Sequential ¹H‐NMR assignment and solution structure of bovine pancreatic ribonuclease A

Abstract

Assignments for 'H-NMR resonances of most of the residues of bovine pancreatic ribonuclease (RNase A) have been obtained by sequence-specific methods. Identification and classification of spin systems have been carried out by two-dimensional phase-sensitive correlated spectroscopy (360 MHz) and single relayed coherence transfer spectroscopy. Sequence-specific assignments have been achieved by phase-sensitive two-dimensional nuclear Overhauser effect spectroscopy. To overcome the problem of spectral overlap use has been made of (a) an exhaustive analysis of partly exchanged RNase A (spectra in DzO), (b) a comparison with the subtilisinmodified enzyme (RNase S) and (c) small spectral perturbations caused by changes in pH and temperature. The secondary structure elements have been identified from the observed sequential, medium and long-range nuclear Overhauser effects together with data from amide-exchange rates. All information collected leads to the conclusion that the crystal and the solution structures are closely similar.Bovine pancreatic ribonuclease (RNase A) [l, 21 is a wellcharacterized enzyme, containing 124 amino acid residues, which catalyses the cleavage of single-stranded RNA. Extensive physical and chemical studies, including X-ray, neutron diffraction and NMR spectroscopy have been carried out on this protein, trying to gain information about its structure and function. Detailed structural information exists for the crystal state of the enzyme itself [3 -61, the subtilisin-modified enzyme RNase S [7], and some of their complexes with mononucleotides [8 -101, dinucleoside monophosphates [I

Periodic properties of proton conformational shifts in isolated protein helices

“…Fig. 11 a shows the conformational shifts estimated in the usual way, i. e. differences between 6 values for residues 1-20 in the protein [35] and random-coil 6 values [36], plotted against the residue number. The 6 differences shown in Fig.…”

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

“…The process was largely facilitated by the previous assignment for the intact protein (Rico et al, 1989;Robertson et al, 1989). In general, the location of most of the signals, i.e., the fingerprint region of the TOCSY spectrum, was followed easily because of their proximity to the corresponding signals of the intact protein.…”

“…Structural properties of RNase dissolved in aqueous TFE that likely contribute to its specific nicking by thermolysin are discussed. Conformational and functional properties of the two nicked RNase molecules are interpreted, taking advantage of the known three-dimensional structure and dynamics of native RNase in the crystal state (Wyckoff et al, 1970;Wlodawer et al, 1982Wlodawer et al, , 1988 or in solution (Rico et al, 1989;Santoro et al, 1993)? concentration.…”

Native-state hydrogen-exchange studies of a fragment complex can provide structural information about the isolated fragments

“…13). An NMR structure is available (14,15) for RNase A with preliminary data for RNase S. Refined 3D x-ray structures of RNase A and RNase S are available at high resolution (16,17). The two proteins have very similar structures and enzymatic activity but exhibit significant differences in their dynamics and stability (18)(19)(20)(21).…”

“…Ribonuclease S (RNase S) is a well studied fragment complementation system. It consists of a noncovalent complex between S peptide (S pep; residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and S protein (S pro; residues 21-124), two proteolytic fragments of the 124-aa residue protein, Ribonuclease A (RNase A; ref. 13).…”