Application Of Mass Spectrometry To Structural Biology

Environmental Health Sciences

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Abstract

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 phosphorylation that are involved in carcinogenesis. 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. The 3-D structures of yeast MLH1 and PMS1 are being probed through the use of differential acetylation. The complex, itself, is currently being expressed. Raf-1: Raf-1 kinase is the best-characterized target of Ras GTPase, the protein product of the ras proto-oncogene. In its active form, Ras can activate Raf-1 leading to stimulation of the MAPK cascade. Activation of this cascade leads to enhanced gene expression and cell proliferation. Oncogenic Ras is constitutively active and can lead to cellular transformation via chronic activation of the MAPK cascade. Raf-1 is subjected to complex regulation, involving many binding partners, and multiple phosphorylations. Raf is normally in an inactive form. It is hypothesized that upon Raf-1 activation, displacement of the N-terminal domains from the kinase domain occurs, but the structures of and the mechanism by which Raf-1 is regulated remain inadequately defined. Understanding the topology of Raf-1 in its inactive and active states will provide information that may be useful in abrogating Raf-1 activity. There are no structural models for full-length Raf-1 as the full-length protein is not amenable to classical structural investigations. The structures of the two N-terminal domains, however, have been determined and the kinase domain can be homology modeled based on the existing structures of other kinases. Our approach involves forming intramolecular chemical cross-links in wild type Raf-1 and a Raf-1 variant that is a mimic of the activated state and identifying the cross-linked residues by mass spectrometry. The data from these experiments are used as inter-residue distance or domain docking constraints during the model building process. To date, we have obtained a purified Raf-1 GST fusion protein and began initial cross-linking studies. We have found cross-linking conditions that show covalent dimer formation via MALDI-MS, but have not yet been able to definitively ID any cross-linked peptides. To enhance the yield of cross-linked peptides, we are using lysine reactive cross-linker that also has biotin as a ?molecular pendant? that will allow us to selectively enrich our cross-linked peptides. We are also probing the structure of the complex between the human anthrax toxin receptor and the Bacillus anthracis protective antigen. This complex is formed as the first step in anthrax infection. Currently we are engaged in expressing the appropriate proteins.