5212 zyxwvutsrqpon Biochemistry zyxwvu 1994, 33, 5212-5220 Tertiary Interactions in the Folding Pathway of Hen Lysozyme: Kinetic Studies Using Fluorescent Probes? Laura S. Itzhaki,t*l Philip zyxwvutsr A. Evans,'.: Christopher M. Dobson,ll and Sheena E. Radfordll Cambridge Centre for Molecular Recognition and Department zyxwvu of Biochemistry, Cambridge University, Tennis Court Road, Cambridge CB2 1 QW, U.K., and Oxford Centre for Molecular Sciences and New Chemistry Laboratory, Oxford University, South Parks Road, Oxford OX1 3QT, U.K. Received September 30, 1993; Revised Manuscript Received February 8, 1994" ABSTRACT: The refolding kinetics of hen lysozyme have been studied using a range of fluorescent probes. These experiments have provided new insight into the nature of intermediates detected in our recent hydrogen- exchange labeling studies [Radford, s. E., et al. (1992) Nature 358,302-3071, which were performed under the same conditions. Protection from exchange results primarily from the development of stabilizing side- chain interactions, and the fluorescence studies reported here have provided a new perspective on this aspect of the refolding process. The intrinsic fluorescence of the six tryptophan residues and its susceptibility to quenching by iodide have been used to monitor the development of hydrophobic structure, and these studies have been complemented by experiments involving binding to a fluorescent hydrophobic dye 1 -aniline- naphthalenesulfonic acid (ANS). Formation of fixed tertiary interactions of aromatic residues has been monitored by near-UV circular dichroism, while development of a competent active site has been probed by binding to a competitive inhibitor bearing a fluorescent label, zyxwv 4-methylumbelliferyl-N,N'-diacetyl-P- chitobiose. The combination of these techniques has enabled us to monitor the development both of the hydrophobic core of the protein and of interactions between the two folding domains. If the behavior of the tryptophans is representative of the hydrophobic residues of the protein in general, it seems that collapse is already substantial in species formed within the first few milliseconds of refolding and is highly developed in later intermediates which nonetheless appear to lack many fixed tertiary interactions. Some of the details of the native structure, including the active site which is formed at the interface between the folding domains, develop only in the slowest stages of folding, even though in a subset of molecules stable native-like structure exists in both domains at an early stage of folding. These late events probably involve fine adjustments of side-chain packing and formation of specific ionic interactions that occur in the native state. Investigation of the pathways by which globular proteins fold is a key approach to understanding their structural organization. Much evidence suggests that folding proceeds, at least in most cases, via transient partially ordered intermediates, so that pathways can be characterized in terms of the structural, kinetic, and thermodynamic properties of these species (Kim zyxwvutsrqp & Baldwin, 1990; Matthews, 1991; Creighton, 1993). A range of chemical and spectroscopic methods, including stopped-flow optical methods, hydrogen- exchange labeling (Baldwin & Roder, 1992; Englander & Mayne, 1992; Baldwin, 1993), and protein engineering techniques (Jennings et al., 1992; Fersht & Serrano, 1993), have provided key information about different aspects of folding pathways. The ephemeral nature of intermediates and the extreme rapidity of at least a number of the steps occurring during folding make the acquisition of the detailed information required to describe folding at the molecular level a demanding problem. The information provided by these different techniques is complementary, and thus, by utilizing a combination of methods to study the same system under We acknowledge support by the Royal Society and by the SERC and MRC through the Oxford Centre for Molecular Sciences and the Cambridge Centre for Molecular Recognition. S.E.R. is a Royal Society 1983 University Research Fellow. C.M.D.is an International Research Scholar of the Howard Hughes Medical Institute. * Author to whom correspondence should be addressed. zyxwvutsrq t Cambridge University. Present address: University Chemical Laboratories, Cambridge 11 Oxford University. @Abstractpublished in Advance ACS Abstracts, March 15, 1994. University, Lensfield Road, Cambridge CB2 lEW, U.K. 0006-2960/94/0433-52 12$04.50/0 identical conditions, a much fuller picture of the events occurring during folding can be drawn than is possible from these methods used in isolation. We have applied this general approach to study the folding of hen lysozyme. This small monomeric protein contains many of the structural features commonly found in globular proteins, including a- and 31°-helices, P-sheets, loops, and turns (Figure 1). Pulsed hydrogen-exchange labeling experiments have revealed that, under the conditions used, the folding process involves a number of well-defined steps and population of specific intermediates (Radford et al., 1992). Specifically, there are two distinct folding domains, so that the a-helical part of the native structure folds, at least in the majority of molecules, more rapidly than a second domain, which includes a triple-stranded @-sheet, a 31°-helix, and a long loop (Figure 1). Further, neither folding domain is stabilized in a single kinetic step: two phases of comparable amplitude (ca. 40%) with average time constants close to zyxw 5 and 65 ms can be resolved for residues in the a-domain, while time constants of approximately 10 and 340 ms are characteristic of amides in the @-domain,the fast phase in this case accounting for only about 25% of the total amplitude. This kineticdiversity reflects the existence of parallel folding pathways which presumably are a consequence of conformational heterogeneity at a crucial, very early stage of refolding. All of these studies have been performed under conditions in which the four disulfide cross- links of the protein remain intact, and one possibility is that slow kinetic phases arise from populations with one or more disulfides in an inappropriate orientation (Chaffotte et al., 0 1994 American Chemical Society