Title

Expression of Human Indoleamine 2,3-Dioxygenase (hIDO1) Variants with Enhanced Spectroscopic Properties for Biological Studies

Presentation Type

Poster

Abstract

The heme containing protein, Indoleamine 2,3-Dioxygenase (hIDO1), is the rate limiting enzyme for tryptophan metabolism and initiates what is known as the kynurenine pathway, which accounts for 90% of human tryptophan catabolism. High levels of hIDO1 have been identified in malignant tumor cells, which diminish the amount of tryptophan available for downstream biosynthesis such as T Cell production and maturation. In order to engineer an hIDO1 inhibitor that will provide maximal benefits, it is crucial to understand the dynamics of this enzyme during catalysis. Wild type hIDO1 is a protein made from 403 amino acids. The published crystal structure shows an unresolved region near the active site, from amino acid residues 361 to 380 and the suspected structure that these 19 residues form is a loop. We hypothesize that this loop is the gate keeper for substrate entry, thereby involved in turn over efficiency and a key to understanding protein dynamics. In order for this research to take place, we constructed an hIDO1 clone in the vector, pQE30, which possesses a T5 promoter that is essential for a (Trp-) auxotrophic strain of ecoli. Of the 403 amino acid residues which form hIDO1, six of which are tryptophans. We have successfully created tryptophan to either phenylalanine or tyrosine mutants, Quik-change Site-Directed Mutagenesis Kit from Agilent. Our approach for studying dynamics is tryptophan fluorescence by characterizing wild type hIDO1, tryptophan mutants and the tryptophan insertion into the loop. In effort to test the hypothesis of the unresolved loop involvement, we are taking time-resolved fluorescence measurements and comparing wild type hIDO1 to the tryptophan mutant which contains additional tryptophan residue, (L374W) near the active site and part of the loop structure in order to observe changes in protein
dynamics.

Category

Physical Sciences

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Apr 17th, 3:00 PM Apr 17th, 4:00 PM

Expression of Human Indoleamine 2,3-Dioxygenase (hIDO1) Variants with Enhanced Spectroscopic Properties for Biological Studies

South UC Ballroom

The heme containing protein, Indoleamine 2,3-Dioxygenase (hIDO1), is the rate limiting enzyme for tryptophan metabolism and initiates what is known as the kynurenine pathway, which accounts for 90% of human tryptophan catabolism. High levels of hIDO1 have been identified in malignant tumor cells, which diminish the amount of tryptophan available for downstream biosynthesis such as T Cell production and maturation. In order to engineer an hIDO1 inhibitor that will provide maximal benefits, it is crucial to understand the dynamics of this enzyme during catalysis. Wild type hIDO1 is a protein made from 403 amino acids. The published crystal structure shows an unresolved region near the active site, from amino acid residues 361 to 380 and the suspected structure that these 19 residues form is a loop. We hypothesize that this loop is the gate keeper for substrate entry, thereby involved in turn over efficiency and a key to understanding protein dynamics. In order for this research to take place, we constructed an hIDO1 clone in the vector, pQE30, which possesses a T5 promoter that is essential for a (Trp-) auxotrophic strain of ecoli. Of the 403 amino acid residues which form hIDO1, six of which are tryptophans. We have successfully created tryptophan to either phenylalanine or tyrosine mutants, Quik-change Site-Directed Mutagenesis Kit from Agilent. Our approach for studying dynamics is tryptophan fluorescence by characterizing wild type hIDO1, tryptophan mutants and the tryptophan insertion into the loop. In effort to test the hypothesis of the unresolved loop involvement, we are taking time-resolved fluorescence measurements and comparing wild type hIDO1 to the tryptophan mutant which contains additional tryptophan residue, (L374W) near the active site and part of the loop structure in order to observe changes in protein
dynamics.