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Published online before print July 6, 2004, 10.1110/ps.04742704
Protein Science (2004), 13:2236-2243. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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In vivo folding of recombinant metallo-{beta}-lactamase L1 requires the presence of Zn(II)

Gopalraj Periyannan, Patrick J. Shaw, Tara Sigdel and Michael W. Crowder

Miami University, Department of Chemistry and Biochemistry, Oxford, Ohio 45056, USA

Reprint requests to: Michael W. Crowder, Miami University, Department of Chemistry and Biochemistry, 112 Hughes Hall, Oxford, OH 45056, USA; e-mail: crowdemw{at}muohio.edu; fax: (513) 529–5715.

(RECEIVED March 16, 2004; FINAL REVISION May 3, 2004; ACCEPTED May 8, 2004)


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 References
 
Metallo-{beta}-lactamase L1, secreted by pathogenic Stenotrophomonas maltophilia, is a dinuclear Zn(II)-containing enzyme that hydrolyzes almost all known penicillins, cephalosporins, and carbapenems. The presence of Zn(II) ions in both metal binding sites is essential for full enzymatic activity; however, the mechanism of physiological metal incorporation is unknown. To probe metal incorporation, L1 was over-expressed in minimal media with (mmL1+Zn) and without (mmL1–Zn) Zn(II) added to the media, and the resulting proteins were purified and characterized. The mmL1+Zn sample was bound by a Q-Sepharose column, exhibited steady-state kinetic properties, bound Zn(II), existed as a tetramer, and yielded fluorescence emission and CD spectra similar to L1 overexpressed in rich media. On the other hand, the mmL1–Zn sample did not bind to a Q-Sepharose column, and gel filtration studies demonstrated that this protein was monomeric. The mmL1–Zn sample exhibited a lower kcat value, bound less Zn(II), and yielded fluorescence emission and CD spectra consistent with this enzyme being folded improperly. Taken together, these data demonstrate that the proper folding of L1 requires the presence of Zn(II) and suggest that in vitro, thermodynamic metal binding studies do not accurately reflect physiological metal incorporation into L1.

Keywords: metallo-{beta}-lactamase; Zn(II); folding; CD spectroscopy; fluorescence; in vivo metal binding

Abbreviations: CcrA, metallo-{beta}-lactamase from Bacteroides fragilis • CD, circular dichroism • {varepsilon}, extinction coefficient • HEPES, N-2-Hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid • ICP-AES, inductively coupled plasma–atomic emission spectroscopy • IPTG, isopropyl-{beta}-thiogalactoside • L1, metallo-{beta}-lactamase from Stenotrophomonas maltophilia • LB, Luria-Bertani • MALDI-TOF, matrix-assisted laser desorption ionization time of flight • mmL1, L1 overexpressed in minimal media • SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04742704.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 References
 
{beta}-Lactam-containing antibiotics constitute the largest family of inexpensive, clinically useful antibiotics (Neu 1992). Bacterial resistance to {beta}-lactam antibiotics is most often the result of the production of {beta}-lactamases, which are bacterial enzymes that hydrolyze the carbon–nitrogen bond in the four-membered ring of the {beta}-lactam-containing antibiotics (Cricco and Vila 1999; Crowder and Walsh 1999; Frere et al. 1999; Wang et al. 1999). The ring-opened antibiotic is no longer an effective antimicrobial agent. There have been over 300 different {beta}-lactamases reported in the literature, and these enzymes have been grouped into four classes based on a number of different molecular and physical properties (Ambler 1980; Bush 1989a,b,c; Bush et al. 1995; Frere et al. 1999). One class of {beta}-lactamase, class B (Ambler 1980) or group 3 (Bush et al. 1995), is distinct in that it requires 1–2 Zn(II) ions per monomer for full catalytic activity, is insensitive to clavulanic acid and other classical {beta}-lactamase inhibitors, and has no clinical inhibitors (Rasmussen and Bush 1997; Bush 1998; Cricco et al. 1999). These metallo-{beta}-lactamases have been reported in several different bacterial strains, and several of these are pathogens including Stenotrophomonas maltophilia (Walsh et al. 1994; Sanschargrin et al. 1998), Bacteroides fragilis (Rasmussen et al. 1990), Serratia marcescens (Osano et al. 1994; Ito et al. 1995), and Klebsiella pneumoniae (Senda et al. 1996).

