Exploring the Minimally Frustrated Energy Landscape of Unfolded ACBP Valéry Ozenne 1, † , Jeffrey K. Noel 2, † , Pétur O. Heidarsson 3 , Søren Brander 3 , Flemming M. Poulsen 3 , Malene Ringkjøbing Jensen 1 , Birthe B. Kragelund 3 , Martin Blackledge 1 and Jens Danielsson 3,4 1 - Protein Dynamics and Flexibility, Institut de Biologie Structurale Jean-Pierre Ebel, CNRS-CEA-UJF UMR 5075, 6, 38000, France 2 - Center for Theoretical Biological Physics, Rice University, Houston, TX 77005-1827, USA 3 - Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen, Denmark 4 - Department of Biochemistry and Biophysics, Arrhenius Laboratories of Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden Correspondence to Jens Danielsson http://dx.doi.org/10.1016/j.jmb.2013.10.031 Edited by C. Kalodimos Abstract The unfolded state of globular proteins is not well described by a simple statistical coil due to residual structural features, such as secondary structure or transiently formed long-range contacts. The principle of minimal frustration predicts that the unfolded ensemble is biased toward productive regions in the conformational space determined by the native structure. Transient long-range contacts, both native-like and non-native-like, have previously been shown to be present in the unfolded state of the four-helix-bundle protein acyl co-enzyme binding protein (ACBP) as seen from both perturbations in nuclear magnetic resonance (NMR) chemical shifts and structural ensembles generated from NMR paramagnetic relaxation data. To study the nature of the contacts in detail, we used paramagnetic NMR relaxation enhancements, in combination with single-point mutations, to obtain distance constraints for the acid-unfolded ensemble of ACBP. We show that, even in the acid-unfolded state, long-range contacts are specific in nature and single-point mutations affect the free-energy landscape of the unfolded protein. Using this approach, we were able to map out concerted, interconnected, and productive long-range contacts. The correlation between the native-state stability and compactness of the denatured state provides further evidence for native-like contact formation in the denatured state. Overall, these results imply that, even in the earliest stages of folding, ACBP dynamics are governed by native-like contacts on a minimally frustrated energy landscape. © 2013 Elsevier Ltd. All rights reserved. Introduction The unfolded state of a protein is an essential reference point for understanding the molecular processes in which the protein is involved, such as folding leading to function, folding upon binding of intrinsically disordered proteins, misfolding, and aggregation [1–3]. The unfolded polypeptide chain is not merely a randomly sampled statistical coil but, rather, possesses secondary structure pro- pensity (SSP) and transient long-range contacts. These structural biases can potentially affect the productive on-pathway events in protein folding [4–7]. Studies of unfolded states are complicated by the fact that, under native conditions, the fraction of unfolded protein is typically very low. The unfolded population can be increased by mutagenesis, deletions [8], or more often using chaotropic chemi- cals or acid to shift the protein equilibrium toward an unfolded state [9]. Acyl co-enzyme binding protein (ACBP) is a small (86 residues) four-helix-bundle, fast-folding protein (Fig. 1) [10]. The unfolded ensemble of ACBP has 0022-2836/$ - see front matter © 2013 Elsevier Ltd. All rights reserved. J. Mol. Biol. (2014) 426, 722–734 Article