Slow Folding of Muscle Acylphosphatase in the Absence of Intermediates Nico A. J. van Nuland 1 , Fabrizio Chiti 1 , Niccolo' Taddei 2 Giovanni Raugei 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 folding of a 98 residue protein, muscle acylphosphatase (AcP), has been studied using a variety of techniques including circular dichroism, ¯uorescence and NMR spectroscopy following transfer of chemically denatured protein into refolding conditions. A low-amplitude phase, detected in concurrence with the main kinetic phase, corresponds to the folding of a minor population (13%) of molecules with one or both pro- line residues in a cis conformation, as shown from the sensitivity of its rate to peptidyl prolyl isomerase. The major phase of folding has the same kinetic characteristics regardless of the technique employed to monitor it. The plots of the natural logarithms of folding and unfolding rate constants versus urea concentration are linear over a broad range of urea concentrations. Moreover, the initial state formed rapidly after the initiation of refolding is highly unstructured, having a similar circular dichroism, intrinsic ¯uorescence and NMR spectrum as the protein denatured at high concentrations of urea. All these results indicate that AcP folds in a two-state manner without the accumulation of intermedi- ates. Despite this, the folding of the protein is extremely slow. The rate constant of the major phase of folding in water, k H 2 O f , is 0.23 s 1 at 28  C and, at urea concentrations above 1 M, the folding process is slower than the cis-trans proline isomerisation step. The slow refolding of this protein is therefore not the consequence of populated intermediates that can act as kinetic traps, but arises from a large intrinsic barrier in the folding reaction. # 1998 Academic Press Keywords: acylphosphatase; folding; proline isomerisation; real-time NMR; two-state model *Corresponding author Introduction The mechanism by which proteins fold to their native conformation is currently an area of active research in structural biology (Dill et al., 1995; Miranker & Dobson, 1996). One of the major apparent differences emerging from folding studies of a number of proteins is concerned with the pre- sence or absence of intermediates accumulating during the folding process. While many small pro- teins appear to fold in a two-state fashion, poly- peptides longer than 100 residues appear generally to fold by multiphasic processes with one or more relatively stable intermediates forming prior to the ®nal native state. The distinction between two- state or multi-state folding is not necessarily a fun- damental one, as this simply depends on whether one or more intermediates are suf®ciently stable to accumulate and to allow experimental detection (Miranker & Dobson, 1996). Nevertheless, the investigation of two-state folding reactions has implications of fundamental importance for the analysis of data for determining thermodynamic and kinetic parameters of protein folding. Another important issue is that fast and highly ef®cient folding has been associated with two-state folding 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; ANS, 8-anilino-1-naphthalenesulphonic acid; far-UV CD, far ultraviolet circular dichroism; HFIP, hexa¯uoroisopropanol; NMR, nuclear magnetic resonance; PPI, peptidyl prolyl isomerase. E-mail address of the corresponding author: chris.dobson@chem.ox.ac.uk Article No. mb982009 J. Mol. Biol. (1998) 283, 883±891 0022 ± 2836/98/440883±09 $30.00/0 # 1998 Academic Press