S. maltophilia is an important pathogen in nosocomial infections of immunocompromised patients suffering from cancer, cystic fibrosis, drug addition, and AIDS and in patients with organ transplants and on dialysis (Muder et al. 1987; Khardori et al. 1990; Laing et al. 1995; Zhang et al. 2000). This organism is inherently resistant to most antibiotics due to its low outer membrane permeability (Garrison et al. 1996) and to {beta}-lactam containing antibiotics due to the production of a chromosomally expressed group 2e {beta}-lactamase (L2) and a group 3c {beta}-lactamase (L1) (Walsh et al. 1994, 1997). L1 has been cloned, overexpressed, and partially characterized by kinetic and crystallographic studies (Crowder et al. 1998; Ullah et al. 1998). The enzyme exists as a homotetramer of ~118 kDa in solution and in the crystalline state. The enzyme tightly binds two Zn(II) ions per subunit and requires both Zn(II) ions for full catalytic activity. The Zn1 site has three histidine residues and one bridging hydroxide as ligands, and the Zn2 site has two histidines, one aspartic acid, one terminally bound water, and the bridging hydroxide as ligands. Recently, Wommer et al. (2002) reported that dissociation constants (KD) for Zn(II) binding were substrate dependent, and the KD value for Zn(II) binding to the first metal binding site was 2.6 nM in the absence of substrate and 5.7 pM in the presence of the substrate. The KD value of Zn(II) binding to the second metal binding site was 6 nM in the absence of substrate and 120 nM in the presence of substrate.

Recently, the metal content of several of the metallo-{beta}-lactamases to exhibit full catalytic activity has been questioned. Mononuclear Zn(II)-containing model complexes have been reported to perform {beta}-lactamase activity (Kurosaki et al. 2000). Paul-Soto et al. (1998, 1999) have reported that metallo-{beta}-lactamase CcrA is active as a mononuclear or dinuclear Zn(II)-containing enzyme. Wommer et al. (2002) subsequently suggested that {beta}-lactamase II and L1 are apo-enzymes in vivo and function as mononuclear Zn(II)-containing enzymes only in the presence of substrate. They continued that dinuclear Zn(II)-containing forms of these enzymes were not physiologically-relevant. These hypotheses were extrapolated from in vitro metal binding and activity studies on apo-enzymes. The studies in this work were designed to test the validity of this extrapolation and to offer information on biological metal incorporation into L1.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 References
 
Overexpression and purification of L1 in different growth conditions
To test the biological incorporation of Zn(II) into metallo-{beta}-lactamase L1, the enzyme was overexpressed in three different growth media: (1) in rich, LB media (LB-L1), (2) in minimal media (Rajagopalan et al. 2000) containing ZnCl2 (mmL1+Zn), and (3) in minimal media with no added ZnCl2 (mmL1–Zn). The production of L1 in all three cultures was induced by the addition of IPTG, and 100 µM ZnCl2 was added to the culture of mmL1+Zn at induction. The resulting cells from all three preparations were lysed by using a French press, and the dialyzed, soluble protein mixtures were loaded onto Q-Sepharose columns. LB-L1 and mmL1+Zn eluted from the Q-Sepharose columns at ~100–150 mM NaCl, as previously reported for LB-L1 (Fig. 1BGo; Crowder et al. 1998). On the other hand, mmL1–Zn eluted from the Q-Sepharose column before the salt gradient was started (Fig. 1AGo), suggesting that mmL1–Zn does not bind (or weakly binds) to Q-Sepharose. To verify that fraction 10 from Figure 1AGo was in fact L1, a sample from this fraction and a sample of purified L1 were treated with trypsin and analyzed with MALDI-TOF mass spectrometry. The samples yielded identical peptide fragments, indicating that fraction 10 is L1. Additionally, MALDI-TOF MS of the purified protein from fraction 10 had the same molecular weight as LB-L1 and mmL1+Zn (data not shown).



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  		Figure 1. SDS-PAGE gels of the elution profiles of mmL1–Zn (A) and mmL1+Zn from Q-Sepharose column (B).

