Structural Characterization of the Transition State for Folding of Muscle Acylphosphatase Fabrizio Chiti 1 , Niccolo' Taddei 2 , Nico A. J. van Nuland 1 Francesca Magherini 2 , Massimo Stefani 2 , Giampietro Ramponi 2 and Christopher M. Dobson 1 * 1 Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, South Parks Road Oxford OX1 3QT, UK 2 Dipartimento di Scienze Biochimiche, Universita' degli Studi di Firenze, Viale Morgagni 50, 50134 Firenze Italy The transition state for folding of a small protein, muscle acylphospha- tase, has been studied by measuring the rates of folding and unfolding under a variety of solvent conditions. A strong dependence of the folding rate on the concentration of urea suggests the occurrence in the transition state of a large shielding of those groups that are exposed to interaction with the denaturant in the unfolded state (mainly hydrophobic moieties and groups located on the polypeptide backbone). The heat capacity change upon moving from the unfolded state to the transition state is small and is indicative of a substantial solvent exposure of hydrophobic groups. The solvent-accessibility of such groups in the transition state has also been found to be signi®cant by measuring the rates of folding and unfolding in the presence of sugars. These rates have also been found to be accelerated by the addition of small quantities of alcohols. Tri¯uor- oethanol and hexa¯uoroisopropanol were particularly effective, suggesting that stabilisation of local hydrogen bonds lowers the energy of the transition state relative to the folded and unfolded states. Finally, a study with a competitive inhibitor of acylphosphatase has provided evi- dence for the complete loss of ligand binding af®nity in the transition state, indicating that speci®c long-range interactions at the level of the active site are not yet formed at this stage of the folding reaction. A model of the transition state for acylphosphatase folding, in which b-turns and one or both a-helices are formed to a signi®cant extent but in which the persistent long-range interactions characteristic of the folded state are largely absent, accounts for all our data. These results are broadly consistent with models of the transition states for folding of other small proteins derived from mutagenesis studies, and suggest that solvent perturbation methods can provide complementary information about the transition region of the energy surfaces for protein folding. # 1998 Academic Press Keywords: acylphosphatase; folding transition state; protein folding; sugars; tri¯uoroethanol *Corresponding author Introduction A complete de®nition of the folding process of a protein requires the characterisation of the structure and relative stabilities of all the species forming before the ®nal and unique native conformation. The energy surface or landscape for a polypeptide chain is complex and at any stage involves a distribution of conformers. In cases where relatively well de®ned intermediates exist, much effort has been expended to charac- terise their structural and dynamic properties (Roder et al., 1988; Udgaonkar & Baldwin, 1988; Present address: N. A. J. van Nuland, Departamento de Quõ Âmica-Fõ Âsica, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain. F.C. is on leave from the Dipartimento di Scienze Biochimiche di Firenze, Universita' di Firenze. Abbreviations used: AcP, acylphosphatase; far-UV CD, far ultraviolet circular dichroism; HFIP, 1,1,1,3,3,3- hexa¯uoro-2-propanol; P i , inorganic phosphate; TFE, 2,2,2-tri¯uoroethanol. E-mail address of the corresponding author: chris.dobson@chem.ox.ac.uk Article No. mb982010 J. Mol. Biol. (1998) 283, 893±903 0022 ± 2836/98/440893±11 $30.00/0 # 1998 Academic Press