Still Looking for the Ivory Tower

Abstract

Following graduate training, which was disrupted by my changing schools and serving in the Navy in World War II, I arrived in Berkeley in 1948 as an instructor in the Biochemistry Department. Despite numerous academic reorganizations and a host of struggles over the University-imposed Loyalty Oath, dismissal of a faculty member because of political affiliations, free speech for students, and my resistance to mandatory retirement, I survived with the help of great graduate students, postdoctoral fellows, undergraduates, superb research assistants, and a supportive wife. Studies on structure of tobacco mosaic virus led to our investigating an ultracentrifuge anomaly and the construction of a synthetic boundary cell. In turn, this resulted in about 15 years of research on the ultracentrifuge and its application to the study of biological macromolecules. Among the latter, the discovery of large ribonucleoprotein complexes, now known as ribosomes, and chromatophores in photosynthetic microorganisms attracted the most attention. But it was the development of the photoelectric absorption optical system and the incorporation of the Rayleigh interferometer onto the ultracentrifuge that had the greatest impact on our further research. These tools, when applied to our initial research on E. coli aspartate transcarbamoylase (ATCase), led to the discovery of distinct subunits for catalysis and regulation and the global conformational change in the enzyme associated with its role in regulation. For almost 35 years we have been using the techniques of protein chemistry and molecular biology in studies of structural and conformational changes in the enzyme, the genes encoding the different polypeptides, subunit interactions, and assembly of the enzyme from six catalytic and six regulatory chains. Hybrids constructed from inactive mutants were used to demonstrate shared active sites requiring the joint participation of amino acid residues from adjoining polypeptide chains. ATCase is still being studied as a model for understanding allostery as a regulatory mechanism. Circularly permuted polypeptide chains are being used to study the folding and assembly pathways, and the recently determined crystal structure of the active nonallosteric catalytic subunit has led to new questions regarding the activated form of ATCase.

Solution NMR and Computational Methods for Understanding Protein Allostery

“…Thus, changes in the concentrations of protons 1 , metal ions 2 , inhibitors 3, 4 , or metabolites 5 as well as changes in temperature 6 can manifest as alterations in biological processes. Enzymes that respond to allosteric ligands possess at least two spatially distinct binding sites – the active site for the substrate and the effector site for the allosteric ligand.…”

“…Allostery is an essential regulatory mechanism in biology whereby enzyme catalytic activity or protein ligand affinity can be modulated in response to changes in the local environment. Thus, changes in the concentrations of protons, metal ions, inhibitors, , or metabolites as well as changes in temperature can manifest as alterations in biological processes. Enzymes that respond to allosteric ligands possess at least two spatially distinct binding sitesthe active site for the substrate and the effector site for the allosteric ligand.…”

Direct and Simultaneous Measurement of the Stiffness and Internal Friction of a Single Folded Protein

“…The domain behaves like a Hookean solid, having no dissipation with the network intact, whereas it has viscous elements, if the network is broken. Understanding the effects of such local changes on the global properties of folded macromolecules is crucial for unraveling the mechanism of allostery 26,27 and could provide an instructive design principle for synthesizing nanomachines using molecular dynamics simulations. 9 While our stiffness measurements match well with previous efforts, 5,6 the friction coefficient of a single protein's folded state is not yet measured.…”