Human angiogenin is a member of the pancreatic ribonuclease superfamily that induces blood vessel formation. Its three-dimensional solution structure has been determined to high resolution by heteronuclear NMR spectroscopy, 30 structures were calculated, based on a total of 1441 assigned NOE correlations, 64 coupling constants and 50 hydrogen bonds, The backbone atomic rms difference from the mean coordinates is 0.067 +/- 0.012 nm and 0.13 nm from the previously determined crystal structure, The side-chain of Gln117 was found to obstruct the active site as observed in the crystal state. There was no evidence of an alternative open form of angiogenin, although two sets of chemical shifts were observed for some residues, mainly around the active site and in the C-terminal segment. The topology of the ribonucleolytic active site is described with a particular emphasis on the conformation and protonation of active-site His residues. The side-chain of His114 adopts two main conformations in solution. In contrast to pancreatic ribonuclease A, His13 was shown to be more basic than His114, with pK(a) values of 6.65 and 6.05 respectively. The His47 residue is located in an environment very resistant to protonation with a pk(a) lower than 4.

To investigate the backbone dynamics of proteins N-15 longitudinal and transverse relaxation experiments combined with {H-1, N-15} NOE measurements together with molecular dynamics simulations were carried out using ribonuclease T-1 and the complex of ribonuclease T-1 with 2'GMP as a model protein. The intensity decay of individual amide cross peaks in a series of (H-1, N-15)HSQC spectra with appropriate relaxation periods was fitted to a single exponential by using a simplex algorithm in order to obtain (NT1)-N-15 and T-1 relaxation times. The relaxation times were analyzed in terms of the ''model-free'' approach introduced by Lipari and Szabo. In addition, a nanosecond molecular dynamics (MD) Simulation of ribonuclease T-1 and its 2'GMP complex in water was carried out. The angular reorientations of the backbone amide groups were classified with several coordinate frames following a transformation of NH vector trajectories. In this study, NH librations and backbone dihedral angle fluctuations were distinguished. The NH bond librations were found to be similar for all amides as characterized by correlation times of librational motions in a subpicosecond scale. The angular amplitudes of these motions were found to be about 10 degrees-12 degrees for out-of-plane displacements and 3 degrees-5 degrees for the in-plane displacement. The contributions from the much slower backbone dihedral angle fluctuations strongly depend on the secondary structure. The dependence of the amplitude of local motion on the residue location in the backbone is in good agreement with the results of NMR relaxation measurements and the X-ray data. The protein dynamics is characterized by a highly restricted local motion of those parts of the backbone with defined secondary structure as well as by a high flexibility in loop regions. Comparison of the MD and NMR data of the free liganded enzyme ribonuclease T-1 clearly indicates a restriction of the mobility within certain regions of the backbone upon inhibitor binding. (C) 1996 John Wiley & Sons, Inc.

A sensitive method to assign Hβ protons stereospecifically as well as to determine rotamer populations about χ1, in two 3D experiments is presented. The SOFT-HCCH-COSY experiment allowed us to measure the3J(Hβ,C′) couplings, using constant time evolution of Cα in t2 and Caliphatic-selective decoupling during t3. The SOFT-HCCH-E.COSY experiment allowed us to measure the3J(Hα,Hβ) couplings, using constant time evolution of Cα in t2, a small flip angle1H 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.

A 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.

Uniformly 15N‐enriched ribonuclease T1 (RNase T1) was obtained from Escherichia coli by recombinant techniques. Heteronuclear 1H, 15N‐shift correlation spectra were recorded utilizing proton detection. Direct 1H, 15N connectivities were established applying the heteronuclear multiple‐quantum coherence technique. Additional 1H, 1H‐TOCSY or 1H, 1H‐NOESY transfer steps allowed for sequential assignments. Nitrogen atoms without directly bonded protons were detected by means of the heteronuclear multiple‐bond correlation experiment. Signals emerging from 15NH and 15NH2 groups were distinguished by heteronuclear triple‐quantum filtering methods. 119 nitrogen resonances out of the expected 127 were assigned unambiguously; in addition, previously obtained proton assignments were extended. Preliminary 1H, 15N NMR investigations were performed on the RNase‐T1–3′GMP inhibitor complex. Results were interpreted with respect to nucleotide binding.