Professor
Thomas C. Jenkins Department of Biophysics
Krieger School of Arts & Sciences

lecomte_jtj@jhu.edu

116 Jenkins Hall
3400 N. Charles Street
Baltimore, MD 21218

Office: 410-516-7019
Lab: 410-516-7699

NMR Spectroscopy and Protein Structure
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 other proteins, organic substrates, and nucleic acids also depends critically on non-covalent interactions. These incompletely understood forces and their role in native state thermodynamics 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 cofactor, 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 alpha structure and the latter alpha and beta 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 and a related protein from the cyanobacterium Synechococcus sp. PCC 7002. This protein provides an opportunity to study the evolution of oxygen binding through site-directed mutagenesis exploration.  A central question in the hemoglobin field concerns the ability of small ligands such as dioxygen to penetrate the tightly packed protein and access the heme group.  NMR spectroscopy is applied to characterize the internal motions of the protein.

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.

Selected Publications
Davis, R.B. Jr. and J.T.J. Lecomte (2008) Structural propensities in the heme binding region of apocytochrome b5.  II.  Heme conjugates. Biopolymers–Peptide Science 90:556–566.

Davis, R.B. Jr. and J.T.J. Lecomte (2008) Structural propensities in the heme binding region of apocytochrome b5.  I.  Free Peptides. Biopolymers–Peptide Science 90:544–555. doi:10.1002/bip.209956

Lecomte, J.T.J., K. Mukhopadhyay, and M.P. Pond (2008) Structural and thermodynamic encoding in the sequence of rat microsomal cytochrome b5. Biopolymers 89:428–442.

Landfried, D.A., D.A. Vuletich, M.P. Pond, and J.T.J. Lecomte. (2007) Structural and thermodynamic consequences of b heme binding for monomeric apoglobins and other apoproteins. Gene 398:12–28.

Knappenberger, J.A., and J.T.J. Lecomte. (2007) Loop anchor modification causes the population of an alternative native state in an SH3-like domain. Protein Sci. 16:863–879.

Vuletich, D.A., C.J. Falzone, and J.T.J. Lecomte. (2006) Structural and dynamic repercussions of heme binding and heme-protein cross-linking in Synechococcus sp. PCC 7002 hemoglobin. Biochemistry 45:14075–14084.

Knappenberger, J.A., S.A. Kuriakose, B.C. Vu, H.J. Nothnagel, D.A. Vuletich, and J.T.J. Lecomte. (2006) Proximal influences in two-on-two globins: Effect of the Ala69Ser replacement on Synechocystis sp. PCC 6803 hemoglobin. Biochemistry 45:11401–11413.

Davis Jr, R.B., and J.T.J. Lecomte. (2006) A dynamic N-cap motif in cytochrome b5: Evidence for a pH-controlled conformational switch. Proteins 63:336–348.

Vuletich, D.A., and J.T.J. Lecomte. (2006) A phylogenetic and structural analysis of truncated hemoglobins. J. Mol. Evol. 62:196–210.

Lecomte, J.T.J., D.A. Vuletich, and A.M. Lesk. (2005) Structural divergence and distant relationships in proteins: Evolution of the globin family. Curr. Opin. Struct. Biol. 15:290–301.

Lecomte, J.T.J., B.C. Vu, and C.J. Falzone. (2005) Structural and dynamic properties of Synechocystis sp. PCC 6803 Hb revealed by reconstitution with Zn-protoporphyrin IX. J. Inorg. Biochem. 99:1585–1592.

Moody, E.M., J.T.J. Lecomte, and P.C. Bevilacqua. (2005) Linkage between proton binding and folding in RNA: A thermodynamic framework and its experimental application for investigating pKa shifting. RNA 11:157–172.