The primary structures of proteins contain the chemical characteristics that determine the native folded conformation having the global free energy minimum for the system in a given microenvironment. Analysis of the pathway(s) of polypeptide chain folding and the nature of the intramolecular interactions that stabilize the final native format are active areas of research in many laboratories. Modern methodologies and technical advances in more classical approaches are now offering a real chance for solving these problems with a high degree of precision. From the standpoint of biological system, the synthesis and thermal stability of enzymes of the hyperthermophilic bacteria and Archaea raise further questi ons about the nature of forces involved in intramolecular stabilization. The experimental system focuses on proteins derived from the hyperthermophile Pyrococcus funosus, an obligate anaerobe isolated from geothermally heated marine sediments off the island of Vulcano, Italy. These organisms grow optimally at 100 C and their enzymes are also maximally active at this temperature. Alpha amylase, a serine protease, a DNAase, and a `dithioglycolase` are the subjects of this study. Work with these proteins will involve their purification from natural and from genetically engineered sources. The purified proteins will be analyzed by enzymologic, spectroscopic, and thermodynamic procedures. Crystallization of some of these enzymes will be attempted. From these studies, a data base will be developed that would allow correlations between structural and catalytic properties characteristic of these thermoactive enzyme systems. This information will be analyzed within the framework of the issue of protein folding and stability. Moreover, in the long run, such understanding may aid in the production of stable enzymes with economic and environmental applications. %%% The primary target of this research is to better our understanding of the process of protein folding. Since the biological specificity and activity of all proteins depends on a precisely folded 3D architecture of the molecule, understanding the rules that govern this process is of fundamental importance both to basic science as well as to biotechnology and medicine. A number of years ago Dr. Anfinsen proposed that the amino acid sequence within a protein chain contained all the thermodynamic information necessary to dictate the final folded state of that protein. Much remains to be understood about the rules governing the mechanics specificity and kinetics of the protein folding pathway(s). The relatively recent introduction of the experimental system of Archaebacteria offers new experimental material for the study of the problem of protein folding and stability. Enzymes from these thermophilic bacteria are stable at high temperatures (up to 100 C); most of the times they are actually inactive at conventional temperatures (15 37 C) and develop their activity only as temperature is raised. The P.I. plans to purify certain enzymes (an amylase, a protease and a DNAase) from such thermophilic archaebacteria and study their catalytic, structural and thermodynamic properties. If successful, these studies should further our understanding of the rules of protein folding and enzyme stability, and might, at some day, contribute towards the engineering of enzymes with improved properties.
