Downhill versus Barrier-Limited Folding of BBL 3. Heterogeneity of the Native State of the BBL Peripheral Subunit Binding Domain and Its Implications for Folding Mechanisms Giovanni Settanni and Alan R. Fersht⁎ Centre for Protein Engineering, Medical Research Council, Hills Road, Cambridge CB2 0QH, UK Received 5 September 2008; received in revised form 3 February 2009; accepted 4 February 2009 Available online 13 February 2009 Protein folding studies are generally predicated on Anfinsen's dogma that there is a unique native state of a protein. However, this is not always the case. NMR measurements of BBL, for example, find a decrease in helicity of helix 2 surrounding His166 on its protonation, which, with other experimental data, suggests that the native state can occupy two or more conformations. Here, we analysed the native structure of BBL as a function of pH, temperature and ionic strength, along with a truncated BBL construct, by extensive all-atom molecular dynamics simulations in explicit solvent, corresponding to at least 400 ns of trajectories collected for each set of conditions. The native state was heterogeneous under a variety of conditions, consisting of two predominant conformations. This equilibrium changed with conditions: protonation of His166 at low pH shifted the equilibrium in favour of a less ordered conformer, while high ionic strength at neutral pH shifted the equilibrium to a more ordered conformer. Furthermore, high temperature and truncation of the sequence also shifted the equilibrium toward the less ordered conformer. Importantly, conforma- tional heterogeneity in a native structure that changes with conditions will lead to deviations from the classic two-state behaviour during the barrier- limited unfolding of a protein. In particular, some regions of the protein will appear to unfold asynchronously and some residues will have anomalous thermal titration curves and unusual baseline behaviour monitored microscopically by NMR spectroscopy and macroscopically by calorimetry and other techniques. Such data could otherwise be interpreted as evidence for barrier-free downhill folding. Any biological significance of downhill folding of BBL appears to be ruled out by recent crystallographic studies on the reaction cycle of the BBL-equivalent domain in a pyruvate dehydro- genase multienzyme complex in which the domain remains of constant structure. © 2009 Elsevier Ltd. All rights reserved. Edited by F. Schmid Keywords: protein folding; downhill; conformational heterogeneity; simulation Introduction The pioneering studies of Anfinsen led to the concept that proteins have a unique three-dimen- sional native conformation that can be reached spontaneously from its unfolded state in a relatively short time from milliseconds to hours. 1 Conven- tional studies on protein unfolding thus assume that there is an energy barrier between a single native- state structure and a denatured-state ensemble such that there is a cooperative unfolding transition in which all residues of the protein change their structure synchronously. This approximation holds very well in practice for many proteins, and there are always distinct states in equilibrium, such as the native and denatured ensembles. Another scenario *Corresponding author. E-mail address: arf25@cam.ac.uk. Abbreviations used: NOE, nuclear Overhauser enhancement; MD, molecular dynamics; H2, helix 2; H1, helix 1; 3 10 H, 3 10 helix; tBBL, truncated BBL. doi:10.1016/j.jmb.2009.02.014 J. Mol. Biol. (2009) 387, 993–1001 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.