In situ structural biology : quantitative, comprehensive, and ultra-sensitive protein footprinting in living cells
Keywords: Biochemistry.

Most cellular processes occur through changes in protein conformation, interactions, and/or ligand binding. Molecular understanding of these events would ideally be achieved by probing them quantitatively and at high structural resolution in living cells, but this is not possible with existing structural techniques. Emerging technologies for high-throughput protein footprinting provide a possible approach in which a protein's per-residue solvent exposure reports on its local conformation, but the goals of comprehensive coverage of all amino acids, quantitative accuracy, and sufficient sensitivity remain challenging, even for measurements made on purified protein. I provide an overview of footprinting approaches in Chapter 1, presenting the possibilities enabled by the technology and describing the substantial hurdles that have stood in the way of it being a viable technology for in-cell analysis of protein conformation. In Chapter 2, I present an in-cell footprinting technique that can quantitatively monitor solvent accessibility at virtually all of a protein's residues using our cysTRAQ label, enabling sensitive detection of footprinted peptides by mass spectrometry. CysTRAQ footprinting exploits three technical innovations, the development and benchmarking of which are described in detail. The first innovation is the development of a shotgun footprinting approach that enables comprehensive coverage of a protein of interest. Using commercially-available arrayed oligonucleotides, we generate hundreds of single cysteine mutants in an expression plasmid in one day and pool the resulting cysteine libraries for in-cell labeling. Second, we designed mass-tagged cysteine alkylating agents that are cheap, compact, and cell-permeable. These cysTRAQ reagents enable precise kinetic footprinting analysis, employing a novel strategy for LC-MS quantification of modified cysteine probes in tandem mass spectra. Finally, we devised a strategy for > 300-fold purification of cysTRAQ-labeled peptides from the vast excess of unmodified peptides, improving both signal to background in isotope ratio data and amino acid coverage in target proteins. This enrichment is mediated by the cysTRAQ reagent's compact hydroxamate moiety, which we present as a novel affinity tag enabling purification by immobilized ytterbium affinity chromatography. Combining these technical advances, we demonstrate the use of cysTRAQ footprinting on a bacterial ribose-binding protein (RBP) in live cells. We describe two experimental formats suited to different types of biological questions: one in which cysTRAQ footprinting encodes the absolute solvent exposure at a given site on the protein and one in which it encodes the relative solvent exposure differences between two conformational states. We compared RBP's footprints in three different environments, finding that labeling rates on purified protein correlated well with rates in the E. coli periplasm (its native environment) and in the cytoplasm. These rates spanned more than three orders of magnitude, providing data on both exposed and buried regions of the protein. Importantly, periplasmic data from both the absolute and the relative cysTRAQ encoding schemes pinpointed RBP's dynamic ligand-binding interface--the residues with ribose-dependent changes in solvent exposure are located in the mouth and hinge regions of RBP previously implicated in ribose binding. This high-throughput approach provides quantitative measures of conformation at single amino acid resolution at more than 50% of the residues in the protein. We also demonstrate how footprinting can guide the design of real-time fluorescent sensors of conformational change. Finally, Chapter 3 describes some of the difficult questions that can be addressed using this novel approach to in-cell measurements of protein structure and reactivity.