A Deuterium‐Hydrogen Exchange Study of Inhibitor‐Induced Conformational Changes in Ribonuclease A

Abstract

Deuterium‐hydrogen exchange has been used to detect possible conformational changes accompanying the interaction of ribonuclease A with two competitive inhibitors: cytidine 3′‐phosphate (Cyd‐3′‐P) and cytidine 2′‐phosphate (Cyd‐2′‐P). An improvement of the cryosublimation technique was employed and combined with mass spectrometry measurements of sample deuterium content. Exchange kinetics were followed from 1 min to 24 h, at pH 6.5, and 38 °C (0.1 M NaCl). Two methods were used to present the data: a graphical exponential‐curve‐fitting and a numerical analysis. They both lead to the same conclusions. Four different classes of hydrogens were found. Among them, the one involving the instantaneously exchanged atoms is increased by about ten hydrogens after the binding of these inhibitors; the number of unexchanged hydrogens (within 24 h) is hardly altered in the presence of Cyd‐3′‐P whereas it is decreased by one third after the fixation of Cyd‐2′‐P. These exchange results suggest that both inhibitors induce in ribonuclease A conformational changes which may be correlated with inhibitor strength.

Effects of sucrose on conformational equilibria and fluctuations within the native‐state ensemble of proteins

“…These HX profiles are consistent with previous observations for these proteins monitored by FT‐IR or mass spectrometry (Nonnenmacher et al 1971; Nabedryk‐Viala et al 1976; Haris et al 1986; de Jongh et al 1995; Dong et al 1996). The HX rate constants were obtained by two exponential decay functions with equation 3 below for fast (<6 h) and slow (>6 h) phases, based on the previous classifications for RNase A and S (Nonnenmacher et al 1971; Wang et al 1995; Chakshusmathi et al 1999) and Cyt c (Foord and Leatherbarrow 1998). For RNase A and S, sucrose reduced very slightly both fast and slow HX (Table 2).…”

“…For example, we have observed that when Cyt c was dissolved in 100% D 2 O, >95% of the amide hydrogens were already exchanged before the first spectrum was acquired (90 sec), and there were no significant differences in the HX rates with or without up to 1.5 M sucrose (data not shown). The previous HX data for RNase A and S dissolved in 100% D 2 O showed much lower fraction values of the initial unexchanged hydrogens than those we reported here (Nonnenmacher et al 1971; Dong et al 1996; Chakshusmathi et al 1999). Our approach of diluting native protein in H 2 O buffer into D 2 O buffer to initiate HX provides the resolution needed to detect the effects of osmolytes on conformational fluctuations in the native state.…”

Effects of ionic strength and state of assembly on kinetics of hydrogen exchange of calf thymus histones

“…There are, however, several examples of binding of a small ligand accelerating exchange [cf. Malin & Englander (1980), Kiener & Waley (1977), Nonnenmacher et al (1971), Printz & Gounaris (1972), and Reynolds et al (1973)].…”

Correlation proton magnetic resonance studies at 250 MHz of bovine pancreatic ribonuclease. II. The pH and inhibitor-induced conformational transitions affecting histidine-48 and one tyrosine residue of ribonuclease A

“…Other lines of evidence have pointed to inhibitor-induced conformational changes in RNase A. rotation studies (Deavin et al, 1966), comparison of deuterium exchange in the absence and presence of inhibitors (Nonnenmacher et al, 1971), and the observation that 2'-CMP significantly reduces the rate of conversion of RNase A to RNase S by subtilisin (Marcus et al, 1968). The present results are in agreement with those of del Rosario and Hammes (1970) on the binding of uridine (or cytidine) 2',3'-cyclic phosphate, namely, that "formation of an initial enzyme-substrate complex [is] followed by a conformational change, with the enzyme-substrate complex able to undergo a conformational change similar to that which the free enzyme undergoes."…”