Protein dynamics. A time-resolved fluorescence, energetic and molecular dynamics study of ribonuclease T1.
Studies using time-resolved fluorescence depolarization were performed on the internal motion of Trp 59 of ribonuclease T1 (EC 3.1.27.3) in the free enzyme, 2'-GMP-enzyme complex and 3'-GMP-enzyme complex. The Trp 59 motion was also studied in the free enzyme using molecular dynamics simulations. Energetic analysis of activation barriers to the Trp 59 motion was performed using both the transition state theory and Kramers' theory. The activation parameters showed a dependence on solvent viscosity indicating the transition state approach in aqueous solution to be inadequate. When taking solvent viscosity contributions into account agreement between the transition state and Kramers' theories was obtained. The results indicate the three enzyme forms to have different conformations with the free enzyme and 3'-GMP-enzyme complex being similar. Comparison of the experimental and theoretical results showed a good agreement on the Trp 59 motion in the free enzyme. Trp 59 appears to vibrate rapidly, with a relaxation time of the order of 1 ps, within free space in the protein matrix and to have a slower motion, with a relaxation time of the order of 100 ps, which is related to breathing of the surrounding protein matrix. Molecular dynamics results indicate high mobility in regions of the enzyme involved in the interaction with the guanine base of the inhibitor or substrate while much lower mobility occurred in residues involved in the catalytic mechanism of ribonuclease T1.
D001236:Aspergillus oryzae / Q000201:enzymology; D004722:Endoribonucleases / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D006163:Ribonuclease T1 / Q000378:metabolism*; D012399:Rotation; D013050:Spectrometry, Fluorescence / Q000379:methods; D013816:Thermodynamics; D013997:Time Factors
D004722:Endoribonucleases; D006163:Ribonuclease T1