Dynamics of Unfolded Polypeptide Chains in Crowded Environment Studied by Fluorescence Correlation Spectroscopy Hannes Neuweiler⁎, Marc Löllmann, Sören Doose and Markus Sauer⁎ Applied Laser Physics and Laser Spectroscopy, University of Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany Proteins have evolved to fold and function within a cellular environment that is characterized by high macromolecular content. The earliest step of protein folding represents intrachain contact formation of amino acid residues within an unfolded polypeptide chain. It has been proposed that macromolecular crowding can have significant effects on rates and equilibria of biomolecular processes. However, the kinetic consequences on intrachain diffusion of polypeptides have not been tested experimen- tally, yet. Here, we demonstrate that selective fluorescence quenching of the oxazine fluorophore MR121 by the amino acid tryptophan (Trp) in combination with fast fluorescence correlation spectroscopy (FCS) can be used to monitor end-to-end contact formation rates of unfolded polypep- tide chains. MR121 and Trp were incorporated at the terminal ends of polypeptides consisting of repetitive units of glycine (G) and serine (S) residues. End-to-end contact formation and dissociation result in “off” and “on” switching of MR121 fluorescence and underlying kinetics can be revealed in FCS experiments with nanosecond time resolution. We revisit previous experimental studies concerning the dependence of end-to-end contact formation rates on polypeptide chain length, showing that kinetics can be described by Gaussian chain theory. We further investigate effects of solvent viscosity and temperature on contact formation rates demonstrating that intrachain diffusion represents a purely diffusive, entropy-controlled process. Finally, we study the influence of macro- molecular crowding on polypeptide chain dynamics. The data presented demonstrate that intrachain diffusion is fast in spite of hindered diffusion caused by repulsive interactions with macromolecules. Findings can be explained by effects of excluded volume reducing chain entropy and therefore accelerating the loop search process. Our results suggest that within a cellular environment the early formation of structural elements in unfolded proteins can still proceed quite efficiently in spite of hindered diffusion caused by high macromolecular content. © 2006 Elsevier Ltd. All rights reserved. *Corresponding authors Keywords: unfolded polypeptides; intrachain diffusion; molecular crowding; fluorescence quenching; fluorescence correlation spectroscopy Introduction A comprehensive understanding of the mecha- nisms of protein folding requires a detailed char- acterization of the underlying kinetic events. The unfolded state represents the starting point for the folding process and is therefore of particular interest in mechanistic protein folding studies. However, although folded proteins can nowadays be eluci- dated experimentally at atomic detail, structure and Abbreviations used: FCS, fluorescence correlation spectroscopy; FRET, fluorescence resonance energy transfer; TTET, triplet-triplet energy transfer; PET, photo-induced electron transfer. E-mail addresses of the corresponding authors: neuweiler@physik.uni-bielefeld.de; sauer@physik.uni-bielefeld.de doi:10.1016/j.jmb.2006.10.021 J. Mol. Biol. (2007) 365, 856–869 0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.