Studies of amyloid fibril formation and the characterization of amyloid - gfp binding

Bishop, Anthony, Charles
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Barquera, Blanca
Colón, Wilfredo
McCallum, Scott
Makhatadze, George, I
García, Angel
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Biochemistry and biophysics
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This electronic version is a licensed copy owned by Rensselaer Polytechnic Institute (RPI), Troy, NY. Copyright of original work retained by author.
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Proteins are complex heteropolymers that fold to a native structure encoded by its amino acid sequence. This process is often in competition with aggregation – the intermolecular association of protein to form insoluble aggregates. One type of aggregate are amyloid fibrils - long thin, highly ordered β-sheet aggregates that can form from a variety of protein sequences. The presence of amyloid fibrils in human tissue are linked to numerous diseases (i.e. Alzheimer’s, Parkinson’s, systemic amyloidosis etc.) and often their role in the disease pathology is not clear. the amyloidogenic protein PAPf39 is present as amyloid fibrils in the semen of healthy men. These fibrils have been shown to greatly increase the infectivity of HIV in vitro and are a subject of ongoing study. Previous work has shown that the formation of PAPf39 is pH dependent, requiring a pH above 5.5 for fibril formation to occur. Removal of the N-terminus of PAPf39 allows fibril formation to occur under both acidic and neutral conditions. Evidence shows that the pH dependence of fibril formation can be explained by a perturbation of the PAPf39 structural ensemble. NMR data in the form of coupling constants and chemical shifts used in conjunction with REMD simulations of monomeric PAPf39 reveal that changes in pH induce the formation of a new β-hairpin in the N-terminus. In addition, neutral pH conditions promote the formation of an α helix near residue 23. Discovering novel binding partners for amyloid fibrils is a subject of great interest. Such compounds could be used to disaggregate, remodel or inhibit the formation of disease-relevant amyloid fibrils. Previous work has shown that sfGFP (superfolder Green Fluorescent Protein) is capable of binding to many different types of amyloid fibril. GFP is an 11 strand β-barrel protein that is fluoresces green upon exposure to blue light. How sfGFP is able to bind to amyloid fibrils is largely unknown. Paramagnetic relaxation enhancement experiments reveal the presence of a binding site centered around three solvent exposed tyrosine residues. Removal of the tyrosine residues, as well as the location of the tyrosine residues on the β-barrel impacts the affinity and the binding mode of GFP for PAPf39 fibrils. Additionally, a novel equilibrium mode assay has been developed for measuring GFP-fibril binding that has potential for examining the affinity of GFP for many other fibril systems.
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Biochemistry and Biophysics Program
Rensselaer Polytechnic Institute, Troy, NY
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