Dynamical Properties of Flexible Peptides are Modified by Covalent Fluorophore Attachment: A Combined Fluorescence and Molecular Dynamics Study

Abstract

Fluorescence spectroscopy techniques like Forster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) have become important tools for the in vitro and in vivo investigation of conformational dynamics in biomolecules. These methods rely on the distance-dependent quenching of the fluorescence signal of a donor fluorophore either by a fluorescent acceptor fluorophore (FRET) or a non-fluorescent quencher, as used in FCS with photoinduced electron transfer (PET). The attachment of fluorophores to the molecule of interest alters the molecular properties and may affect the relevant conformational states and dynamics especially of flexible biomolecules like intrinsically disordered proteins (IDP). Using the 14 amino acid long S-peptide as a model system, we investigate the impact of terminal fluorescence labeling on the molecular properties.We perform extensive molecular dynamics simulations on the labeled and unlabeled peptide and compare the results with in vitro PET-FCS measurements. Experimental and simulated timescales of end-to-end fluctuations were found to be in excellent agreement. Comparison between simulations with and without labels reveal that the π-stacking interaction between the fluorophore labels traps the conformation of S-peptide in a single dominant state, while the unlabeled peptide undergoes continuous conformational rearrangements. Furthermore, we find that the refolding rate of S-peptide is decreased by at least one order of magnitude by the fluorophore attachment. Our approach combining experimental and in silico methods provides a benchmark for the simulation result and reveals the significant effect that fluorescence labeling can have on the conformational dynamics of small biomolecules. The presented protocol is not only useful to compare PET-FCS experiments with simulation results but provides a strategy to minimize the influence on molecular properties when designing fluorescence labeling.