We have determined the pKa values for thiol-thiolate equilibria of Escherichia coli thioredoxin and compared structural properties of reduced and oxidized forms of the protein in solution by Raman spectroscopy. Ionization and hydrogen-bonding states of the two cysteine sulfhydryls (Cys32 and Cys35) in reduced thioredoxin were determined by monitoring the complex Raman SH stretching band (Li & Thomas, 1991) as a function of pH in the range 4.0 < pH < 12.2. The Raman SH markers indicate the following: (i) Both sulfhydryls of the native protein are relatively robust hydrogen-bond donors, but one is a stronger hydrogen-bond donor than the other. (ii) The sulfhydryl which donates the weaker hydrogen bond, assigned tentatively to Cys32, is preferentially titrated to the thiolate ion (S-) as the solution pH is increased from 4 to 7. Water or a hydroxyl oxygen is the probable acceptor for the Cys32 S-H donor. (iii) The sulfhydryl which donates the stronger hydrogen bond, Cys35, resists substantial ionization until roughly 50% of the more acidic sulfhydryl has been titrated. A carbonyl oxygen is proposed as the likely acceptor of the Cys35 S-H donor. (iv) The Raman titration data indicate pK1 = 7.1 +/- 0.2 and pK2 = 7.9 +/- 0.2 for the two thiol-thiolate equilibria. The lower pKa, which is the more strongly perturbed, is assigned tentatively to Cys32.(ABSTRACT TRUNCATED AT 250 WORDS)

The active-site cysteines (Cys 32 and Cys 35) of Escherichia coli thioredoxin are oxidized to a disulfide bridge when the protein mediates substrate reduction. In reduced thioredoxin, Cys 32 and Cys 35 are characterized by abnormally low pK, values. A conserved side chain, Asp 26, which is sterically accessible to the active site, is also essential to oxidoreductase activity. pK, values governing cysteine thiol-thiolate equilibria in the mutant thioredoxin, D26A, have been determined by direct Raman spectrophotometric measurement of sulfhydryl ionizations. The results indicate that, in D26A thioredoxin, both sulfhydryls titrate with apparent pK, values of 7.5 f 0.2, close to values measured previously for wild-type thioredoxin. Sulfhydryl Raman markers of D26A and wild-type thioredoxin also exhibit similar band shapes, consistent with minimal differences in respective cysteine side-chain conformations and sulfhydryl interactions. The results imply that neither the Cys 32 nor Cys 35 SH donor is hydrogen bonded directly to Asp 26 in the wild-type protein. Additionally, the thioredoxin main-chain conformation is largely conserved with D26A mutation. Conversely, the mutation perturbs Raman bands diagnostic of tryptophan (Trp 28 and Trp 31) orientations and leads to differences in their pH dependencies, implying local conformational differences near the active site. We conclude that, although the carboxyl side chain of Asp 26 neither interacts directly with active-site cysteines nor is responsible for their abnormally low pK, values, the aspartate side chain may play a role in determining the conformation of the enzyme active site.Keywords: cysteine; pK,; Raman spectra; structure; thiol; thiolate; thioredoxin The thioredoxin fold is an extensively studied structural motif found in a variety of proteins that interact with cysteine-containing substrates (Edman et al., 1985;Martin, 1995). Both the thioredoxin fold and the local sequence at the active site (-W-C-G-P-C-) are highly conserved among thioredoxins of different species (Holmgren, 1985;Martin, 1995). In Escherichia coli, thioredoxin functions not only as an oxidoreductase, but also as a host regulator of prokaryotic viral assembly (reviewed by Holmgren, 1985). The active-site cysteines 32 and 35 in E. coli thioredoxin are oxidized to form a cystine bridge when the protein mediates the reduction of a suitable substrate. The reduced form of thioredoxin is subsequently regenerated by the enzyme thioredoxin reductase.

The University of Missouri-Kansas City (UMKC) School of Dentistry has piloted two years of an Admissions Enhancement Program (AEP) with students from underrepresented minority groups and/or economically disadvantaged areas of Missouri interested in applying to dental school. The AEP utilizes an innovative online-onsite hybrid format to elevate students' foundational knowledge in biology, chemistry, organic chemistry, and quantitative reasoning. The online component includes interaction with UMKC instructors using tablet technology and Wimba virtual classroom sessions. The onsite component engages students in academic and professional development, enrichment activities targeting skills training, experience in dental labs and clinics, and mentoring in preparing the dental school application, essay writing, and interviewing. Results to date indicate overall program satisfaction among AEP participants and a dental school acceptance rate of 73.7 percent (14/19 students). Participants reported the mock interviews and essay-writing portions contributed to their becoming competitive candidates for the admission process, and the online material enhanced their preparation for the Dental Admission Test (DAT).

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