THE SIR SUPPLIED DR. WOODWARD WITH L-[4-13C]ASPARTIC ACID (500 MG). E. COLI. THIOREDOXIN IS A PROTOTYPE OF AN IMPORTANT AND UBIQUITOUS FAMILY OF OXIDOREDUCTASES. OUR STUDIES ARE AIMED AT FUNDAMENTAL QUESTIONS OF REACTIVITY AND FOLDING WHICH APPLY TO THE FAMILY AS A WHOLE. THIOREDOXIN ACTIVITY INVOLVES THE DISULFIDE-DITHIOL OXIDATION/REDUCTION OF THE TWO ACTIVE SITE CYS RESIDUES. NEAR THE ACTIVE SITE IS ASP26, ALMOST BUT NOT COMPLETELY BURIED AT THE BOTTOM OF A DEEP CLEFT. WE HAVE SHOWN THAT IN THE OXIDIZED (DISULFIDE) FORM OF THIOREDOXIN THE PKA OF ASP26 IS 7.5. NEITHER OUR LAB, NOR OTHER ABS, HAVE BEEN ABLE TO GET AN UNAMBIGUOUS VALUE OF ASP26 PKA IN REDUCED (DITHIOL) THIOREDOXIN BECAUSE OF THE COMPLICATIONS OF THE -SH TITRATIONS OF THE ACTIVE SITE THIOLS. THIS PKA'S OF THE ACTIVE SITE THIOLS AND ASP26 ARE OF FUNDAMENTAL IMPORTANCE IN THE MECHANISM OF THIOREDOXINS. WE WILL USE THE PROCEDURE WORKED OUT IN THE LAB OF DR. F. DAHLQUIST FOR OVEREXPRESSION OF PROTEIN HAVING L-[4-13C] ASPARTIC ACID INCORPORATED AT ALL ASP SITES. DR. DAHLQUIST HAS SHOWN (UNPUBLISHED DATA) THAT THE TITRATION OF ALL ASPARTICS IN LYSOZYME T4 CAN BE DETERMINED FROM PH VERSUS CHEMICAL SHIFT OF 13C RESONANCES. DR. DAHLQUIST IS SUPPLYING US WITH THE AUXOTROPHIC E. COLI STRAIN, IN WHICH ASP-TO-ASN CONVERSION IS ALSO BLOCKED. USING THE STRAIN PROVIDED BY DR. DAHLQUIST, AND WORKING FROM HIS PROCEDURE, WE WILL EXPRESS THIOREDOXIN WITH OUR EXPRESSION PLASMID. WE HAVE A HIGH YIELD EXPRESSION SYSTEM FOR PRODUCTION OF E. COLI THIOREDOXIN. WE HAVE PUBLISHED 5 PAPERS ON WT AND MUTANT THIOREDOXINS OBTAINED FROM THIS SYSTEM, AND SO HAVE ESTABLISHED ALL OF THE PROCEDURES FOR MANIPULATION OF THE STRAINS AND PLASMIDS. MY LAB HAS PUBLISHED NUMEROUS PAPERS USING BASIC NMR TECHNIQUES, AND WE HAVE A 500 AND 600 MHZ BRUKER SPECTROMETERS FOR OUR USE.
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