Sequence and Environmental Determinants of the Protein Energy Landscape

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All proteins sample a diverse array of conformations (folded, unfolded, and excited states) with differing free energies and dynamics depending on the environmental conditions. We can now predict a structural model for the folded state given the amino acid sequence. The sequence of a protein, however, encodes much more than just this native structure – it encodes the entire energy landscape – an ensemble of conformations whose populations (energetics) and dynamics are finely tuned and critical for proper function and cellular health. A major hurdle in going from sequence to function is our lack of understanding of the non-native regions of the landscape. These high-energy conformations are important for directing the stability, dynamics and folding of a protein, and modulations of this ensemble play a role in misfolding, protein signaling, catalytic activity, and allostery. A compromised landscape, due to either changes in the cellular milieu, intrinsic genetic defects, or the cumulative effects of cellular stresses, has been linked to disruption of proteostasis, resulting in varying misfolding diseases and pathologies. Rare and transient conformations are, by their very nature, difficult to study. For decades, biophysical chemists (including the PI) have been probing these fluctuations with high-level technologies using purified proteins in a test tube. The test tube, however, is very different from the cell. In vivo, proteins live in a crowded cellular environment, subject to quality control machinery, cellular modifications and subject to non-equilibrium effects such as protein synthesis and degradation. In order to take full advantage of the wealth of detailed, quantitative biophysical data available from in vitro studies, we need to understand how cellular factors and the cellular environment modulate the energetics and dynamics. Such complex settings, however, are inaccessible to the standard toolbox used for quantitative biophysical studies. The PI is an expert in the area of protein folding and dynamics, having devoted most of her career to developing and utilizing sophisticated technologies to probe rare and transient conformations, both at the single molecule and ensemble level. This current proposal focuses on: 1) understanding how these states are modulated by features in the cell, such as co-translational folding, post-translational modifications, and 2) understanding how the dynamics of conformational changes are controlled at the sequence level. The long- term goal is a is a molecular, quantitative, and predictive understanding of the relationship between sequence and the energy landscape, together with a predictive understanding of how the environment modulates this landscape.