| Noncanonical amino acids are tools for expanding and altering the chemical functionalities available within proteins. Much recent work has focused on developing biosynthetic means for incorporating noncanonical amino acids into proteins, and applications of noncanonical amino acids to many problems in science and engineering are emerging. The first portion of this thesis describes established methods to incorporate noncanonical amino acids into proteins and efforts to exploit the properties of noncanonical amino acids in areas such as protein structure determination, protein and organism evolution, modulation of the immune system, and proteomics. Researchers' creative and successful use of this growing toolkit suggests that noncanonical amino acids will continue to be a valuable asset for dissecting biological problems and imparting proteins with new chemical and physical properties.;Biophysical studies with noncanonical amino acids provide a platform for studying the effects of atom-by-atom manipulations of amino acid side chains on protein properties. The middle portions of this thesis describe work to better understand how protein properties are affected by subtle amino acid side chain manipulations. This work was aided greatly by the establishment of homoisoleucine as a translationally active analog of leucine in bacterial cells. The small side chain differences between leucine, homoisoleucine, and the fluorinated amino acid trifluoroleucine allow for detailed studies on how amino acid side chain size and fluorination affect protein stability and hydration dynamics. Replacement of leucine by homoisoleucine in coiled-coil peptides stabilizes these proteins, as shown by elevation of the coiled coil thermal denaturation temperature. The stabilization observed when homoisoleucine replaces leucine in the peptides is greater than when trifluoroleucine replaces leucine, suggesting that expansion of side chain volume may play a role in protein stabilization irrespective of hydrocarbon or fluorocarbon character.;The final portion of this thesis details efforts to engineer the binding properties and chemical reactivity of antibody fragments with noncanonical amino acids. The properties of the single chain variable fragment form of a model anti-digoxin antibody have been studied after replacement of the protein's methionine residues with methionine analogs containing alkyne, azide, and aliphatic side chains. Experiments with antibody fragments displayed on the surface of Escherichia coli cells revealed that replacement of the methionine residues of the fragment with an analog containing an alkyne side chain reduced the fluorescence levels of cells treated with a fluorescently labeled antigen to background levels, indicating loss of binding function. Replacement of methionine with analogs containing aliphatic and azide side chains left the fluorescence of cells unchanged and reduced by a factor of 0.6, respectively. Fluorescence-activated cell sorting of libraries of cell surface-displayed antibody fragments enabled the isolation of clones functional in multiple amino acid contexts. Cells displaying variants containing alkyne, azide, and aliphatic analogs and treated with fluorescently labeled antigen were more fluorescent than cells displaying the methionine form of the parent antibody fragment by factors of roughly 1.7, 3.5, and 1.3, respectively. Furthermore, the amino acid context used during highthroughput screening experiments appears to affect the frequencies of mutations occurring at various positions within the antibody fragment construct. High-throughput sequencing revealed that populations isolated in different amino acid contexts exhibit mutational rates differing by greater than twenty percent at some residues in the protein. (Abstract shortened by UMI.)... |
