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.

Clouds of Suspicion: Airspace Arrangements, Escalation, and Discord in U.S./NATO-Russian Relations

How have cooperative airspace arrangements contributed to cooperation and discord in the Euro-Atlantic region? This study analyzes the role of three sets of airspace arrangements developed by Euro-Atlantic states since the end of the Cold War—(1) cooperative aerial surveillance of military activity, (2) exchange of air situational data, and (3) joint engagement of theater air and missile threats—in political-military relations among neighbors and within the region. These arrangements provide insights into the integration of Central and Eastern European states into Western security institutions, and the current discord that centers on the conflict in Ukraine and Russia’s place in regional security. The study highlights the role of airspace incidents as contributors to conflict escalation and identifies opportunities for transparency- and confidence-building measures to improve U.S./NATO-Russian relations. The study recommends strengthening the Open Skies Treaty in order to facilitate the resolution of conflicts and improve region-wide military transparency. It notes that political-military arrangements for engaging theater air and missile threats created by NATO and Russia over the last twenty years are currently postured in a way that divides the region and inhibits mutual security. In turn, the U.S.-led Regional Airspace Initiatives that facilitated the exchange of air situational data between NATO and then-NATO-aspirants such as Poland and the Baltic states, offer a useful precedent for improving air sovereignty and promoting information sharing to reduce the fear of war among participating states. Thus, projects like NATO’s Air Situational Data Exchange and the NATO-Russia Council Cooperative Airspace Initiative—if extended to the exchange of data about military aircraft—have the potential to buttress deterrence and contribute to conflict prevention. The study concludes that documenting the evolution of airspace arrangements since the end of the Cold War contributes to understanding of the conflicting narratives put forward by Russia, the West, and the states “in-between” with respect to reasons for the current state of regional security. The long-term project of developing a zone of stable peace in the Euro-Atlantic must begin with the difficult task of building inclusive security institutions to accommodate the concerns of all regional actors.