Tang, Yi (2002) Protein engineering using unnatural amino acids : incorporation of leucine analogs into recombinant protein in vivo. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-08152006-084149
The incorporation of unnatural amino acids into recombinant proteins is an important tool for understanding protein function, engineering robust proteins and introducing useful building blocks for protein-based materials biosynthesis. While site-directed mutagenesis using natural amino acids allows one to vary protein composition and protein functions, the scope of such manipulations is limited to the twenty naturally occurring amino acids. Important chemical functionalities such as alkenes, alkynes, ketones, halides, and azides are not present in the pool of amino acids specified by the genetic code. Developing methods to insert amino acids containing these orthogonal groups, either site specifically or residue specifically, can lead to new tools in protein chemistry and protein engineering. In this thesis, we will describe the incorporation of leucine analogs into recombinant proteins in vivo. Our objectives are to investigate and relax the substrate specificity of E. coli leucyl-tRNA synthetase towards nonproteinogenic amino acids. Our results show that manipulation of synthetase activity and specificity can provide new opportunities for stabilization and chemoselective modification of proteins.
Substitution of leucine residues by 5,5,5-trifluoroleucine at the d-positions of the leucine zipper peptide GCN4-pld increases the thermal stability of the coiled-coil structure. The midpoint thermal unfolding temperature of the fluorinated peptide is elevated by 13°C at 30 uM peptide concentration. The modified peptide is more resistant to chaotropic denaturants, and the free energy of folding of the fluorinated peptide is 0.5 to 1.2 kcal/mol larger than that of the hydrogenated form. A similarly fluorinated form of the DNA-binding peptide GCN4-bZip binds to target DNA sequences with affinity and specificity identical to those of the hydrogenated forn, while demonstrating enhanced thermal stability. Molecular dynamics simulation on the fluorinated GCN4-pld peptide using the Surface Generalized Born implicit solvation model revealed that the coiled-coil binding energy is 55% more favorable upon fluorination. These results suggest that fluorination of hydrophobic substructures in peptides and proteins may provide new means of increasing protein stability, enhancing protein assembly, and strengthening receptor-ligand interactions.
To make fluorination a general method of stabilizing protein structures, we studied in vivo incorporation of trifluoroleucine (Tfl) and hexafluoroleucine (Hfl) in place of leucine using leucine auxotrophic E. coli strains. The target protein is Al, which is a leucine zipper protein that has 74 residues and eight leucines. The leucine residues are buried at the dimer interface and stabilize the protein complex. Tfl supported protein synthesis efficiently and replaced up to 92% of leucines in the protein under normal expression conditions. The yield of fluorinated protein was reduced from 40 mg/L to 20 mg/L. We were able to tune the level of fluorination by altering the concentration of competing leucine in culture media. Tfl-A1 adopted the identical helical secondary structure and dimeric aggregation order. Tm of Tfl-A1 was elevated to 67°C, a 13°C increase over A1. The concentration of urea needed to denature 50% (Cm) of protein was elevated from 2.7 M to 7 M. In contrast to Tfl incorporation, the more hydrophobic amino acid Hfl did not support protein synthesis under similar conditions. From in vitro characterization of leucyl-tRNA synthetase (LeuRS) substrate specificity, Hfl was shown to be activated 4100 times slower than leucine (compared to the 240-fold rate attenuation of Tfl). The decreased rate of tRNA(Leu), aminoacylation by Hfl resulted in insufficient amounts of Hfl-tRNA(Leu) during protein synthesis. We raised the cellular LeuRS activity eightfold at the time of protein induction by overexpressing LeuRS under a constitutive promoter during cell growth. Under these conditions, Hfl was effectively incorporated into A1 at ~80% substitution rate. The presence of the nearly perfluorinated side chains in the protein core enhanced protein stability even further. Tm was increased to 76°C and [Delta]Gf decreased by 3.6 kcal/mol. More remarkably, Cm of Hfl-A1 was not observed within the urea solubility limit.
To further broaden the chemical functionality available for protein engineering, we investigated the proofreading mechanism of leucyl-tRNA synthetase (LeuRS). The aaRSs that activate the hydrophobic amino acids leucine, isoleucine and valine employ a proofreading mechanism that hydrolyzes noncognate aminoacyl adenylates and misaminoacylated tRNAs. Discrimination between structurally similar amino acids by these AARSs is believed to operate by a double-sieve principle, wherein a separate editing domain governs hydrolysis based on the size and hydrophilicity of the amino acid side chain. Leucyl-tRNA synthetase (LeuRS) relies on its editing function to correct misaminoacylation of tRNA(Leu) by isoleucine and methionine. Thr252 of E. coli LeuRS has been shown previously to be important in defining the size of the editing cavity. Here we report the isolation and characterization of three LeuRS mutants with point mutations at this position (T252Y, T252L, and T252F). The proofreading activity of the synthetase is significantly impaired when an amino acid bulkier than threonine is introduced. The misaminoacylation rate of tRNA(Leu) by isoleucine and valine increases with increasing size of the amino acid substituent at position 252, and the noncognate amino acids norvaline and norleucine are inserted efficiently at leucine sites of recombinant proteins under conditions of constitutive overexpression of the T252Y mutant in E. coli. In addition, the unsaturated amino acids allylglycine, homoallylglycine, homopropargylglycine and 2-butynylalanine all support protein synthesis. These results demonstrate that programmed manipulation of the editing cavity can allow in vivo incorporation of novel protein building blocks.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Major Option:||Chemical Engineering|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||23 May 2002|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||24 Aug 2006|
|Last Modified:||26 Dec 2012 02:57|
- Final Version
Restricted to Caltech community only
See Usage Policy.
Repository Staff Only: item control page