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42214-GB4
Manipulating Protein Interfaces by Photocrosslinking of Genetically Incorporated Unnatural Amino Acids
Project activity
(1) Studying crosslinking proteins with genetically incorporated p-benzyolphenylalanine.
Five crosslinking sites (TAG codons) within 3 different model proteins have been verified by sequencing and protein production containing p-benzyolphenylalanine (pBpa). These three proteins of different interface sizes are triosphosphate isomerase (small), malate dehydrogenase (medium), and histidinol dehydrogenase (large). We irradiated pure protein and washes cells to determine the best crosslinking conditions in vitro and in vivo. By monitoring crosslinking by SDS-PAGE analysis we have learned the propensity of different interface sizes toward crosslinking, the specificity of different types of crosslinking locations and the yields one can expect from protein interfaces.
Juxtaposed to the incorporation site for pBpa we have also altered the predicted site of crosslinking to a variety of natural amino acids in order to test microscopic environmental effects on pBpa crosslinking. Natural amino acids, alanine, phenylalanine, and methionine are being place opposite the pBpa to determine how local environment effects rate and product distribution.
Crosslinking within membrane proteins was preformed within the Tsr bacteria chemoreceptor at nine different sites. Three sites were incorporated at the tip of the receptor in the cytosol and six sites in the membrane spanning region. These sites have been confirmed by sequencing and protein production with western blotting with anti-Tsr antibodies. Crosslinking of these sites has been monitored by size changes on SDS-PAGE followed by western blotting. We have also been working with our collaborators on the bacteria swarming assays that are needed to assess functional protein in living cells.
(2) Studies and development of a new synthetase for the site-directed incorporation of fluorinated phenylalanine analogs.
A synthetase that can site-specifically incorporate p-trifluoromethylphenylalanine was used to probe conformational changes on four different proteins. These labeled proteins have allowed us to monitor conformational changes in the proteins by monitoring 19-NMR chemical shifts upon the binding of substrates, inhibitors, etc. We have optimizing the 19F-protein production conditions so that facile, reproducible production of protein can be produce using this synthetase in rich media and autoinduction media. In collaboration with Dave Wemmer at UC Berkeley we are probing protein dynamics with a two component regulatory protein, NtrC. p-Trifluoromethylphenylalanine has been incorporated at five different sites to determine how phosphorylation effects protein conformations and equilibria.
Findings
(1) Studying crosslinking proteins with genetically incorporated p-benzyolphenylalanine.
We have elucidated that the largest influence on pBpa crosslinking ability at protein interfaces is the size of the contacting interface. Small interface size showed no crosslinking because the pBpa incorporation compromised the integrity of the interface preventing oligomerization. Initial studies on differences in local environment (differences in juxtaposed amino acids structure) currently show little effect on pBpa crosslinking effectiveness or rate.
(2) Studies and development of a new synthetase for the site-directed incorporation of p-trifluormethylphenylalanine.
19F NMR is a powerful probe for monitoring protein conformational changes and interactions, however the inability to site-specifically introduce fluorine labels into proteins of biological interest severely limits its applicability. Using methods for genetically directing incorporation of unnatural amino acids, we have inserted trifluoromethyl-L-phenylalanine (tfm-Phe) into proteins in vivo at TAG nonsense codons with high translational efficiency and fidelity. The binding of substrates, inhibitors, and cofactors, as well as reactions in enzymes, were studied by selective introduction of tfm-Phe and subsequent monitoring of the 19F NMR chemical shifts. Subtle protein conformational changes were detected near the active site and at long distances (25Å). 19F signal sensitivity and resolution was also sufficient to differentiate protein environments in vivo. Since there has been interest in using 19F labeled proteins in solid state membrane protein studies, folding studies, and in vivo studies, this general method for genetically incorporating a 19F-label into proteins of any size in Escherichia coli should have broad application beyond that of monitoring protein conformational changes.
We have been successful in the optimization of additional translation machinery to incorporate trifluoromethyl-L-phenylalanine in response to a TAG codon so that it produced large quantities of labeled protein in rich media. The added convenience of using rich media facilitated more studies as well as other scientist utilization of the new incorporation tools.
Training and Development.
The funds from this grant have enabled the training of eleven undergraduates in synthetic chemistry, molecular biology, and protein research (production, isolation, structural study, and enzymatic assay).
Each student is required to present their work to the Department, to the entire College, and nationally at ACS meetings. So far, ten presentations have been made at national meetings as a result of this funding. Every student who was funded by this research has gone on to continue their education in the sciences. To date seven have enrolled in graduate school or medical school (University of Pennsylvania - graduate school, Texas A&M - graduateschool, Temple - medical school, University of Pennsylvania - medical school, The Scripps Research Institute - graduateschool), University of California Berkeley – graduate school, The University of Pittsburg – graduateschool.
