Conformation of Prion Protein Repeat Peptides Probed by FRET Measurements and Molecular Dynamics Simulations

Abstract

We report the combined use of steady-state fluorescence resonance energy transfer (FRET) experiments and molecular dynamics (MD) simulations to investigate conformational distributions of the prion protein (PrP) repeat system. FRET was used for the first time to probe the distance, as a function of temperature and pH, between a donor Trp residue and an acceptor dansyl group attached to the N-terminus in seven model peptides containing one to three repeats of the second decarepeat of PrP from marsupial possum (PHPGGSNWGQ)nG, and one and two human PrP consensus octarepeats (PHGGGWGQ)nG. In multirepeat peptides, single-Trp mutants were made by replacing other Trp(s) with Phe. As previous work has shown PrP repeats do not adopt a single preferred stable conformation, the FRET values are averages reflecting heterogeneity in the donor-acceptor distances. The T-dependence of the conformational distributions, and derived average dansyl-Trp distances, were obtained directly from MD simulation of the marsupial dansyl-PHPGGSNWGQG peptide. The results show excellent agreement between the FRET and MD T-dependent distances, and demonstrate the remarkable sensitivity and reproducibility of the FRET method in this first-time use for a set of disordered peptides. Based on the results, we propose a model involving cation-pi or pi-pi His-Trp interactions to explain the T- (5-85 degrees C) and pH- (6.0, 7.2) dependencies on distance, with HW i, i + 4 or WH i, i + 4 separations in sequence being more stable than HW i, i + 6 or WH i, i + 6 separations. The model has peptides adopting loosely folded conformations, with dansyl-Trp distances very much less than estimates for fully extended conformations, for example, approximately 16 vs. 33, approximately 21 vs. 69, and approximately 22 vs. 106 A for 1-3 decarepeats, and approximately 14 vs. 25 and approximately 19 vs. 54 A for 1-2 octarepeats, respectively. The study demonstrates the usefulness of combining FRET with MD, a combination reported only once previously. Initial "mapping" of the conformational distribution of flexible peptides by simulation can assist in designing and interpreting experiments using steady-state intensity methods, and indicating how time-resolved or anisotropy methods might be used.

Molecular simulations of protein disorderThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

“…Singlemolecule Förster resonance energy transfer (FRET) can also be used to obtain a distribution of distances between two residues with fluorophores (accessible distances in the range of 10-80 Å ) Vendruscolo 2007). These distance distributions are easily incorporated as restraints in the determination of conformational ensembles (Eliezer 2009), or compared with distances from simulations (Gustiananda et al 2004). Similarly, small-angle X-ray scattering (SAXS) provides information about pairwise distance and a measure of radius of gyration .…”

Equilibrium unfolding of the poly(glutamic acid)20 helix

“…Figure 9a shows a plot of F h versus simulation temperature (K) for Models 1, 2, and 3 using the (128 6 60)8 dihedral criteria for F h discussed in Materials and Methods [Eq. (12)]. Plotted on the right of Figure 9a are the F h values determined from CD measurements between pH 3.33 and 4.93 (black circles) obtained from the pH titration shown in Figure 6.…”

“…In addition, the versatility of FRET measurements on ''difficult'' proteins, such as small peptides, membrane proteins, amyloid aggregates, and instrinsically disordered proteins often makes FRET the only experimental method which can provide quantitative structural information on the structures. 9,[11][12][13][14][15] The ability of FRET to provide structural insights in these difficult protein systems is particularly notable in amyloid peptides, whose shift from random coil/a-helix to b-strand conformations during aggregation is not measurable with high resolution NMR or X-ray crystallography methods. 15,[22][23][24][25][26][27][28][29] When combined with theoretical methods,…”

“…12 In the study of protein folding and structure, FRET has been used to measure changes in donor-acceptor distances during the folding of full-length proteins. 11,13,14,[30][31][32][33] Many other studies have demonstrated that FRET is capable of measuring relatively small distance and distance distribution changes during conformational shifts in short peptide systems such as glycopeptide folding, glycosylation of peptide substrates, thermal stretching of unfolded peptides, zinc finger folding, hydrophobic peptide collapse, and a-helix denaturation.…”

Modern Pathology: Protein Mis-Folding and Mis-Processing in Complex Disease

“…A number of researchers are developing new methods for computer based modeling to characterize changes and mutations in human prion protein (HuPrP) [68,[89][90][91]. Many of these models aim to understand how the conformation and oligomerization state of wild type prion protein in normal cellular isoform (PrP C ) can be differentiated from the disease-inducing isoforms of mutant prion protein (PrP SC PrP Sc/fCJD, PrP Sc/FFI, PrP Sc/GSS ) [92].…”