STRUCTURE AND FUNCTION OF THE GROUP III CHAPERONINS, A UNIQUE CLADE OF PROTEIN FOLDING NANOMACHINES

The survival and descent of cells is universally dependent on maintaining their proteins in a properly folded condition. It is widely accepted that the information for the folding of the nascent polypeptide chain into a native protein is encrypted in the amino acid sequence, and the Nobel Laureate Christian Anfinsen was the first to demonstrate that a protein could spontaneously refold after complete unfolding. However, it became clear that the observed folding rates for many proteins were much slower than rates estimated in vivo. This led to the recognition of required protein-protein interactions that promote proper folding. A unique group of proteins, the molecular chaperones, are responsible for maintaining protein homeostasis during normal growth as well as stress conditions. Chaperonins (CPNs) are ubiquitous and essential chaperones. They form ATP-dependent, hollow complexes that encapsulate polypeptides in two back-to-back stacked multisubunit rings, facilitating protein folding through highly cooperative allosteric articulation. CPNs are usually classified into Group I and Group II. Here, I report the characterization of a novel CPN belonging to a third Group, recently discovered in bacteria. Group III CPNs have close phylogenetic association to the Group II CPNs found in Archaea and Eukarya, and may be a relic of the Last Common Ancestor of the CPN family. The gene encoding the Group III CPN from Carboxydothermus hydrogenoformans and Candidatus Desulforudis audaxviator was cloned in E. coli and overexpressed in order to both characterize the protein and to demonstrate its ability to function as an ATPase chaperone. The opening and closing cycle of the Chy chaperonin was examined via site-directed mutations affecting the ATP binding site at R155. To relate the mutational analysis to the structure of the CPN, the crystal structure of both the AMP-PNP (an ATP analogue) and ADP bound forms were obtained in collaboration with Sun-Shin Cha in Seoul, South Korea. The ADP and ATP binding site substitutions resulted in frozen forms of the structures in open and closed conformations. From this, mutants were designed to validate hypotheses regarding key ATP interacting sites as well as important stabilizing interactions, and to observe the physical properties of the resulting complexes by calorimetry.

Phase transitions in gene networks evolved under different selection rules

Mathematical models of gene regulation are a powerful tool for understanding the complex features of genetic control. While various modeling efforts have been successful at explaining gene expression dynamics, much less is known about how evolution shapes the structure of these networks. An important feature of gene regulatory networks is their stability in response to environmental perturbations. Regulatory systems are thought to have evolved to exist near the transition between stability and instability, in order to have the required stability to environmental fluctuations while also being able to achieve a wide variety of functions (corresponding to different dynamical patterns). We study a simplified model of gene network evolution in which links are added via different selection rules. These growth models are inspired by recent work on `explosive' percolation which shows that when network links are added through competitive rather than random processes, the connectivity phase transition can be significantly delayed, and when it is reached, it appears to be first order (discontinuous, e.g., going from no failure at all to large expected failure) instead of second order (continuous, e.g., going from no failure at all to very small expected failure). We find that by modifying the traditional framework for networks grown via competitive link addition to capture how gene networks evolve to avoid damage propagation, we also see significant delays in the transition that depend on the selection rules, but the transitions always appear continuous rather than `explosive'.