Experimental and Theoretical Studies of Charge-Charge Interactions in Proteins

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The objective of this project is to further understand the role of charge-charge interactions in determining the thermodynamic and kinetic stability of proteins and enzymes. The experimental work will be performed with several model proteins and enzymes, each uniquely suited for the questions to be addressed. The folding kinetics of rationally stabilized variants of cold shock proteins (CspB-Bs and CspB-TB), ubiquitin (UBQ), the activation domain of human procarboxypeptidase A2 (ADA2h), the fibronectin type III domain of human tenascin (TnfIII), human acylphosphatase (ACPh), and the N-terminal RNA-binding domain of human U1A protein (U1A) will be compared with the corresponding wild type proteins. The major question to be addressed is whether there is a predominant kinetic mechanism of stabilization for proteins with optimized surface charge-charge interactions. The role of the stability of individual domains for the function of a model two-domain protein, the yeast 3-phosphoglycerate kinase (PGK), which undergoes large conformational changes during the catalysis, will also be studied. The major question that will be addressed is whether the rational optimization of surface charge-charge interactions of individual domains can be done without affecting the overall activity of PGK. Finally, the role of protein stability in supporting the activity of enzymes at low temperature will be investigated using comparative studies of a model enzyme, S-adenosylmethionine decarboxylase (AdoMetDC) from psychrophilic, mesophilic and thermophilic organisms. This research effort will use methods of rational protein engineering and design to modulate stabilities of the psychrophilic, mesophilic and thermophilic AdoMetDC proteins. Such multidisciplinary approaches will enable to gain insights into the adaptation mechanisms that are related to protein stability. To this end, a combination of various methods such as comparative sequence analysis, computational modeling, protein design, a battery of biophysical methods (differential scanning calorimetry, isothermal titration calorimetry, circular dichroism spectroscopy, fluorescence spectroscopy, NMR spectroscopy, analytical ultracentrifugation, dynamic light scattering, stopped-flow) and biochemical methods to characterize the biophysical mechanisms and structural determinants that lead to modulation of the stability of model proteins and enzymes will be used. This project will provide a systematic study of the mechanism of protein stabilization by uniquely combining a variety of biochemical, biophysical and computational tools. As such, it is expected that the knowledge accumulated from these experiments will lay the foundation for future studies of these fundamentally important issues with broad implications for many different areas including the development of the next generation of biosensors, environmentally friendly catalysts and robust biomaterials. The PI is actively involved in graduate and undergraduate education and curriculum development. In addition, students at all levels (high-school, undergraduate and graduate) will directly participate in both the computational and experimental aspects of all research efforts in the PIs laboratory.