Biophysics and Protein Engineering with Noncanonical Amino Acids

Citation

Van Deventer, James A. (2011) Biophysics and Protein Engineering with Noncanonical Amino Acids. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/VT2B-8B33. https://resolver.caltech.edu/CaltechTHESIS:04272011-162341841

Abstract

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.

Studies of water-protein interactions using designed coiled coils containing surface-exposed leucine, homoisoleucine, or trifluoroleucine residues enabled systematic examination of the roles that side chain size and fluorination play in dictating solvation dynamics. Fluorinated side chains appear to exert a large electrostatic drag on nearby water molecules. These results have important implications for the design and engineering of fluorinated proteins due to the critical role water-protein interactions play in many protein properties and functions.

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 high-throughput 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.

Characterization of soluble fragments indicated that each noncanonical amino acid used in this study modulates the binding kinetics of antibody fragments in a distinct fashion. Perhaps most interestingly, fragments containing the azide-containing analog azidohomoalanine exhibit improved binding kinetics relative to their methionine-containing counterparts. Replacement of methionine by azidohomoalanine in several variants lowers the dissociation constant of the fragment by up to a factor of two. Chemical conjugation of azide-containing fragments to fluorescent dyes and biotin proved facile with strain-promoted cycloaddition reactions. Quantifications of the extent of reaction using fluorescent dyes revealed that approximately 0.4 dyes had been conjugated per protein, and the resulting conjugates were found to retain their binding function in kinetic and Western blotting assays. Experiments in which azide-containing fragments were displayed on the surface of Escherichia coli cells and subjected to strain-promoted cycloadditions demonstrated that the extent of chemical modification and antigen binding can be monitored simultaneously and used to isolate cells displaying functional, modified proteins. These experiments demonstrate how noncanonical amino acids can be used to modulate multiple properties of antibody fragments and illustrate the feasibility of developing and screening libraries of chemically modified proteins. Evolved, functional bioconjugates may be applicable to a variety of outstanding diagnostic and therapeutic problems.

Thesis Files