Leonard D. Spicer

The focus of this laboratory is the study of structure/function relationships in biological macromolecules and their binding interactions. The principal method we use for system characterization is magnetic resonance spectroscopy. One specific area of interest is the structural characterization of functional domains in proteins which regulate the transcription of DNA coding for biosynthetic enzymes. The system under current investigation is the methionine repressor protein metJ, its corepressor S-adenosylmethionine, and the cognate sequence DNA. This protein, which functions as a dimer, exhibits a recently described DNA binding motif involving insertion of two beta strands into the major groove with additional stabilization of the complex arising from helix contacts at the dimer-dimer interface. We are using a full complement of heteronuclear 3D and 4D NMR methods to aid in the assignment of the main chain of the metJ repressor. We have recently reported a thermodynamic analysis of the binding interactions of metJ with its cognate DNA and corepressor SAM. We are now developing methods to measure fast proton exchange rates to complement our planned solution structural characterization. We have just initiated another project in collaboration with scientists at the Pacific Northwest National Laboratory to study macromelecular structures of DNA repair proteins in the nucleotide excision repair pathway. The first components of this critical supramacromolecular assembly we are investigating involve the DNA binding domain of the XPA protein for which we are determining the global fold in solution by NMR. Our program also includes a systematic approach to characterizing the conformational preferences of a number of sequentially related peptides developed by Dr. Barton Haynes' laboratory as candidate vaccines for HIV. The peptides consist of a fusion of two noncontiguous segments of the HIV protein gp120. Our goal is to establish whether structural conformers in solution contribute to peptide immunogenicity. We have finished a careful conformational analysis of the initial four peptides and are now correlating the conformer similarities and differences with immunogenic properties. We have also rationally designed several new peptides based on structural criteria and corresponding structural homology to the heavy fragment of IgA proteins. Initial NMR analysis and immunogenic response to three of the designed mutants indicate the rational design of preferred conformers was successful, but raised some novel questions regarding function of immunogenic peptides. We have also just begun a study of solution conformations of the hypoglycosylated tumor specific epitope repeat unit of human mucin and a promising mutant identified by Dombrowski and Wright. This epitope is common to breast and other adenocarcinomas and regulation of tumor specific lymphoid cells responding to this immunogen may be an important step in tumor control. Another protein under investigation is a functional core packing mutant of thioredoxin. We have fully characterized backbone chain dynamics to assess the impact of this mutation on molecular motions and are currently determining its high resolution tertiary structure. Currently, we are also using this mutant to demonstrate a new approach to global fold determination using a minimum set of long range NMR constraints. Finally, as an essential part of these studies, we are developing and have reported new 3- and 4-dimensional NMR experiments and heteronuclear filters for application to large protein systems and binding complexes.

Finally, the core activities of the NMR Center staff have continued to progress rapidly and enhancements to the state-of-the-art instrumentation have again been incorporated. A new deuteration strategy for assignment and study of large proteins by NMR has been developed and used to characterize one of the largest protein monomer reported to date, human carbonic anhydrase. We have also shown that we can observe the longest range distance constraints to date from NOESY correlations which are important in determining tertiary structure of proteins and we are examining the efficacy of structure determinations based on using these critical but limited constraints.