Description of Research
In a broad sense my research involves the use of biochemical and biophysical techniques to investigate the structure and membrane association of polypeptides. At the present time my research involves three main areas of investigation:
1. Determining the structure and assembly of thin aggregative fimbriae of salmonella
2. Determining hecformation changes associated with the structural inter-conversion and membrane association of human C-reactive protein (hCRP)
3. Investigating the sequence and structural characteristics that govern the aggregation of islet amyloid polypeptide (IAPP).</a>
Determining the Structure of Thin Aaggregative Ffimbriae of Salmonella
Thin aggregative fimbriae (Tafi) are a class of cell surface filaments found in Escherichia and Salmonella spp. Tafi, comprised manly of a single oligomerized protein, are thought to play an important role in the long-term survival and persistence of the respective organism. A recent report demonstrated that E.coli Tafi (curli) exhibit characteristics similar to amyloid fibrils found in a number of human diseases such as Alzheimer’s and Parkinson’s disease. Based on these observations it has been suggested that curli may serve as a valuable model system for studying amyloid formation. Despite their importance, molecular details regarding the arrangement of protein within Tafi and their structure is lacking. In these proposed studies I hope to investigate the structure of the Salmonella thin aggregative fimbril protein AgfA in its oligomeric form using electron paramagnetic resonance (EPR) spectroscopy together with site directed spin labeling (SDSL). We will employ a series of spin labeled single cysteine mutants of AgfA and obtain EPR data to determine the proteins’ organization within the fibril, and to generate a three-dimensional model of AgfA in its oligomeric form. The combination of EPR spectroscopy and SDSL has proven to be a powerful method in elucidating structural details of amyloid fibrils, and, therefore, is ideally suited for studying the structure of Tafi. The proposed experiments will provide us with structural information that may, in the long term, enable the development of novel, fimbriae based, therapies to reduce long-term pathogen survival and persistence.
The long-term objective of this research is to establish structural details of the Salmonella fimbrial protein AgfA. I seek to answer the following questions: (1). What are the main structural features of monomeric AgfA in solution? (2). What are the main structural features of AgfA in its oligomerized form within fimbriae? (3). How are AgfA monomers organized within fimbriae?
Structural Inter-conversion and Membrane Association of Human C-Reactive Proteins
C-reactive protein (CRP) is a major plasma protein that is thought to play an important role in innate immunity and inflammation. Human CRP is a soluble pentamer of non-covalently associated identical monomers, each with 206 amino acid residues. In addition to this native form of CRP, several other forms, including a membrane bound pentameric form, a membrane bound monomeric form, and an aggregated fibrillar form, have also been described. Many of CRPs’ physiological roles are thought to be associated with its ability to interact with lipid membranes and it has been suggested that the various structural forms of CRP are responsible for its multiple functions. I am interested in investigating the membrane bound and aggregated forms of CRP, and the structural changes that accompany these associations, using site-directed spin labeling and electron paramagnetic resonance spectroscopy.
I seek to answer the following questions: (1). Is the native disulfide important for ligand binding? (2). What structural changes accompany interaction of hCRP with lipid membranes? (3). What structural changes accompany the aggregation of hCRP?
Sequence and Structural Features that Govern the Aggregation of Islet Amyloid Polypeptide
The misfolding, aggregation and membrane interaction of the 37-residue IAPP is thought to play a vital role in the pathogenesis of type II diabetes. IAPP is thought to belong to the calcitonin gene related peptide superfamily to which calcitonin and the calcitonin gene related peptide (CGRP) also belong. Although IAPP and CGRP share significant sequence similarity CGRP has not been shown to aggregate. What sequence characteristics prevent the aggregation of CGRP? Like IAPP calcitonin is also known to aggregate and form amyloid fibrils. Are the amyloid fibrils of calcitonin similar to those of IAPP? Does calcitonin interact with membrane in a manner similar to IAPP? Answers to such questions may help us understand the molecular details that govern the aggregation of IAPP and in turn may help us elucidate the mechanism of IAPP toxicity.