All proteins sample a diverse array of conformations (folded, unfolded, and excited states) with differing free energies and dynamics depending on the environmental conditions. A protein’s primary sequence encodes more than just the native structure; it encodes the entire energy landscape – an ensemble of conformations whose energetics and dynamics are finely tuned and modulated. The goal of this proposal is a quantitative and predictive understanding of the relationship between sequence and the landscape, together with an understanding of how a protein’s environment modulates this landscape. A major hurdle in going from sequence to function is our lack of understanding of the non-native regions of the landscape. High-energy conformations are important for directing the stability and folding of a protein, and modulations of this ensemble play a role in misfolding, protein signaling, catalytic activity, and allostery. While many sequences can encode the same structure, their function and dynamics can vary dramatically – due to changes in the landscape. Small variations in a sequence can have effects that range from undetectable to pathological. Soon we will have access to thousands of human genomes, and without an ability to interpret variation, the potential of these data to impact medicine and human health will not be fully appreciated. The experiments outlined here will focus on how modulations in the cellular environment (the ribosome, post-translational modifications and co-translational folding) affect a protein’s energy landscape as well as new approaches to investigate the effect of sequence variation on these landscapes. Aim 1. How do cellular components (the ribosome and ubiquitination) modulate the energy landscape? a. Determine the effect of ubiquitination on the energy landscape of target proteins. b. Determine the effects of the ribosome on the energy landscape of ribosome-bound nascent chains (RNCs), monitoring kinetics and hydrogen exchange. Aim 2: Biophysical studies of co-translational folding a. Determine the role of topology by studying the co-translational folding of circular permutants of HaloTag. b. Monitor protein folding in complex environments using X-ray hydroxyl radical footprinting (XF/MS). c. Probe the temporal coordination between translation and nascent chain folding using single-molecule fluorescence in zero-mode waveguides. Aim 3: ASR studies to probe the sequence determinants of protein landscapes a. Probing the rate-limiting step in protein folding by investigating a family containing kinetically stable and thermodynamically stable proteins. b. ASR analysis on the alpha-lytic protease family.