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.