Two-dimensional 1H NMR investigation of ribonuclease A and ribonuclease-A-pyrimidine-nucleotide complexes

Abstract

Ribonuclease A was studied by two-dimensional 'H NMR spectroscopy. I0 out of 12 alanine and 9 out of 10 threonine spin systems as well as all valine [9], leucine [2] and isoleucine [3] spin systems were identified from the correlated spectroscopy (COSY) and relayed coherence transfer spectroscopy (RCT). Sequence-specific assignments were obtained from nuclear Overhauser effect spectra for proton resonances of 21 amino acid moieties.2' and 3'-pyrimidine-nucleotide -RNase-A complexes were also investigated by two-dimensional NMR. We were able to monitor structural changes in the active center, the vicinity of the active center and in regions far from the catalytic region. Chemical shift changes of resonances of protons near Thr-45 reflected the binding of the same moiety. This in turn is also dependent on the position of the nucleotide phosphate group. Binding of 2' nucleotides led to characteristic changes in protein regions not affected by the binding of 3' nucleotides. These results are interpreted in terms of structural differences between the 2' and 3'-nucleotide -RNase-A complexes; the structure of the complex of the native 3' nucleotide inhibitor being more closely related to that of the free protein.Ribonuclease A from bovine pancreas (RNase A) is a wellcharacterized protein of 124 amino acids and a molecular mass of 13 700 Da. The enzyme hydrolyses single-strand RNA at the 3'-OH position of pyrimidine nucleotides via a cyclic 2',3'-diester intermediate [I -21. X-ray investigations and neutron diffraction studies have shown that the enzyme is a highly structured molecule with three helical regions and two fi sheets with three and four strands respectively. It consists of two domains that are connected by a hinge region. Together they form a large cleft for substrate binding [3-61. RNase A binds 2' and 3' pyrimidine nucleotides as competitive inhibitors, the binding constant being larger for 2' nucleotides. Cytidine nucleotides are bound more tightly than uridine nucleotides.RNase A and its complexes have long been used as a simple model for protein-nucleotide interactions and have been intensively studied by physical methods. Extensive Xray and a great number of ' H and heteronuclear NMR studies (3'P,'3C,'5N) have beenapplied to thissystem these data the structure of the active site of the enzyme and specific interactions between the protein and the nucleotide have been proposed.

Proton NMR assignments and regular backbone structure of bovine pancreatic ribonuclease A in aqueous solution

“…Rigorous interpretation of both types of experiments requires assignment of the resonances in the 'H NMR spectrum of RNase A. The present work confirms the assignments of Hahn and Riiterjans (1985) and continues the sequential assignments of the backbone resonances of RNase A to near completion.…”

“…The two leucine residues proved more elusive; only one showed a /3CH-5CH RELAY, and the /3CH resonance could not be aligned with any observable aCH-/3CH COSY crosspeaks. We observed NOEs between two 5CH resonances of one leucine (later assigned to L51) and a putative aCH, as did Hahn and Riiterjans (1985). A single COSY fingerprint peak could be aligned with this aCH resonance.…”

“…Work since then has led to assignments for many of the aromatic protons (Bradbury & Scheraga, 1966;Meadows et al, 1968;Patel et al, 1975; Lenstra et al, 1979; Tanokura, 1983) as well as a number of carbon resonances (Walters & Allerhand, 1980; Howarth & Lian, 1984). Hahn and Riiterjans (1985) made sequence-specific 'H NMR as-1 Abbreviations: COSY, two-dimensional chemical shift correlation spectroscopy; NMR, nuclear magnetic resonance; DQF-COSY, doublequantum-filtered COSY; FID, free induction decay; NOE, nuclear Overhauser enhancement; NOESY, two-dimensional NOE spectroscopy; RELAY, relayed COSY; RNase A, bovine pancreatic ribonuclease A (EC 3.1.27); DSS, 2,2-dimethyl-2-silapentane-5-sulfonate. In discussions of NOESY data, dAB(iJ) designates the distance between proton types A and B located in amino acid residues i and j, respectively, where N, a, 0, and y denote the amide protons, aCH, /3CH, and yCH, respectively.…”

2D NMR and structural model for a mitochondrial signal peptide bound to a micelle

“…Unfortunately, many CaH, to NaH,-+3 or NaHi+4 crosspeaks, indicative of «-helical structure, overlapped with other crosspeaks but several were well resolved [R3 « to L6 NH (/, í+3), S16 ß to L19 NH (/, i+3), and R3 « to S7 NH (i, /+4)]. Additional support for the two «-helical sections is provided by the C^H-NaH/+, connectivities, where often these crosspeaks are more intense in «-helical regions (Hahn & Ruterjans, 1985). Either SI6 ß to R17 (i, i+l) or S20 ß to Y21 (/', /+1) displayed strong crosspeaks.…”

3′‐Azido‐3′‐deoxythymidine binding to ribonuclease A: Model for drug‐protein interaction

“…The enzyme hydrolyzes single‐stranded RNA at the 3′‐OH position of pyrimidine nucleotide through a cyclic 2′,3′‐diester intermediate. RNase A is the most studied protein and much is known about it concerning the enzyme kinetics,o X‐ray and neutron diffraction structure, and stability and mechanistic analysis 1–8. However, much remains to be learned, including the role of electrostatic interaction in structural stability, drug binding, and catalysis.…”