Professor of the Graduate School Division of Biochemistry, Biophysics and Structural Biology
We are interested in the structure, function, assembly and interactions of biological macromolecules, with particular emphasis on the regulatory enzyme, aspartate transcarbamylase (ATCase).
Revisiting allostery. Recent x-ray diffraction studies resulting in a high-resolution structure of the free, nonallosteric, catalytic trimer of wild-typeATCase and mutant forms of the holoenzyme call into question the widely accepted structure of the so-called, relaxed (R) conformation of the holoenzyme. Previous crystallographic studies had designated the complex of ATCase with a bisubstrate ligand as the R state of the enzyme. However, it is now known from these recent studies that changes in the conformation may be the result of the actual binding event rather than the allosteric transition whereby the enzyme is converted from an inactive, taut (T) state to the activated R conformation. Various mutant forms with greatly altered allosteric equilibria serve as excellent targets for resolving this perplexing problem and for demonstrating the role of flexibility in controlling enzyme activity.
Formation of active enzyme from circularly permuted and fragmented polypeptide chains. Instead of the polypeptide starting at residue 1 as in wild-type ATCase, it is formed biosynthetically with N- and C-termini at many different locations in the chain and the wild-type termini linked through a covalent bond. These circularly permuted molecules serve as models to relate in vivo folding of growing polypeptide chains to in vitro refolding studies of denatured proteins. We have developed a method for producing randomly circularly permuted genes, inserting them in an appropriate vector, and allowing E. coli to select those permuted chains, which assemble into enzyme. Parallel studies with fragmented polypeptide chains are aimed at understanding the formation of domains and the effect of deletions of helical regions on stability and the folding and assembly pathways.
Quantitative Urea Gradient Gel Electrophoresis for Studies of Dissociation and Unfolding of Oligomeric Proteins. [N. N. Kalnine and H. K. Schachman (2002) Biophysical Chemistry (in press)]
In vivo assembly of aspartate transcarbamoylase from fragmented and circularly permuted catalytic polypeptide chains. [X. Ni and H. K. Schachman (2001) Protein Sci. 10, 519-527]
Random circular permutation leading to chain disruption within and near a-helices in the catalytic chains of aspartate transcarbamoylase: Effects on Assembly, Stability and Function. [P. T. Beernink, Y. R. Yang, R. Graf, D. S. King, S. S. Shah, and H. K. Schachman (2001) Protein Sci. 10, 528-537]
Binding of bisubstrate analog promotes large structural changes in the unregulated catalytic trimer of aspartate transcarbamoylase: Implications for allosteric regulation. [J. A. Endrizzi, P. T. Beernink, T. Alber, and H. K. Schachman (2000) Proc. Natl. Acad. Sci. USA 97, 5077-5082]
Last Updated 2003-09-05