 
In order to determine whether the Q-Sepharose binding behavior was due to differences in the quaternary structures of mmL1+Zn and mmL1–Zn, the molecular weight of each sample was determined. Fraction 10 from the Q-Sepharose column (Fig. 1AGo) was concentrated and loaded onto a Sephacryl S-200 gel filtration column. The elution of mmL1–Zn from the gel filtration column was consistent with this enzyme being a monomer of ca. 28 kDa. On the other hand, LB-L1 and mmL1+Zn eluted from the column as tetramers from the gel filtration column, as previously reported (Crowder et al. 1998). The first interpretation of this result is that monomeric L1 binds differently to Q-Sepharose than does tetrameric L1. However, we previously reported that the monomeric M175D mutant, which binds 2 equivalents of Zn(II), elutes from the Q-Sepharose column identically as wild-type, LB-L1 (Simm et al. 2002). Therefore, the differential binding of the L1 samples to Q-Sepharose cannot be attributed to differences in quaternary structure.

Metal content and steady-state kinetics
To probe whether the metal content of the L1 samples was different, ICP-AES was used. Metal analyses of as-isolated LB-L1, mmL1+Zn, and mmL1–Zn samples revealed that these enzymes bind 2.0 ± 0.1, 1.5 ± 0.2, and 0.17 ± 0.03 equivalents of Zn(II), respectively (Table 1Go). The slightly lower metal content of the mmL1+Zn, as compared to LB-L1, is likely due to the higher Zn(II) bioavailability in LB as compared to minimal media + Zn(II). The zinc content of the media was measured by ICP-AES, and LB contains 9.2 µM Zn(II) while the minimal media with added metal mix contains 2.4 µM Zn(II). Even though the media used to prepare mmL1+Zn contained 10 times more Zn(II) than LB (100 µM versus 9.2 µM), we predict that the bio-availability of Zn(II) in LB is higher. LB media contains peptides that readily coordinate Zn(II) and make it soluble. The minimal media, however, contains amino acids that would not be expected to increase the solubility of Zn(II). Support for this prediction is found in the fact that mmL1–Zn contains 10 times less Zn(II) than LB-L1 despite there being only fourfold less Zn(II) in the media used to prepare these enzymes. The small amount of Zn(II) in mmL1–Zn can be attributed to 2.4 µM Zn(II) in the media used and the nearly impossible task of removing all Zn(II) from buffers or media despite using Chelex 100 (Wommer et al. 2002). It is not clear why the small amount of mmL1–Zn that binds Zn(II) is found in the fraction that does not bind to the Q-Sepharose instead of in the normal fraction that elutes as LB-L1 and mmL1+Zn. It is possible that the mmL1–Zn sample is a mixture of partially loaded L1 (sample that contains less than 2 equivalents of Zn(II)) and apo-L1, and neither of these forms bind to Q-Sepharose. Fractions 22–24 (see Fig. 1AGo), where LB-L1 normally elutes, from the mmL1–Zn preparation were pooled and concentrated. This sample bound <0.009 equivalents of Zn(II), indicating that there is little or no L1 that binds Q-Sepharose made in the mmL1–Zn preparation.


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  		Table 1. Steady-state kinetic constants and metal content of L1 samples
 
To determine whether the kinetic properties of the L1 samples were different, steady-state kinetic studies were performed in the presence and absence of Zn(II) using nitrocefin as the substrate. These studies show that LB-L1 and mmL1+Zn exhibit similar kinetic properties, with kcat values of 41 and 45 s–1, respectively, and Km values of 4 and 5 µM, respectively (Table 1Go), when the assays were conducted in the presence of 100 µM ZnCl2. In the absence of added Zn(II) in the assay buffer, LB-L1 and mmL1+Zn exhibit kcat values of 38 and 34 s–1 and Km values of 5 and 6 µM, respectively. On the other hand, mmL1–Zn exhibits kcat and Km values of 1.7 s–1 and 7 µM, respectively (Table 1Go), when the assays were conducted in the presence of 100 µM ZnCl2. In the absence of added Zn(II) in the assay buffer, mmL1–Zn exhibits a kcat value of 0.8 s–1 and a Km value of 9 µM. The mmL1–Zn sample contained 8% of the Zn(II) bound by LB-L1 and exhibited ~4% of the total activity (kcat, Table 1Go), suggesting that the activities of all of the L1 samples may be due to the same enzyme form.

