Lanterna Rally

Author: John Tuan Ngo

Publisher:

Published: 2012

Total Pages: 256

ISBN-13:

Proteins in living cells can be made receptive to bioorthogonal chemistries through metabolic labeling with appropriately designed noncanonical amino acids (ncAAs). In the simplest approach to metabolic labeling, an amino acid analog replaces one of the natural amino acids specified by the protein's gene (or genes) of interest. This approach, often termed "residue-specific incorporation," allows the ncAA to be incorporated in controlled proportions into positions normally occupied by the natural amino acid residue. Chapter I of this thesis describes how this strategy has been used to track cellular protein synthesis with reactive ncAAs. In procedures similar to isotopic labeling, translationally active ncAAs are incorporated into proteins during a "pulse" in which newly synthesized proteins are tagged. The set of tagged proteins can be distinguished from those made before the pulse by bioorthogonally ligating the ncAA side chain to probes that permit detection, isolation, and visualization of the labeled proteins. Chapter II of this thesis describes how the selectivity of the method can be enhanced through the use of mutant aminoacyl tRNA synthetases (aaRSs) that permit incorporation of ncAAs not used by the endogenous biomachinery. Expression of a mutant synthetase in a portion of cells within a complex cellular mixture restricts labeling to that subset of cells. In multicellular environments, this approach permits the identification of the cellular origins of labeled proteins. The work in Chapter III illustrates how the extent of temporal and spatial resolution of protein labeling can be enhanced through controlled expression of mutant synthetases. Use of characterized promoters to direct transcription of mutant synthetase genes can limit labeling to relevant cells and physiological states in settings of increased complexity. Chapter IV presents a novel strategy with which ncAAs can be uniquely incorporated at the N-terminal positions of nascent proteins while excluded from insertion at internal positions. This approach permits "site-selective" tagging of cellular proteins, and its use in tagging and visualization of cell-cycle dependent protein synthesis is described. The work described throughout this thesis was designed with the objective of providing powerful and versatile methods for the study of protein synthesis in complex multicellular systems, including live animals. Thus, Chapter V considers how these strategies might be used to dissect protein synthesis in living animals.