Conformational studies of SEM1 monomer and characterization of GFP-amyloid binding

Abstract

Amyloid fibrils are highly ordered insoluble protein aggregates that are associated with a number of diseases, including Alzheimer’s Disease, Parkinson’s Disease, and Huntington’s Disease. These fibrils can be formed from many different protein sequences. More recently, amyloids have been found that are not associated with any disease state and may even have physiological function. Firstly, we focus on SEM1, an amyloid system found in the semen of healthy men that has been shown to increase human immunodeficiency virus (HIV) infectivity. Previous work has shown that SEM1 fibrillation is pH dependent, requiring a pH greater than 6 to occur. We completed molecular dynamics simulations to examine the monomeric structural conformations of SEM1 at pH 2 and pH 7 and compared it to NMR observables such as chemical shift perturbations and J-Coupling constants. We observed an interaction between an N-terminal α-helix and C-terminal β-hairpin that is reduced in pH 2 as compared to pH 7. We also examined 3 different SEM1 mutants and observed fibrillation at pH 5.5 but not at pH 2. Secondly, we work to characterize a novel discovery in our lab that green fluorescent protein (GFP) binds specifically to amyloid fibrils. We tracked the kinetics of fibril formation through the use of GFP and confocal microscopy and discovered that GFP was capable of detecting small particles unable to be seen by traditional dyes such as ThT or Congo Red. We also developed GFP as a novel inhibitor of fibril formation. Through the development of GFP mutants we explored the binding mechanism of GFP to fibrils and suggest a dual effect of surface aromatics and electrostatics. Moving forward, we have worked to develop GFP as a new tool in detecting endogenous plaque found in Alzheimer’s Disease.