Coulomb Forces Control the Density of the Collapsed Unfolded State of Barstar Hagen Hofmann 1 ⁎, Ralph P. Golbik 1 , Maria Ott 2 , Christian G. Hübner 3 and Renate Ulbrich-Hofmann 1 1 Institute of Biochemistry and Biotechnology, Kurt-Mothes Strasse 3, 06120 Halle, Germany 2 Institute of Physics, Martin-Luther University Halle-Wittenberg, 06120 Halle, Germany 3 Institute of Physics, University of Lübeck, 23538 Lübeck, Germany Received 27 September 2007; received in revised form 26 November 2007; accepted 27 November 2007 Available online 4 December 2007 Although it has been recently shown that unfolded polypeptide chains undergo a collapse on transfer from denaturing to native conditions, the forces determining the dynamics and the size of the collapsed form have not yet been understood. Here, we use single-molecule fluorescence resonance energy transfer experiments on the small protein barstar to characterize the unfolded chain in guanidinium chloride (GdmCl) and urea. The unfolded protein collapses on decreasing the concentration of denaturants. Below the critical concentration of 3.5 M denaturant, the collapse in GdmCl leads to a more dense state than in urea. Since it is known that GdmCl suppresses electrostatic interactions, we infer that Coulomb forces are the dominant forces acting in the unfolded barstar under native conditions. This hypothesis is clearly buttressed by the finding of a compaction of the unfolded barstar by addition of KCl at low urea concentrations. © 2007 Elsevier Ltd. All rights reserved. Edited by C. R. Matthews Keywords: barstar; collapse; protein folding; single-molecule fluorescence Introduction Studying the properties of unfolded polypeptide chains is necessary for a better understanding of the conformational search leading to biologically active proteins. Unfortunately, the nonphysiological con- ditions required to populate the unfolded states of proteins impede their characterization under native conditions. Therefore, methods commonly used to study the structure and size of denatured proteins such as CD or small-angle X-ray scattering are limited in yielding information on unfolded sub- populations. 1–3 In contrast, single-molecule spectro- scopy allows the separation of the signals from folded and unfolded subpopulations in a heteroge- neous mixture, thus avoiding ensemble averaging. 4 Hence, this technique is well suited for studying unfolded proteins under nativelike conditions, even in the presence of an overwhelming excess of native molecules. Recent single-molecule studies have shown an expansion of the unfolded polypeptide chain with increasing concentrations of denaturants such as guanidinium chloride (GdmCl). 4–11 This ex- pansion seems to be continuous rather than discrete and was interpreted as a second-order phase transi- tion 6 in analogy to the well-studied swelling of poly- mers on transfer from “poor” to “good” solvents. The structural features of the collapsed polypeptide chain are not well understood and it is under debate whether rudimentary secondary structure elements have already been present in this state. 8,12 Such structures might serve as nucleation sites, guiding the polypeptide chain through efficient pathways of the energy folding landscape to the native state. However, a complete understanding of the col- lapse process as well as the subsequent folding reaction requires not only a structural but also an energetic characterization of the unfolded polypep- tide chain. The model of this study is a variant of barstar (10.2 kDa), a protein containing 90 amino acid resi- dues. Barstar is the biological inhibitor of barnase, a *Corresponding author. E-mail address: hagen.hofmann@biochemtech.uni-halle.de. Abbreviations used: GdmCl, guanidinium chloride; pWT, pseudo-wild type; pWT*, labelled pseudo-wild type; FRET, fluorescence resonance energy transfer; spFRET, single-pair FRET. doi:10.1016/j.jmb.2007.11.083 J. Mol. Biol. (2008) 376, 597–605 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.