5644 zyxwvutsrqpo Biochemistry zyxwvu 1993, 32, 5644-5649 Side-Chain Mobility of the P-Lactamase zyxw A State Probed by Electron Spin Resonance Spectroscopy+ Linda J. Calciano, Walter A. Escobar, Glenn L. Millhauser, Siobhan M. Miick, Joa Rubaloff, A. Paul Todd, and Anthony L. Fink' Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064 Received September 30, 1992; Revised Manuscript Received February 26, I993 ABSTRACT: 8-Lactamase from Bacillus licheniformis forms a stable compact intermediate state at low pH and moderate salt concentration (the A state), with properties consistent with a molten globule. A single cysteine residue was introduced into this class A @-lactamase by site-directed mutagenesis at position 166. A spin label was attached to the thiol of this cysteine residue via a disulfide bond as a probe of the side-chain mobility. The mutant protein and the spin-labeled derivative exhibited similar conformational properties to the wild-type enzyme at acidic pH. The A state induced by chloride or trichloroacetate (TCA) anions was characterized by circular dichroism and esr. The A state at pH 0.5 (0.32 M HCl), or at pH 2 in the presence of 8 mM TCA or 0.4 M C1-, had comparable amounts of secondary structure to the native state but lacked significant tertiary structure, as judged by the lack of near-UV circular dichroism. Analysis of the esr spectral line widths showed that the mobility of the spin label in the A state was similar to that in the native state and much less mobile than in the unfolded state, indicating significant constraints on the side-chain mobility in this region of the molecule in the A state. The implications of this finding to the structure of the A state are discussed. Intermediate states of proteins have now been observed for many proteins under both equilibrium and kinetic conditions (Ptitsyn, 1987; Kuwajima, 1989; Goto et al., 1990a; Chris- tensen zyxwvutsrqp & Pain, 1991). In some cases these are quite compact and have been called molten globules (Ohgushi & Wada, 1983). Characteristic features of these partially folded states include the presence of significant secondary structure, little nativelike tertiary structure, and substantial exposed hydro- phobic surface area (Baldwin, 1991). Relativelylittleis known about the detailed structures of such intermediates, with the exceptionthat at least some of the secondary structure present is also present in the native state (Dolgikh et al., 1985; Damaschun et al., 1986; Baum et al., 1989; Hughson et al., 1990; Jeng & Englander, 1991). The apparent lack of significanttertiary structurein compact intermediate states suggests that there is minimal interaction between side chains, in contrast to the native state where side chains are normally tightly packed and interdigitated. One means of probing the mobility of side chains is to replace them with a spin label and use electron spin resonance spectroscopy (esr) to measure the mobility of the label. This method has been pioneered by Hubbel and co-workers (Todd et al., 1989), who have used it to determine structural characteristics of colicin E l , The basis of this approach is to attach a spin label to a unique Cys residue introduced by site-directed mutagenesis. Measurements of the esr spectrum of the spin label can then be used to determine its local environment, including its mobility, from the shape of the spectral signal. Analysis of the line shape provides information about the motional amplitude and frequency of the nitroxide and thelocal solvent polarity (Todd et al., 1989). Themobility of the nitroxide, as determined from the esr spectrum, reflects both the motion of the spin label relative to the protein and that of the protein itself. This research was supported by a grant from the National Science Foundation (to A.L.F.). We have previously shown that 8-lactamase from Bacillus cereus forms a stable compact intermediate with molten globule-like properties under conditions of low pH and low to moderate anion concentration (Goto et al., 1990a). The homologous 0-lactamase from Bacillus licheniformis was chosen for the present study because a high-resolution crystallographic structure is available (Moews et al., 1990) and becausewe have previously generated site-specificmutants using the cloned gene (Ellerby et al., 1990; Escobar et al., 1991). In this report we show that the class A p-lactamase from B. licheniformis behaves very similarly at low pH to that from B. cereus, even though the isoelectric points are quite different (4.9 and 8.4, respectively). Replacement of Glu-166 by Cys (E166C) has negligible effect on the low-pH conformational properties of p-lactamase; the absence of Cys in the wild-type enzyme thus allowed us to introduce a single spin label at position 166 in the mutant protein. The presence of the spin label also had little effect on the low pH conformational behavior of the protein. EXPERIMENTAL PROCEDURES Materials. The cysteine- 166 mutant of B. licheniformis p-lactamase was constructed and expressed in Bacillus subtilis as previously described (Escobar et al., 1991). Methaneth- iosulfonatespin label [( 1 zyxw -oxyl-2,2,5,5-tetramethyl-3-pyrroline- 3-methy1)methanethiosulfonate) (MTSSL) (Berliner et al., 1982)] was obtained from Reanal (Budapest, Hungary). Hydroxy-TEMPO was obtained from Aldrich. Trichloro- acetic acid was obtained from Fisher Scientific, ultrapure guanidine hydrochloride (GdmHC1) from ICN Biochemicals, and ultrapure urea from Boehringer Mannheim Biochemicals. The concentration of stock solutions of urea was determined by measuring the refractive index according to Nozaki (1 972). All solutions used for circular dichroism (CD) and high- performance liquid chromatography (HPLC) were made with HPLC-grade water from Fisher Scientific and filtered through a 0.22-bm Millipore filter. All pH measurements were made with a Beckman F71 pH meter using a microcombination 0006-2960/93/0432-5644$04.00/0 0 1993 American Chemical Society