| | | | Thomas C. Jenkins
Department of Biophysics
110 Jenkins Hall
3400 N. Charles Street
Baltimore, MD 21218 410-516-7245 phone
410-516-4118 fax | | | | |
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| Johns Hopkins University
Department of Biophysics
3400 North Charles Street
Baltimore, MD 21218
410-516-8215 Office
410-516-4118 Fax
lattman@jhu.edu | This laboratory is engaged in a variety of projects involving protein crystallographic studies, protein folding, drug design, electrostatics, and protein-RNA interactions. - We use the mutants of the enzyme staphylococcal nuclease as a system to probe catalysis, and to analyze factors that control or the stability of the folded state. In 1995 Drs. Susan Green and Apostolos Gittis analyzed a deletion mutant that transforms nuclease from a monomer to a tight dimer, and that sheds light on possible general mechanisms for the evolution of oligomers. Dimerization is achieved by a helix swapping mechanism, by which the C-terminal a helices of the two monomers are exchanged, and occupy binding sites in the other monomer. This mutant helps to confirm the mechanism of domain swapping proposed by Eisenberg as a general method of oligomerization. In addition, these experiments illustrate how protein folds are modified or decorated during evolution.
- In collaboration with Prof. Bertrand García-Moreno we are probing the physical basis of the dielectric behavior of proteins by studying mutants in which ionizable groups are buried in the protein core. The mutants Val66->Lys, Val66->Glu and others have been studied by potentiometric titration, solution physical chemistry, and X-ray diffraction. The pKs of these buried groups are shifted by about 4-5 units, implying an effective dielectric constant in the range 10-20. The crystal structure of Val66->Glue shows a string of 4 water molecules running from the molecular surface to the carboxylate group of Glu66., suggesting that partial solvation by means of a “proton wire” may be the physical basis of this high dielectric constant. Dr.Gittis is leading the charge on this work.
- In a related project we are exploring the hypothesis that protein molecules in their native state are randomly and transiently penetrated by water molecules, and thus contain, on average, more water molecules than is currently believed. This results in a protein molecule whose interior is more polar than expected, and which is subject to significant fluctuations in solvent content. This view of protein structure has major implications for the quantitative understanding of binding and electrostatics. This work is now being pursued by graduate student Leila Reynald.
- Dr. Robert Kelly has pioneered out a search for new classes of compounds that inhibit the gelation of sickle cell hemoglobin (HbS), and which could thus serve as lead compounds for the development of therapeutic agents. The target site chosen is the axial contact region between adjacent molecules in the long filaments of HbS molecules that comprise the sickled fiber. The program DOCK has been used to screen a library of 100,000 small molecules to find molecules that might possibly bind to the target site. This preliminary set of molecules has been further screened using more sophisticated molecular mechanics programs, and some ligands are being test in vitro. Some promising candidates have been uncovered.
- In a collaboration between the laboratory of Prof. David Draper and ours, Graeme Conn and Apostolos Gittis have determined the crystal structure of a complex between the large subunit ribosomal protein L11 and a 58 base piece of ribosomal RNA. L11 is the binding site for elongation factor G, and is the target for the thiostrepton group of antibiotics. By studies of variants of this system we hope to gain new insight into the energetics of RNA tertiary structure. Graduate student Adetokunbo Lukan is now heavily involved in following up this work.
- Gittis, A.G., W.E. Stites, and E.E. Lattman. 1993. The phase transition between a compact denatured state and a random coil state in staphylococcal nuclease is first-order. J. Mol. Bio. 232:718-724.
- Keefe, L.J., S. Quirk, A. Gittis, J. Sondek, and E. Lattman. 1994. Accommodation of insertion mutations on the surface and in the interior of staphylococcal nuclease. Protein Sci. 3:391-401.
- Libson, A., A. Gittis, and E. Lattman. 1994. The Crystal Structures of Binary Ca2++ and pdTp complexes and the ternary complex of the Asp21->Glu mutant of staphylococcal nuclease. Biochemistry 33:8007-16.
- Griko, Y.V., A. Gittis, E.E. Lattman, and P. Privalov. 1994. Residual structure in a staphylococcal nuclease fragment: Is it a molten globule and is its unfolding a first order phase transition? J. Mol. Biol. 243:93-99.
- Green, S.M., A.G. Gittis, A.K. Meeker, and E.E. Lattman. 1995. One-step evolution of a dimer from a monomeric protein. Nat. Struct. Biol. 2:746-751.
- García-Moreno E., B., J.J. Dwyer, A.G. Gittis, E.E. Lattman, D.S. Spenser, and W.E. Stites. 1997. Experimental measurement of the effective dielectric in the hydrophobic core of a protein. Biophys. Chem. 64:211-224.
- Conn, G.L., D.E. Draper, E.E. Lattman, and A.G. Gittis. 1999. Crystal structure of a conserved ribosomal protein - RNA complex. Science 284:1171-1174.
- Dwyer, J.L., A.G. Gittis, D. Karp, E.E. Lattman, D.S. Spencer, E. Wesley, W.E. Stites, and B. Garcia-Moreno E. 2000. High apparent dielectric constants in the interior of a protein reflect water penetration. Biophys J. 79:1610-1620.
- Conn, G.L., A.G. Gittis, E.E. Lattman, V.K. Misr, and D.E. Draper. 2001. A Compact RNA Tertiary Structure Contains a Buried Backbone-K+ Complex. Nat. Struct. Biol. (submitted).
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