Molecular Mechanisms and Design of Protein Folding, Function, and Aggregation

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Proteins are exquisite molecular machines - they perform a vast range of functions, from acting as catalysts, to specifically recognizing and binding all kinds of molecules, to structural support roles. The primary amino acid sequence of a protein determines how it folds to its 3D structure, which in turn determines its function.  Despite extensive study, the details of the molecular mechanisms governing protein folding, aggregation and function remain obscure. Defining these mechanisms is of tremendous importance because such knowledge will ultimately enable the precise prediction and control of the specific properties of any protein: in natural processes, in disease and in biotechnology. It will give us the ability to engineer or design proteins for any desired application! Natural and engineered proteins are widely used in research and industry and are transforming biotechnology through their applications in material science, and as catalysts and drugs. Explosive growth in knowledge of protein sequences, structures and functions and in computational modeling of proteins has set the stage for uncovering the molecular mechanisms that determine protein properties. For example, based on our experimental measurements of protein solubility, we recently developed new structure-based methods to predict and design the solubility of engineered target binding proteins known as Adnectins. As for many proteins, Adnectins form insoluble inclusion bodies (IBs) when expressed in bacteria, and the level of IB formation is related to the solubility of the pure protein. Our findings for Adnectins are important for controlling solubility when developing Adnectins for medical applications. In addition, we advanced a method for high resolution structure analysis of IBs using quenched H/D exchange nuclear magnetic resonance spectroscopy (NMR). Applying this method revealed native-like structure in IBs, which has important implications for developing IBs as functional materials. In a separate project, we developed a combined NMR and molecular dynamics computational method that revealed at atomic resolution the mechanism of pH-dependent switching function in Hisactophilin, which controls cell movement. This powerful method is widely applicable to defining local structural stability and the population of alternative states that control protein structure and function. Our discoveries for these and other proteins (including numerous mutants which we design to systematically grow our knowledge) are obtained using a battery of experimental (spectroscopy, calorimetry, light scattering) and computational tools.  We will build on our previous exciting discoveries to advance knowledge of how proteins work and how they can be harnessed for a myriad of applications; this research will provide valuable collaborative interdisciplinary and international training for many highly qualified personnel in developing proteins for further great benefit to society.