A. PMS1 NTD:DNA To gain insight into the binding surface involved in the interaction of Pms1-NTD with DNA in the presence of a non-hydrolyzable ATP analog, a series of experiments involving limited proteolysis and OPF were performed. The results from these reactions were combined with mutation results from the Kunkel lab and have been mapped onto the solved structure of yPms1 NTD. It appears the DNA may bend and fit into a concave area close to a flexible loop which may clamp down on the DNA. These results will aid in our understanding of the mechanism of mismatch repair. B. Calbindin: Calbindin-D28k is a ubiquitous protein of the calmodulin family of Ca2+ binding proteins, and has been proposed to act both a sensor and a buffer protein. It is believed to function in both the Apo- and Calcium- loaded states. The structure of holo-Calbindin-D28k has been solved by NMR, but only in the absence of a known disulfide linkage. We have performed differential surface modification, acetylation and histidine modification, combined with mass spectrometry to probe the tertiary structural changes that occur both as a result of disulfide bond formation in the holo-form and the transition to the apo-form structure. We have identified specific regions of the protein that undergo significant conformational changes. These changes occur primarily in the linker regions. We have also determined other conformational changes, such as changes in salt-bridges. A structural picture of both apo-Calbindin-D28k and the disulfide-bridged holo-form have been modeled. (Biochemistry in press) C. Anthrax Protective Antigen: We have continued our structural studies of the Bacillus anthracis Protective antigen and of epitopes on PA recognized by antibodies. We are studying the conformational change undergone by PA at low pH using differential oxidative surface mapping. Dynamic structural information can be obtained through analysis of amino acid oxidation both before and after a conformational change. In the present study, PA at pH 7.5 and 5.5 was exposed to hydroxyl radicals generated by ionizing radiation. Mass spectrometry was then used to both identify and quantitate the extent of oxidation of differentially modified residues. Several residues were found to be more readily oxidized at pH 7.5, most of which clustered toward the bottom plane of the prepore heptamer. Only two amino acids had greater oxidation rates at pH 5.5, and each are found on the outer periphery of the prepore. A comparison of OPF results with the current solved structure of the prepore and model of the pore was performed and yielded mixed consistency with the current model in that some modified residues were found buried in the prepore structure and/or pore model of the PA63 heptamer. The results of this study provide empirical structural information that should be useful in modeling an improved structure. We have now started additional structural studies based on the PA heptamer bound to the antrax toxin receptor. D. Factor Xa dimer. We investigated the structure of the FXa dimer that is involved in blood clotting, using differential surface acetylation of Lys residues. These reactions were carried out in the presence of 400 M PS and 5 mM Ca+2, conditions where the dimer is stable. Controls were FXa in the presence of 3 mM Ca+2 and in the presence of 400 M PS and 3 mM Ca+2. Distinct changes in extent of acetylation of specific residues were observed upon addition of PS and upon increasing Ca+2 concentration from 3 to 5 mM. Our results are consistent with a model where the Xa catalytic domain is a tightly coupled structure and that binding to a substrate or dimer face elicits changes at one or more of the other faces. These data and the resulting model form the basis of a manuscript that is near submission. These data are important in understanding the conformational changes that take place during the blood coagulation process. Our results are important in that they also demonstrate that differential surface modification studies can be made in the presence of membrane mimics. E. Sjogrens Syndrome.
The aim of this stage of our study is to provide a better understanding of the structure and function of LaSSB and Ro52 autoantigens and their role in the autoimmune response. The LaSSB antigen is known to interact with a wide variety of RNAs, however, the ultimate function of LaSSB is currently unknown. The Ro52 protein is the main SSA autoantigen, and is predicted to belong to the RING-B-box-coiled-coil (RBCC) family of proteins. We are using differential surface modification and cross-linking combined with MS to help characterize LaSSB and Ro auto-antigens associated with Sjoegrens Disease. The flash photolysis system is being used to gain tertiary structural information regarding LaSSB and Ro52. F. Technical advances in differential surface modification. We have developed a novel amine modification reagent, trimethylglycyine N-hydroxysuccinimide ester, for use in differentially derivatizing protein amino functionalities without changing the formal charge on the amine group. This modification reagent should reduce precipitation of proteins due to changes in the pI of the amine groups during the course of differential surface modification studies, a major potential problem in these studies. To increase the extent of modification observed during OFP studies, we have set up a laser flash photolysis system that has a significantly higher hydroxyl radical yield than does the gamma irradiation source. Hydroxyl radical reaction times are on the order of millisecs. which are sufficiently short so that conformational changes should not occur in the proteins under our reaction conditions. G. Alzheimers disease (AD) Glycosylation studies of the amyloid precursor protein implicated in Alzheimers Disease. We have been able to characterize the occupancy and heterogeneity of occupancy at three O-glycosylation sites on the protein. A major O-glycosylation site was found near the alpha-secretase cleavage site and it is possible that correct O-glycosylation may play a role in proper cleavage. Incorrect cleavage is thought to be an important step in plaque formation during the course of the disease. We have also found that the N-glycosylation pattern of anti-A-beta antibodies (Abs) differ from that of the general pool of serum Abs, specifically in an increased abundance of bisecting and terminally sialilated glycans which may exhibit anti-inflammatory properties.
