic)-selective decoupling during t3. The SOFT-HCCH-E.COSY experiment allowed us to measure the 3J(H(alpha),H(beta)) couplings, using constant time evolution of C(alpha) in t2, a small flip angle H-1 excitation pulse in the second mixing time, and double-band-selective decoupling (aliphatic and carbonyl carbons) during t3. The method was applied to ribonuclease T1
Assignment of the H-1, C-13 and N-15 resonances of the PPIase domain of the trigger factor from Mycoplasma genitalium
No full textDETERMINATION OF H(N),H-ALPHA AND H(N), C' COUPLING-CONSTANTS IN C-13,N-15-LABELED PROTEINS
No full textSensitive three-dimensional NMR experiments, based on the E.COSY principle, are presented for the measurement of the 3J(H(N),H(alpha) and 3J(H(N),C') coupling constants in uniformly C-13- and N-15-labeled proteins. They employ gradient coherence selection in combination with the sensitivity enhancement method in HSQC-type spectra (Cavanagh et al., 1991; Palmer et al., 1991). In most cases, the two measured coupling constants unambiguously define the phi-angle for protein structure determination. The method is applied to uniformly C-13,N-15-labeled ribonuclease T1
CONFORMATION OF VALINE SIDE-CHAINS IN RIBONUCLEASE T-1 DETERMINED BY NMR-STUDIES OF HOMONUCLEAR AND HETERONUCLEAR (3)J COUPLING-CONSTANTS
No full textA conformational analysis of the valine side chains of ribonuclease T-1 (RNase T-1) was performed using NMR spectroscopy, in particular homonuclear (H-1,H-1 and C-13,C-13) and heteronuclear (H-1,N-15 and H-1,C-13) 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, (3)J(H alpha H beta),(3)J(NH beta),(3)J(C'H beta),(3)J(H alpha C gamma 1),(3)J(H alpha C gamma 2),(3)J(C'C gamma 1) and (3)J(C'C gamma 2), were evaluated in a quantitative manner. The analysis of (3)J 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 (3)J coupling constants ((3)J(H alpha)H(beta), (3)J(NH beta), (3)J(C'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 (3)J coupling informations agree with the conformation in the crystal structure of ribonuclease T-1 (Martinez-Oyanedel et al., 1991)
DETERMINATION OF H(N),H-ALPHA AND H(N), C' COUPLING-CONSTANTS IN C-13,N-15-LABELED PROTEINS
No full textSensitive three-dimensional NMR experiments, based on the E.COSY principle, are presented for the measurement of the 3J(H(N),H(alpha) and 3J(H(N),C') coupling constants in uniformly C-13- and N-15-labeled proteins. They employ gradient coherence selection in combination with the sensitivity enhancement method in HSQC-type spectra (Cavanagh et al., 1991; Palmer et al., 1991). In most cases, the two measured coupling constants unambiguously define the phi-angle for protein structure determination. The method is applied to uniformly C-13,N-15-labeled ribonuclease T1
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