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Isotopic labeling of heme in dehaloperoxidase and CYP102A2 for NMR studies

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
David Irby Bryson (Creator)
Institution
The University of North Carolina at Greensboro (UNCG )
Web Site: http://library.uncg.edu/
Advisor
Gregory M. Raner

Abstract: "13C NMR has been shown to be a powerful tool for probing the electronic distribution in paramagnetic hemes and hemoproteins. Methods that can measure the coordination state and electronic structure of the paramagnetic hemes and hemoproteinscan be very useful for mechanistic strudies. The drawback with this technique is the low natural abundance of 13C in the macrocycle, which hinders sensitivity. Advances in the use of recombinant expression systems have provided us with the resources to selectively label the heme cofactor of paramagnetic heme-containing proteins and enzymes. However, current methodologies of replacing the prosthetic heme with 13C labeled heme in P450 apoproteins are not compatible with cytochromes P450 due to their denaturation upon the removal of the cofactor. We have developed a recombinant expression system, using the hemoprotein dehaloperoxidase as a model, that allows us to selectively label the heme cofactor biosynthetically by supplementing aminolevulinic acid (ALA) deficient E. coli Hu227 cells with synthesized isotopomers of ALA. We have also generated a recombinant plasmid encoding for CYP102A2 that will be compatible with our novel expression system, and have preliminary data indicating this method should be very useful for generating cytochrome P450 samples for 13C analysis."--Abstract from author supplied metadata.

Additional Information

Publication
Thesis
Language: English
Date: 2007
Keywords
13C NMR, tool, electronic distribution, paramagnetic hemes, hemoproteins,
Subjects
Nuclear magnetic resonance spectroscopy
Heme