Novel Techniques for Characterization of Biomolecular Behavior Across Multiple Biological Scales

<p>The discovery and development of the central dogma of molecular biology in the late 1950's unlocked a now booming field of medical innovation centered about the study of biomolecules. Among these, proteins play perhaps the most active role, acting as molecular devices to carry out the majority dynamic processes of life maintenance including energy metabolism, DNA replication, cellular signaling, and many other important functions. Proteins have historically been studied in small subsets; however, recent years have shown a paradigm shift toward studying complex systems of proteins and interactions in physiologically relevant conditions. This shift is driven by advances in both sequencing and computing technology that have enabled novel approaches in integrative biology. Here we present two such techniques that combine computational and experimental methods to elucidate protein interactions with previously indiscernible temporal resolution across multiple scales.</p> <p>The first of these is a tool to measure protein binding affinity and kinetics. Fluorescence Rolling Correlation Spectroscopy (FRCS) is a novel algorithm and software that utilizes principles of diffusometry to enable facile characterization of binding kinetics across the entire dynamic range of typical protein-protein interactions (ka ϵ(1E4,1E6) M^-1s^-1, kd ϵ(1E-4,1E1) s^-1). The second is a methodology of non-canonical amino acid (ncAA) proteome labeling using Azidohomoalanine (Aha) to selectively label nascent proteins <i>in vivo</i> for enrichment or imaging. We further characterize labeling dynamics of Aha with both <i>in vivo </i>and <i>in silico</i> models. Each of tools is presented with open source software and models to enable future work in the study of protein behavior.</p>