Conformational stability of ribonuclease T1 determined by hydrogen‐deuterium exchange

Abstract

The hydrogen-deuterium exchange kinetics of 37 backbone amide residues in RNase T1 have been monitored at 25,40, 45, and 50 "C at pD 5.6 and at 40 and 45 "C at pD 6.6. The hydrogen exchange rate constants of the hydrogen-bonded residues varied over eight orders of magnitude at 25°C with 13 residues showing exchange rates consistent with exchange occurring as a result of global unfolding. These residues are located in strands 2-4 of the central P-pleated sheet. The residues located in the a-helix and the remaining strands of the P-sheet exhibited exchange behaviors consistent with exchange occumng due to local structural fluctuations. For several residues at 25"C, the global free energy change calculated from the hydrogen exchange data was over 2 kcal/mol greater than the free energy of unfolding determined from urea denaturation experiments. The number of residues showing this unexpected behavior was found to increase with temperature. This apparent inconsistency can be explained quantitatively if the cis-trans isomerization of the two cis prolines, Pro-39 and Pro-55, is taken into account. The cis-trans isomerization equilibrium calculated from kinetic data indicates the free energy of the unfolded state will be 2.6 kcal/mol higher at 25 "C when the two prolines are cis rather than trans (Mayr LM, Odefey CO, Schutkowski M, Schmid FX. 1996. Kinetic analysis of the unfolding and refolding of ribonuclease T1 by a stopped-flow double-mixing technique. Biochemistry 35: 5550-5561). The hydrogen exchange results are consistent with the most slowly exchanging hydrogens exchanging from a globally higher free energy unfolded state in which Pro-55 and Pro-39 are still predominantly in the cis conformation. When the conformational stabilities determined by hydrogen exchange are corrected for the proline isomerization equilibrium, the results are in excellent agreement with those from an analysis of urea denaturation curves.Keywords: hydrogen-deuterium exchange; NMR; protein folding; ribonuclease TI The principal factors governing the conformational stability of proteins in solution are still not fully understood. While traditional methods of denaturant titration and calorimetry provide valuable information on global stability, no direct information on the conformational dynamics in solution is gained. The internal mobility of proteins can be measured, however, by following the hydrogendeuterium exchange (HX) kinetics of individual backbone amide hydrogens. Hydrogen exchange experiments take advantage of the fact that buried or hydrogen-bonded protons exchange with solvent several orders of magnitude more slowly than exposed or nonhydrogen-bonded protons. More importantly, large differences are also observed in the exchange kinetics of hydrogen-bonded residues. These differences are a direct reflection of the local stability in the region of the hydrogen bond, thus allowing an assessment of Reprint requests to: Frank M. Raushel, Department of Chemistry, Texas A & M University, College Station, Texas 77843; e-mail: r...

Neutralizing Positive Charges at the Surface of a Protein Lowers Its Rate of Amide Hydrogen Exchange without Altering Its Structure or Increasing Its Thermostability

“…Equation 7 expresses the free energy of the transition from a closed to an open state (under EX2 conditions) as a function of the observed rate of H/D exchange. $$\mathrm{\Delta }{\mathrm{normalG}}_{\text{HD}}=-RT\text{ ln }{K}_{\text{op}}=-RT\text{ln}(k_{obs}/k_{\text{int}})$$ The rate of hydrogen exchange has, therefore, been used to measure the conformational stability of folded proteins and the free energy of folding for individual residues in proteins 20,76,77. We found, however, that the least thermally stable (and most highly charged) rungs of the charge ladder were more protected from H/D exchange than the more stable (and less charged) rungs.…”

Influences of temperature and threshold effect of NaCl concentration on Alpias vulpinus OCT

“…There are several reports in the literature of NaCl or KCl effects on proteins stabilization, such as RNase T1 [7,25], stem bromelain and Escherichia coli Trp repressor [10,26]. However, it is often difficult to quantify the relative contributions of salt interactions to the overall protein structures stabilization.…”

Tumor suppressor INK4: comparisons of conformational properties between p16 INK4A and p18 INK4C 1 1Edited by P. E. Wright

“…In some cases, the inconsistency can be attributed to the cis-trans isomerization of a proline residue (Bai et al, 1994;Mullins et al, 1997). However there is no cis-proline observed in NMR studies of both p16 and p18.…”