Although the field of genomics has recently sequenced thousands of genes from multiple species, it is much slower at providing information on assigning functions to these genes. Proteins are generally the effector molecules that ascribe roles to genes, and most of the functions of proteins arise through their three-dimensional structures and their interactions with other molecules. Conventional genetic and structural biology techniques have, respectively, been the most powerful avenues for determining the organization of cell signaling networks and the molecular details of protein structure and protein/protein interactions. Understanding protein structure and the protein:protein interactions of the cell at a basic level (i.e. identification of the molecules involved, determination of their molecular architecture, and elucidation of how these molecules interact with one another) is imperative for understanding how the disruption of a single element can result in human disease. X-ray crystallography and nuclear magnetic resonance spectroscopy are the current methods of choice for obtaining high resolution structural information. Mass spectrometry (MS) is an emerging technique that is showing tremendous potential for both identification of protein complexes and elucidation of protein structure, in particular for proteins that are not amenable to classical structural techniques. The advantages of MS sensitivity, low sample consumption, and the ability to analyze inhomogeneous mixtures can overcome the obstacles that hamper other structural methods. MS was actually developed about 100 years ago, but its utility in biological research is just now being realized. Using MS, we can probe the structure of a protein with chemical reagents and then assess inter-residue distances and solvent accessibility. These data can aid in the determination of the protein structure and, hence, as to how the protein works. Moreover, MS can also be employed to simultaneously determine what other proteins a particular protein of interest interacts with and how these interactions are formed. ? ? Protein:protein and protein:DNA interactions are often at the center of biological processes, both beneficial and harmful. The primary goal of this project is to determine structural features of protein interactions that are critical in biologically functional or in pathological processes. As examples, we are currently studying DNA repair enzyme interactions with DNA, the interaction surfaces in DNA mismatch repair enzymes, and structural transformations of proteins concomitant with oxidation that affect biological processes.? ? MLH1:PMS1 heterodimer: DNA mismatch repair (MMR) is critical for the maintenance of the genetic material, and the major features have been conserved throughout evolution. MutL proteins are primary components of MMR with MutL homolog 1 (MLH1) and Post meiotic segregation 1 (PMS1) critical components in yeast. These proteins form a heterodimer via the C-terminal domain. Upon DNA and ATP binding, further dimer contacts are thought to form in the N-termini. Little is known about their tertiary structure. It is hypothesized that mutations in these proteins cause changes in their structures, which may lead to loss of MMR activity and ultimately carcinogenesis. ? ? Hepatitus C mAb: Hepatitis C virus infects over 170 million people worldwide, and in most cases, the infectionsdevelop into chronic hepatitis, which is one of the most prevalent causes of liver cirrhosis and represents the most frequent indication for liver transplantation. Hepatitis C virus (HCV) is a small, enveloped positive-strand RNA virus belonging to the Flaviviridae family. The genome of HCV is ~ 9500 nucleotides and contains a single large open reading frame encoding a single polypeptide of ~3010aa. This polyprotein is subsequently processed co-and posttranslationally, generating the structural proteins Core, E1, E2, and p7, and five nonstructural proteins. HCV-E2 is a 50kDa glycoprotein that shows large variations among HCV genotypes and contains a hypervariable 27 aa sequence at its amino terminus.? Specific aims: 1.To characterize the structure of the HCV-E2 protein and of the E1E2 protein complex by peptide mapping using mass spectrometry; 2.Determine epitopes of the E2 protein, recognized by in vivo neutralizing, human mAbs. Status:a)Peptide mapping is being performed on HCV-E2 protein and the E1E2 protein complex and have partially confirmed the sequences of the proteins; b)Initial experiments have been performed on 3 neutralizing mAbs CBH2, CBH5 and CBH7. MS/MS experiments are being performed to characterize the structure of the epitope fragments.? ? Mapping sites of interaction of Sindbis coat glycoprotein in the intact virus: We have used surface modification for structural analysis of the Sindbis virus using biotin-tagged succinimide-based label targeted to primary amines. This study found that the structure of an intact, infectious virus could be studied by surface labeling and mass spectrometry, without the virus losing any infectivity. Sufficient data was generated to suggest that the previously published X-ray crystal structure of the isolated E1 glycoprotein does not accurately represent the conformation of the E1 glycoprotein in the live virus, especially for the N-terminal domain of the E1 glycoprotein. Additionally, surface-exposed lysines in the E2 glycoprotein (for which there is no structure available) were probed, which can be used as constraints for computational modeling of the E2 glycoprotein structure.? ? One of the key research areas at the interface of biology and chemistry is how to investigate the dynamics of proteins and their interactions. We are utilizing mass spectrometry to study protein structures and changes in protein structures by using oxidative surface mapping. For protein structure analysis, we can identify the most solvent accessible residues in aprotein by identifying residues that have been oxidized by 137Cs generated hydroxyl radicals. This information is useful for providing structural information about the protein. This technique can also be used to follow changes in protein structure as a result of hydroxyl radical-surface mapping which mimics oxidative damage on protein structure. This method measures the rate of oxidative damage at many different oxidation targets in the protein simultaneously, in a pseudo-zero order kinetic regime. This rate constant has been shown to be a function of the chemical identity of the oxidation target (which does not change) and the accessibility of the target to the radical. By monitoring the apparent rate of oxidation, we can follow when each oxidation target site becomes more or less exposed to radical as a function of increasing oxidative damage. This technique has been demonstrated on the Bacillus subtilis (a non-toxic model for bacillus biochemistry) protein Spo0F which is a key protein in the sporulation pathway. We have also used this approach to follow changes in conformation in the Bacillus anthracis heptameric pore protein PA63 which undergoes conformational change as it enters the acidic endosomal environment during the anthrax infection process.? ? We have initiated a project to characterize protein:protein interactions relevant to the chronic inflammatory autoimmune disease Sjogren's Syndrome. We are currently charcterizing the structure of? Ro52, the main SSA autoantigen.
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