Proteome-wide amino acid substitution and biochemical selection to understand protein biology

Abstract

Proteins are important macromolecules that carry out most biological functions and are enabled to do so by the diverse chemistries of the twenty amino acids. Changes in the amino acid sequence of a protein can change structure, function, and other biochemical properties, sometimes resulting in disease. While genome sequencing has identified numerous mutations that cause changes to the encoded amino acid sequence of proteins, our understanding of the functional consequences is limited. During my thesis work I have contributed to the development and application of mass spectrometry-based methods for understanding the effects of amino acid substitutions at the proteome scale. The work I describe here includes: 1) a screen of noncanonical amino acids to generate random amino acid substitutions across the E. coli proteome for use in functional studies; 2) a high throughput selection method for understanding the impact of pH on protein solubility; and 3) a proteome-wide study of the impact of amino acid substitutions on the critical post-translational modification of phosphorylation. Taken together, these projects provide high throughput methods for directly measuring proteins and understanding how amino acid substitutions impact their own biochemical properties and more broadly, the proteome.