NR AWUJ
AU Mukhopadhyay,S.; Krishnan,R.; Lemke,E.A.; Lindquist,S.; Deniz,A.A.
TI A natively unfolded yeast prion monomer adopts an ensemble of collapsed and rapidly fluctuating structures
QU Proceedings of the National Academy of Sciences of the United States of America 2007 Feb 20; 104(8): 2649-54
PT journal article; research support, n.i.h., extramural; research support, non-u.s. gov't
AB The yeast prion protein Sup35 is a translation termination factor, whose activity is modulated by sequestration into a self-perpetuating amyloid. The prion-determining domain, NM, consists of two distinct regions: an amyloidogenic N terminus domain (N) and a charged solubilizing middle region (M). To gain insight into prion conversion, we used single-molecule fluorescence resonance energy transfer (SM-FRET) and fluorescence correlation spectroscopy to investigate the structure and dynamics of monomeric NM. Low protein concentrations in these experiments prevented the formation of obligate on-pathway oligomers, allowing us to study early folding intermediates in isolation from higher-order species. SM-FRET experiments on a dual-labeled amyloid core variant (N21C/S121C, retaining wild-type prion behavior) indicated that the N region of NM adopts a collapsed form similar to "burst-phase" intermediates formed during the folding of many globular proteins, even though it lacks a typical hydrophobic core. The mean distance between residues 21 and 121 was approximately equal to 43 A. This increased with denaturant in a noncooperative fashion to approximately equal to 63 A, suggesting a multitude of interconverting species rather than a small number of discrete monomeric conformers. Fluorescence correlation spectroscopy analysis of singly labeled NM revealed fast conformational fluctuations on the 20- to 300-ns time scale. Quenching from proximal and distal tyrosines resulted in distinct fast and slower fluctuations. Our results indicate that native monomeric NM is composed of an ensemble of structures, having a collapsed and rapidly fluctuating N region juxtaposed with a more extended M region. The stability of such ensembles is likely to play a key role in prion conversion.
MH Amino Acid Sequence; Computer Simulation; Fluorescence Resonance Energy Transfer; Molecular Sequence Data; Peptides/chemistry; Prions/*chemistry/*metabolism; Protein Denaturation; *Protein Folding; Protein Structure, Tertiary; Saccharomyces cerevisiae/*chemistry; Saccharomyces cerevisiae Proteins/*chemistry/*metabolism
AD Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
SP englisch
PO USA