Correlated atomic displacements in crystals of yeast initiatortRNA and aspartate aminotransferase analyzed by x-ray diffuse scattering
Kolatkar, Anand Ratnakar
Phillips, George N., Jr.
Doctor of Philosophy
Biological macromolecules do not function as static molecules. A variety of biological functions are the result of fluctuations in protein and nucleic acid structure. The method of X-ray diffuse scattering analysis has been extended to provide information about intermolecular and intramolecular disorder in protein and nucleic acid crystals. Yeast initiator transfer ribonucleic acid (tRNA) carries the initial methionine to the ribosome during translation initiation. Results from analysis of the yeast initiator tRNA crystal diffuse scattering indicate that anisotropic, lattice-coupled motions contribute to the overall disorder in the crystals. The lattice-coupled disorder implies a flexing motion between the anti-codon and acceptor arms. This type of flexing has been implicated in tRNA's role in protein synthesis on the ribosome. Diffuse scattering analysis also shows that the distal half of the anti-codon arm of tRNA undergoes isotropic short-range motion correlated over a distance of approximately 3 A. This distance corresponds well with the base-pair stacking distance in RNA helices. These results are consistent with nearest-neighbor base pairs moving isotropically and as coherent units in the tRNA anti-codon arm. Aspartate aminotransferase (AspAT) is an important enzyme in the catabolism of amino acids and catalyzes the removal of their amino groups. Crystallographic evidence suggests that the small domain of one of the subunits in the biologically active dimer of AspAT in acetate buffer is disordered. Analysis of the X-ray diffuse scattering from AspAT crystals shows that the small domain moves isotropically and as a rigid body with an amplitude consistent with the crystallographically determined average B-value for the small domain. A preliminary attempt has been made to map the three-dimensional diffuse scattering from a set of simulated still diffraction photographs. This technique has been applied to actual diffraction data from AspAT crystals. The results show that an important diffuse scattering feature is weak but discernible in the reconstructed 3-D volume. X-ray diffuse scattering analysis provides unique experimental evidence for motions in biological macromolecules in crystals. Information obtained about global as well as local intramolecular motion is useful in understanding the role of molecular flexibility in biological function.