Conformations within Soluble Oligomers and Insoluble Aggregates Revealed by Resonance Energy Transfer

A fluorescently labeled 20-residue polyglutamic acid (polyE) peptide 20 amino acid length polyglutamic acid (E-20) was used to study structural changes which occur in E-20 as it co-aggregates with other unlabeled polyE peptides. Resonance energy transfer (RET) was performed using an o-aminobenzamide donor at the N-terminus and 3-nitrotyrosine acceptor at the C-terminus of E-20. PolyE aggregates were not defined as amyloid, as they were nonfibrillar and did not bind congo red. Circular dichroism measurements indicate that polyE aggregation involves a transition from alpha-helical monomers to aggregated beta-sheets. Soluble oligomers are also produced along with aggregates in the reaction, as determined through size exclusion chromatography. Time-resolved and steady-state RET measurements reveal four dominant E-20 conformations: (1) a partially collapsed conformation (24 angstrom donor-acceptor distance) in monomers, (2) an extended conformation in soluble oligomers (>29 angstrom donor-acceptor distance), (3) a minor partially collapsed conformation (22 angstrom donor-acceptor distance) in aggregates, and (4) a major highly collapsed conformation (13 angstrom donor-acceptor distance) in aggregates. These findings demonstrate the use of RET as a means of determining angstrom-level structural details of soluble oligomer and aggregated states of proteins. (C) 2009 Wiley Periodicals, Inc. Biopolymers 93: 299-317, 2010.