I will first reiterate the functions of the NMR facility of the LMC: The first is as a resource for the entire chemist community of the LMC and NCI Frederick to use as a routine use facility to walk up and collect NMR data on synthetic compounds prepared for projects under the auspices of other PI's and 2) It is used to tackle more challenging problems related to the structure and 2-dimensional conformations of drug molecules or other biopolymers (peptide, glycopeptides, oligonucleotides and oligosaccharides) studied in the various sections of the lab. In the past year I have spearheaded the reorganization of some instrumentation here at NCI fRederick to more efficiently handle the workload for NMR here on campus. This project relates to number (2) in that I use part of my time to do NMR-based conformational analysis that is a critical part of my work and of the entire LMC. Thus the creation of this new project is to define the effort dedicated to original research in the area of conformational analysis of molecules of biological significance to the LMC and NCI as a whole. In the past fiscal year, we have completed a comprehensive study of three difluorinated nucleosides by NMR X-ray crystallography and molecular modeling. Using various programs with different force fields and parameter sets, we defined these structures very precisely and were able to propose a model for how fluorine affects the conformational parameters of nucleosides. We have performed a variety of structural studies on the highly interesting glycopeptide Antiproliferative Factor (APF), a sialylated glycopeptide that is the causative agent of a necrotic disease of the bladder called interstitial cystitis. This molecule also has potent anti tumor activity in a variety of cancers including bladder cancer, and it will become a useful tool in the design of anti cancer therapeutics. The peptide is virtually unstructured in water solution but assumes some structure in membrane mimicking environments (trifluoroethanol). Several other analogues have been studied by NMR in an attempt to explain the unusual biological effects that occur with very minor changes to the structure. This work has progressed where nearly 50 peptide-segment analogues of the glycopeptide were synthesized and an SAR study was published in J. Med. Chem. this year. Due to the untimely death of Christopher Michejda, the leader of this SAR project, I have taken over the responsibility of supervising his former post doc who will continue this work into 2009. Dr. Piotr Kaczmarek has been working in my group and has prepared several more analogues for study. He is now working on modifications to the sugar portion of the molecule and several analogues are near completion. A separate project that was started in mid 2007 is to explore the binding site of a Single Chain Variable Fragment (scFV) of an immunoglobulin to hen eggwhite lysozyme (HEL) that was designed, cloned and comprehensively analyzed by Sandra Smith-Gill and her colleagues. Due to difficulties in obtaining crystal or NMR structures of the scFV-HEL complex, they undertook the expression of a series of mutants that contained 5-fluoro tryptophan in place of other aromatic amino acids and we are examining the 19F NMR spectra in collaboration with this group. We have collected spectra of the free proteins (six mutants), and have performed several titration studies with HEL at various temperatures according to the stability profile of each protein. We have shown that the spectra are completely resolved only in the presence of the enzyme at specific concentrations, and we have begun to mapped the binding site by observing changes in chemical shift of the 19F signals: Only 3 of 6 show marked changes with addition of enzyme, suggesting they are involved in binding. In addition, we have completed the assignments of the tryptophan residues by collecting spectra of deletion mutants prepared by the Smith-Gill lab. Relaxation data collected on the mutants has allowed us to calculate the T1 and T2 relaxation times and relate these to motion in the protein at he fluorinated sites. A manuscript draft is available and is very close to submission
