A.PMS1 NTD:DNA To gain insight into the Pms1-NTD DNA-binding surface, 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 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.Base Excision Repair: We are identifying additional co-factors in BER and developing means of enriching BER intermediates. An NaBH4 cross-linked Schiff base dRP lyase intermediate protein-DNA complex was used as bait to identify additional proteins that have dRP lyase activity or influence removal of the cytotoxic dRP from BER intermediates in the absence of pol-B in a pol-B null mouse cell extract. MS identified the high-mobility group box 1 protein (HMGB1) as specifically interact-ing with the BER intermediate. It is hypothesized that HMGB1 binds to the initial BER in-termediate immediately upon formation and that HMGB1 simulates repair due to its stimulatory effect on apurinic/apyrimidinic endonuclease (APE). But, in the presence of high levels of APE activity, HMGB1 may reduce single nucleotide BER activity by inhibiting pol-s dRP lyase activity. Also HMGB1 may stimulate long patch BER where LP BER strand-displacement DNA synthesis is proficient.? C.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 the structure of the apo-form has not been solved, but it is known that there is a significant conformational difference between the two forms and that this conformational change influences its role in the cell. Differential surface modification combined with mass spectrometry is being used to probe the tertiary structural changes that occur upon calcium binding. A structural picture of apo-Calbindin-D28k has been modeled. ? D. 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. (Biochemistry, in press.)? E. Factor Xa dimer? We investigated the structure of the FXa dimer 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.? ? F. 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 currently modifying surface exposed residues and performing cross-linking experiments in combination with MS and have observed 37 of 60 lysine residues in LaSSB and 13 of 27 lysine residues in Ro52 have been observed as unmodified or dif-ferentially modified. Using three crosslinkers of various lengths, distance constraints be-tween specific cross-linked amino acids have been determined for LaSSB We have performed computational protein-protein docking studies of the individually solved LaSSB protein domain structures and the experimental results obtained from MS acetylation studies as constraints in modeling the structure. Homology models of the individual domains within Ro52 have also been developed, and we are currently developing a full-length model of Ro52 based on incorporation of our chemical modification results in a manner similar to that used for LaSSB.? Alzheimers disease (AD) is one of the most prevalent forms of neurodementia affect-ing the aging population. Accumulation of -amyloid (A) in the brain of the elderly is generally believed to be a key event in the onset and development of AD. In the past decade, significant progress has been made in understanding the mechanisms underlying AD pathology, but an efficient therapy still remains to be established. Active immunization with pre-aggregated A(1-42) showed that the antibodies raised against this immunogen were able to disaggregate the -amyloid plaques in transgenic mouse models, resulting in restoration of cognitive functions. Initial clinical trials were discon-tinued because several AD patients developed severe secondary inflammation The discovery of circulating anti-A autoantibodies represents a promising alternative prospect for the early diagnsis and prevention of A-induced neurodegeneration.? The specific goal of this project is to characterize anti--amyloid antibodies in an effort to gain insight to the mechanism of Alzheimers disease and/or potential protective mechanisms of the autoantibodies. Mouse MAb 6E10 is a commercially available Ab used extensively in Alzheimers research. We have identified 95% of the light chain and 82% of the heavy chain sequences of this Ab. We identified pronounced sequence microheterogeneities for the light chain CDR2 region indicating that changes at the protein level derived from somatic hypermutation of the Ig VL genes in mature B-cells might contribute to unexpected structural diversity. Also, major glycoforms at the conserved heavy chain glycosylation site were determined by structural MS analysis to be core-fucoslylated, biantennary, complex glycans.
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