Protein structure and function; Protein folding; Protein-protein and protein-DNA interactions; Transcriptional control of prokaryotic gene expression with emphasis on the quorum sensing system of bioluminescent bacteria; Mechanism of flavoprotein monooxygenases in general and bacterial luciferase in particular; Biochemistry of bioluminescent systems; High level overexpression of recombinant proteins in Escherichia coli.

Web Site: Laboratory Home Page

Research Interests

Research in our group is divided into two areas, both geographically and topically. One group, housed in the College of Medicine of the Arizona Health Sciences Center, focuses on the molecular details of the quorum sensing system by which luminous marine bacteria control the expression of bioluminescence. Quorum sensing, first discovered in bioluminescent marine bacteria, has more recently been discovered in numerous nonluminous bacteria, and is especially common in pathogenic bacteria. Quorum sensing is a mechanism by which bacteria can control the expression of certain genes or families of genes as a population that they do not express as individuals, thus permitting bacteria to behave as if they were multicellular organisms. The cells constitutively produce a small molecule autoinducer, a 3-oxo-N-acylhomoserine lactone, which is diluted into the growth medium. As the local concentration of cells producing a specific autoinducer increases, so will the concentration of the autoinducer, and at a critical concentration, the autoinducer activates a transcriptional activator which stimulates transcription of a set of genes that encode both control functions and functions that allow the cells to produce visible light. It is easy to understand why cells would not express bioluminescence as single cells single-cell luminescence would have no biological consequence as there would be insufficient light to be detected by any other organism. Many pathogenic organisms use the same control mechanism to solve the same problem; individual or small numbers of cells would not survive if they were to express the pathogenic functions, but as a large population, they can overpower the target biological system.

The second component of our research program, housed in Biological Sciences West, investigates protein folding and subunit assembly processes. Our model system is the bacterial luciferase heterodimer which provides a nearly ideal model system with which to study protein folding as the enzyme assay is exceedingly simple and fast, and the bioluminescent signal is linear over an enormous range of enzyme concentrations. Furthermore, it is possible to assay the enzyme in vivo without perturbing the living cell. We have carried out extensive studies of the folding of the individual alpha and beta subunits in vitro and have begun to extend the results of these studies to an investigation of the biosynthetic folding of the protein as it occurs on the ribosome. The long term objective of these studies is to understand how proteins fold within the context of the living cell. More details of these and other projects can be found on our home page.

Selected Publications

Baldwin, T. O. 1999. Protein folding in vivo: The importance of ribosomes. Nature Cell Biology 1: 154-155. Noland, B. W., L.J. Dangott, and T.O. Baldwin. 1999. Folding, stability and physical properties of the alpha subunit of bacterial luciferase. Biochemistry 38: 16136-16145.

Fedorov, A. N. and T.O. Baldwin. 1999. Process of biosynthetic protein folding determines the rapid formation of native structure. J. Mol. Biol. 294: 579-586.

Baldwin, T. O., M.M. Ziegler, V.A. Green, and M.D. Thomas, 2000. Overexpression of bacterial luciferase and purification from recombinant sources. Methods in Enzymology 305: 135-152.

Clark, A. C., B.W. Noland, and T.O. Baldwin. 2000. A rapid chromatographic method to separate the subunits of bacterial luciferase in urea-containing buffer. Methods in Enzymology 305: 157-164.

Baldwin, T. O. and V.A. Green. 2000. Purification of firefly luciferase from recombinant sources. Methods in Enzymology 305: 180-188.

Apuy, J. L., Z-Y. Park, P.D. Swartz, L.J. Dangott, D.H. Russell, and T.O. Baldwin. 2001. Pulsed-alkylation mass spectrometry for the study of protein folding and dynamics: Development and application to the study of a folding/unfolding intermediate of bacterial luciferase. Biochemistry 40: 15153-15163.

Apuy, J. L., X. Chen, D.H. Russell, T.O. Baldwin, and D.P. Giedroc. 2001. Ratiometric pulsed alkylation/mass spectrometry of the cysteine pairs in individual zinc fingers of MRE-binding transcription factor-1 (MTF-1) as a probe of zinc chelate stability. Biochemistry 40: 15164-15175.

Sparks, J. M. and T.O. Baldwin. 2001. Functional implications of the unstructured loop in the (b/a)₈ barrel structure of the bacterial luciferase a subunit. Biochemistry 40: 15436-15443.

Inlow, J. K., and T.O. Baldwin. 2002. Mutational analysis of the subunit interface of Vibrio harveyi bacterial luciferase. Biochemistry 41: 3905-3915.

Noland, B. W. and T.O. Baldwin. 2003. Demonstration of two independently folding domains in the a subunit of bacterial luciferase by preferential ligand binding-induced stabilization. Biochemistry 42: 3105-3112.

Contact Information

Mailing:
Dr. Thomas O. Baldwin, Professor and Head
Department of Biochemistry & Molecular Biophysics
University of Arizona
1041 E. Lowell Street
Biosciences West 362A1
Tucson AZ 85721-0088

Telephone: 520-621-9185
Fax: 520-626-9204

tbaldwin@u.arizona.edu

Biological Sciences West
1041 East Lowell Street
P.O. Box 210088 · Tucson, AZ 85721-0088
Tel: (520) 621-5110
FAX (520) 626-9204

Life Sciences South
1007 East Lowell Street
P.O. Box 210106 · Tucson, AZ 85721-0106
FAX (520) 621-3709