Most water-soluble proteins are capable of folding spontaneously into the three-dimensional structure necessary for their function. This organized state of the molecule, the native state, is stabilized relative to the inactive, unfolded state by a collection of non-covalent interactions. The association of proteins with their physiological partners, such as organic substrates, nucleic acids, and other proteins also depends critically on non-covalent interactions. These poorly understood forces and their role in native state thermodynamics and in binding processes are the focus of the Lecomte group. The goals of the research are to develop a theoretical and empirical basis for the interpretation of structural data and to derive strategies to design proteins of arbitrary properties. One line of investigation is directed to a physical understanding of temperature, pH, and salt effects on protein structure and stability. Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique to characterize in detail the response of individual protein sites to changes in external conditions. The Lecomte group applies NMR spectroscopy to map the consequences of these changes. This information is used to explain the resistance of proteins to thermal and acid denaturation. In a parallel research effort, hemeproteins are utilized to analyze the determinants of molecular recognition. The simplest hemeproteins contain a single prosthetic group, iron-protoporphyrin IX, embedded in the protein matrix and held by one or two coordination bonds to the iron atom. Natural b hemoproteins generally have a strong affinity for heme, which they bind rapidly in vitro. To understand the origin of heme affinity, two hemeproteins were selected, myoglobin and cytochrome b5. These proteins have unrelated functions: myoglobin stores oxygen in red muscles and cytochrome b5 serves as an electron transport protein; they also have distinct folds, the former containing α structure and the latter α and β structure in a complex topology. By applying NMR methods, it was possible to produce working models of the proteins free of heme. It was thus discovered that in both cases the empty heme-binding site assumes on one side a well-defined conformation resembling closely that in the heme-bound form, and on the other a dynamic set of isoenergetic conformations. The NMR description illustrates a balance of enthalpic and entropic contributions to the binding free energy and suggests how new hemeproteins could be engineered. Design approaches derived from cytochrome b5 and myoglobin are applied to generate artificial proteins of controlled chemical properties. The Lecomte group is also interested in the structural, functional, and evolutionary properties of ancient proteins. Cyanobacteria are oxygenic photosynthetic organisms that are thought to be responsible for the modification of the atmosphere 3.5 to 2 billion years ago. As such, they are excellent sources of intriguing proteins. For example, Synechocystis sp. PCC 6803 is a unicellular cyanobacterium whose genome has been entirely sequenced. It contains a single gene encoding a protein with amino acid sequence distantly related to that of hemoglobin, the oxygen carrier of vertebrates. The globin from Synechocystis sp. PCC 6803 binds heme tightly and has unusual chemical properties. Research is ongoing to determine the structure and the function of this cyanobacterial hemoglobin. This protein provides an opportunity to study the evolution of oxygen binding through site-directed mutagenesis exploration. Molecular recognition and protein design are at the forefront of modern biochemical research. Typical projects in the group address these problems through training in molecular biology, protein preparation and purification, optical spectroscopy, NMR spectroscopy with the state-of the-art spectrometers of the NMR facility, and interpretation of the results with computer programs.
Landfried, D.A., Vuletich, D.A., Pond, M.P., and Lecomte, J.T.J. (2007) Structural and Thermodynamic Consequences of b Heme Binding for Monomeric Apoglobins and Other Apoproteins. Gene 398:12–28. doi:10.1016/j.gene.2007.02.046 Knappenberger, J.A., and Lecomte, J.T.J. (2007) Loop Anchor Modification Causes the Population of an Alternative Native State in an SH3-like Domain. Protein Science 16:863–879. doi:10.1110/ps.062469507 Vuletich, D.A., Falzone, C.J., and Lecomte, J.T.J. (2006) Structural and Dynamic Repercussions of Heme Binding and Heme-Protein Cross-Linking in Synechococcus sp. PCC 7002 Hemoglobin. Biochemistry 45:14075–14084. doi:10.1021/bi061532g Knappenberger, J.A., Kuriakose, S.A., Vu, B.C., Nothnagel, H.J., Vuletich, D.A., and Lecomte, J.T.J. (2006) Proximal Influences in Two-on-Two Globins: Effect of the Ala69Ser Replacement on Synechocystis sp. PCC 6803 Hemoglobin.Biochemistry 45:11401–11413. doi:10.1021/bi060691x Davis Jr, R.B., and Lecomte, J.T.J. (2006) A Dynamic N-cap Motif in Cytochrome b5. Evidence for a pH-controlled Conformational Switch. Proteins: Structure, Function, and Bioinformatics, 63:336–348.doi:10.1002/prot.20759 Vuletich, D.A., and Lecomte, J.T.J. (2006) A Phylogenetic and Structural Analysis of Truncated Hemoglobins. Journal of Molecular Evolution 62:196–210. doi:10.1007/s00239-005-0077-4 Lecomte, J.T.J., Vuletich, D.A, and Lesk, A. M. (2005) Structural Divergence and Distant Relationships in Proteins: Evolution of the Globin Family. Current Opinion in Structural Biology 15:290–301.doi:10.1016/j.sbi.2005.05.008 Lecomte, J.T.J., Vu, B.C., and Falzone,C.J. (2005) Structural and Dynamic Properties of Synechocystis sp. PCC 6803 Hb Revealed by Reconstitution with Zn-Protoporphyrin IX. Journal of Inorganic Biochemistry 99:1585–1592.doi:10.1016/j.jinorgbio.2005.04.018 Moody, E.M., Lecomte, J.T.J., and Bevilacqua, P.C. (2005) Linkage Between Proton Binding and Folding in RNA: A thermodynamic framework and its experimental application for investigating pKa shifting. RNA 11:157–172. doi:10.1261/rna.7177505 Scott, N.L., and Lecomte, J.T.J. (2005) Protein Structure: Unusual Covalent Bonds. Nature Encyclopedia of Life Sciences John Wiley & Sons, Ltd: Chichester, UK. http://www.els.net/ doi:10.1038/npg.els.0003015
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