An increasing number of proteins are known to form amyloid fibrils in vivo, and the formation of these fibrils is implicated in several diseases. One of these proteins, human (B-2-microglobulin (B2m), can form amyloid fibrils in the presence of Cu (ll), and these fibrils are presumed to be the main pathogenic process underlying dialysis-related amyloidosis (DRA). Like other amyloid systems, p2m fibril formation proceeds by partial protein unfolding, subsequent oligomerization, and eventual elongation to form mature fibrils. While aspects of general amyloid formation are understood, molecular-level information is lacking for most amyloid systems; however, this information is critical for the rational design of therapeutics against amyloid diseases like DRA. We plan to obtain amino acid-level information of the unfolding and oligomerization process associated with B2m fibrillogenesis. To do so, we will apply, optimize, and develop 3 mass spectrometry (MS)-based tools with the necessary temporal and spatial resolution. (1). Metal-catalyzed oxidation (MCO) reactions with MS detection will provide information about the evolution of Cu (ll)-B2m interactions during unfolding and oligomerization. Improvements will also be made to the MCO/MS method in order to simplify the analysis and increase the temporal resolution of this technique. (2). A new 'detuned' MCO/MS method will measure the tertiary structural changes that occur around Cu (ll) during (B2m unfolding and oligomerization. The scope of this new method will be studied, including its potential to provide information about the relative distances of amino acids from Cu (ll). (3). Radical-based protein footprinting will be used to study changes in B2m structure during unfolding and oligomerization. Peroxynitrite will be investigated as a new protein footprinting reagent. We hypothesize that Cu (ll) binding destabilizes the N-terminal region of B2m. This destabilization leads to partial unfolding, and the subsequent structural changes that lead to oligomerization will be elucidated using our 3 complementary structural analysis tools.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM075092-03
Application #
7254045
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Edmonds, Charles G
Project Start
2005-07-01
Project End
2009-06-30
Budget Start
2007-07-01
Budget End
2008-06-30
Support Year
3
Fiscal Year
2007
Total Cost
$217,581
Indirect Cost
Name
University of Massachusetts Amherst
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
153926712
City
Amherst
State
MA
Country
United States
Zip Code
01003
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