Conformational stability of ribonuclease T1 determined by hydrogen‐deuterium exchange

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 β‐pleated sheet. The residues located in the α‐helix and the remaining strands of the β‐sheet exhibited exchange behaviors consistent with exchange occurring 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.

[1]  H. Dyson,et al.  Comparison of the hydrogen-exchange behavior of reduced and oxidized Escherichia coli thioredoxin. , 1995, Biochemistry.

[2]  A. D. Robertson,et al.  Temperature and pH dependences of hydrogen exchange and global stability for ovomucoid third domain. , 1996, Biochemistry.

[3]  L Mayne,et al.  Primary structure effects on peptide group hydrogen exchange. , 1972, Proteins.

[4]  P. Alexander,et al.  Assessment of stability differences in the protein G B1 and B2 domains from hydrogen-deuterium exchange: comparison with calorimetric data. , 1995, Biochemistry.

[5]  J. Markley,et al.  Hydrogen exchange in unligated and ligated staphylococcal nuclease. , 1993, Biochemistry.

[6]  S. L. Mayo,et al.  Guanidinium chloride induction of partial unfolding in amide proton exchange in RNase A. , 1993, Science.

[7]  A. Fersht,et al.  Relationship between equilibrium amide proton exchange behavior and the folding pathway of barnase. , 1995, Biochemistry.

[8]  A. D. Robertson,et al.  Thermodynamics of unfolding for turkey ovomucoid third domain: Thermal and chemical denaturation , 1993, Protein science : a publication of the Protein Society.

[9]  J Clarke,et al.  An evaluation of the use of hydrogen exchange at equilibrium to probe intermediates on the protein folding pathway. , 1996, Folding & design.

[10]  U. Hahn,et al.  Folding of RNase T1 is decelerated by a specific tertiary contact in a folding intermediate , 1992, Proteins.

[11]  U. Hahn,et al.  Folding of ribonuclease T1. 2. Kinetic models for the folding and unfolding reactions. , 1990, Biochemistry.

[12]  S W Englander,et al.  Isotope effects in peptide group hydrogen exchange , 1993, Proteins.

[13]  F. Raushel,et al.  Are turns required for the folding of ribonuclease T1? , 1996, Protein science : a publication of the Protein Society.

[14]  L Mayne,et al.  Protein folding studied using hydrogen-exchange labeling and two-dimensional NMR. , 1992, Annual review of biophysics and biomolecular structure.

[15]  C. Dobson,et al.  Hydrogen exchange in native and denatured states of hen egg‐white lysozyme , 1992, Proteins.

[16]  D. W. Bolen,et al.  Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. , 1988, Biochemistry.

[17]  C. Pace,et al.  Investigation of ribonuclease T1 folding intermediates by hydrogen-deuterium amide exchange-two-dimensional NMR spectroscopy. , 1993, Biochemistry.

[18]  C. Woodward,et al.  Hydrogen exchange identifies native-state motional domains important in protein folding. , 1993, Biochemistry.

[19]  U. Hahn,et al.  Folding of ribonuclease T1. 1. Existence of multiple unfolded states created by proline isomerization. , 1990, Biochemistry.

[20]  A. Chaffotte,et al.  Structure of a rapidly formed intermediate in ribonuclease T1 folding , 1992, Protein science : a publication of the Protein Society.

[21]  H. Rüterjans,et al.  Ribosomal protein interactions in yeast. Protein L15 forms a complex with the acidic proteins. , 1988 .

[22]  N. Kallenbach,et al.  Hydrogen exchange and structural dynamics of proteins and nucleic acids , 1983, Quarterly Reviews of Biophysics.

[23]  I. Brand,et al.  Properties of a 19-kDa Zn2+-binding protein and sequence of the Zn2+-binding domains. , 1988, European journal of biochemistry.

[24]  D. W. Bolen,et al.  Unfolding free energy changes determined by the linear extrapolation method. 2. Incorporation of delta G degrees N-U values in a thermodynamic cycle. , 1988, Biochemistry.

[25]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[26]  U. Hahn,et al.  Replacement of a cis proline simplifies the mechanism of ribonuclease T1 folding. , 1990, Biochemistry.

[27]  Yawen Bai,et al.  Protein stability parameters measured by hydrogen exchange , 1994, Proteins.

[28]  U. Heinemann,et al.  Ribonuclease T1 with free recognition and catalytic site: crystal structure analysis at 1.5 A resolution. , 1991, Journal of molecular biology.

[29]  J. D. Bernal.  Structure of Proteins , 1939, Nature.

[30]  W. Saenger,et al.  Structurally conserved water molecules in ribonuclease T1. , 1991, The Journal of biological chemistry.

[31]  A. Hvidt,et al.  Hydrogen exchange in proteins. , 1966, Advances in protein chemistry.

[32]  E A Merritt,et al.  Raster3D Version 2.0. A program for photorealistic molecular graphics. , 1994, Acta crystallographica. Section D, Biological crystallography.

[33]  Protein internal flexibility and global stability: effect of urea on hydrogen exchange rates of bovine pancreatic trypsin inhibitor. , 1993, Biochemistry.

[34]  C. Pace,et al.  Thermodynamics of ribonuclease T1 denaturation. , 1992, Biochemistry.

[35]  C. Pace,et al.  Energetics of ribonuclease T1 structure. , 1994, Biochemistry.

[36]  F. Schmid,et al.  Kinetic analysis of the unfolding and refolding of ribonuclease T1 by a stopped-flow double-mixing technique. , 1996, Biochemistry.