In the last reporting period we have pursued the study of solution properties and hydration of gamma crystallins. These proteins represent the major protein component in the nucleus of vertebrate eye lenses. They are at present extremely high concentrations, in some species even approaching the close packing limit. Their aggregation is associated with the formation of cataract. The biophysical properties by which gamma crystallins can remain soluble at such high concentrations are poorly understood. In collaboration with Dr. Graeme Wistow (NEI) we studied crystallins from different species. Applying density contrast sedimentation velocity and density contrast sedimentation equilibrium analytical ultracentrifugation, in conjunction with structure- based hydrodynamic modeling and structure-based prediction of the hydration shell, we were able to demonstrate unusually low degrees of hydration for gamma crystallins from the most extreme environments, fish gamma M crystallins, driven by a large number of methionine residues at the protein surface. In contrast, human gamma D crystallin exhibits normal levels of protein hydration. These results shed light on the balance between hydration contributions to protein stability, and the effect of macromolecular crowding. In a separate project in collaboration with Dr. Mark Mayer, we have continued to study the homo- and hetero-oligomerization of amino terminal domains of different glutamate receptor isoforms. The quaternary structure of these receptors controls their ion gating properties. To characterize their interactions we have applied sedimentation velocity analytical ultracentrifugation enhanced with fluorescence detection, for which the high-affinity interactions of glutamate receptors serves as a model system. Finally, we have participated in several other studies: In collaboration with Dr. Amy Hudson, we have established the binding mode of human herpes virus 7 immuno-evasive molecule U21 to host MHC class 1 molecules, allowing us to gain more experience in the application of multi-signal sedimentation velocity techniques;in collaboration with the laboratory of Dr. Alexander Wlodawer we have expanded the multi-method modeling capabilities of our SEDPHAT software to permit the global analysis of fluorescence spectroscopy and isothermal titration calorimetry data of Fabs binding trimeric HIV-1 gp41;in collaboration with Dr. Geoffrey Howlett we have further refined our computational model for the evolution of the length distribution of amyloid fibrils;with Dr. David Margulies we have studied molecules constituting the peptide loading complex, a project potentially providing evolving into a model application for global multi-method analysis; and in collaboration with Dr. George Patterson we have embarked on the characterization of self-association of fluorescent proteins, which sheds light on the potential utility of these molecules in cellular imaging, and at the same time allows us to gain more experience with fluorescent optical detection in the analytical ultracentrifuge.
Showing the most recent 10 out of 39 publications