Fluorescence spectroscopy
The presence of five total tryptophans, with three of the Trp residues in close proximity to the metal binding site of L1 (Ullah et al. 1998), allows for the study of metal and substrate binding and localized protein structure by fluorescence emission spectroscopy (Spencer et al. 2001; Carenbauer et al. 2002; G. Periyannan and M.W. Crowder, unpubl.). By using a 295-nm excitation wavelength, LB-L1 yields the most intense fluorescence emission peak at 322 nm, mmL1+Zn has slightly lower emission intensity, and mmL1–Zn exhibits nearly a fourfold decrease in emission intensity with an emission maximum shifted slightly to higher wavelengths (Fig. 2Go). The addition of Zn(II) to LB-L1 or mmL1–Zn resulted in no changes in the corresponding emission spectrum (Fig. 2Go). On the other hand, the addition of Zn(II) to as-isolated mmL1+Zn to a total of 2 equivalents of Zn(II) resulted in an emission spectrum with similar intensity to that of L1-LB (Fig. 2Go).



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  		Figure 2. Fluorescence emission spectra of L1 samples and the effect of added Zn(II). The spectra were obtained using 0.25 µM L1 samples (monomer) in 50 mM HEPES (pH 7.0) at 25°C. The emission spectrum of the buffer was subtracted from each of the spectra. The excitation wavelength used in these studies was 295 nm.

 
To further test Zn(II) binding to L1, apo-L1 was prepared from LB-L1 as previously reported (Crowder et al. 2001). The resulting apoenzyme contained <<0.1 equivalents of Zn(II) and exhibited a kcat of 0.026 s–1 (Crowder et al. 2001). Interestingly, the fluorescence emission spectrum of apo-L1 is more intense than that of mmL1–Zn (Fig. 2Go). The addition of Zn(II) to apo-L1, a total of 2 equivalents, resulted in a large increase in fluorescence emission intensity (Fig. 2Go). These results suggest that Zn(II) can reversibly bind to apo-L1 and bind to mmL1+Zn to afford proteins with similar fluorescence properties as LB-L1. However, mmL1–Zn has different fluorescence properties than any of the other L1 samples and exhibits less fluorescence emission than apo-L1. Significantly, the addition of Zn(II) to this sample does not afford a protein with similar fluorescence properties as LB-L1.

Circular dichroism
The fluorescence emission studies suggested that there is a structural difference between mmL1–Zn and all of the other L1 samples studied. To probe the secondary structure of the L1 samples, CD spectroscopy was used. The CD spectrum of LB-L1 is typical of an {alpha}+{beta} protein and is similar to that previously reported (Crowder et al. 1998). The addition of Zn(II) to this sample results in no change in the spectrum, particularly at 220 nm that is often attributed primarily to {alpha}-helix (data not shown; Greenfield 1999). The CD spectrum of mmL1+Zn is similar to that of LB-L1 (Fig. 3Go); although, there is a reduction in the peak at 195 nm. Analyses with CDSSTR of this CD spectrum demonstrate 38% {alpha}-helix, 37% {beta}-strand/turn, and 25% unordered secondary structure. Addition of Zn(II) to the mmL1+Zn sample to total 2 equivalents results in a large increase in the peak at 195 nm and no change in the 220 nm feature. This observation suggests that Zn(II) binding to L1 (in this case mmL1+Zn) results in a change in {beta}-components in the enzyme. This result is not surprising since the Zn(II) binding sites in L1 sit in between two sets of {beta}-sheets (Ullah et al. 1998). Comparison of this spectrum to that of LB-L1 suggests that there is no difference in the secondary structures of these two enzymes. However, the CD spectrum of mmL1–Zn is significantly different than those of the other L1 samples. This spectrum has a very small feature at 195 nm and large rotations between 210 and 220 nm, suggesting a significantly different structure containing relatively large amounts of random coil in this sample (Greenfield 1999). CDSSTR analyses of this CD spectrum demonstrate 15% {alpha}-helix, 48% {beta}-strand/turn, and 36% unordered structure. Significantly, the addition of Zn(II) to this sample to total 2 equivalents results in a further decrease in the 195 nm feature and a slight increase in the 220 nm feature, suggesting a larger percentage of random coil in mmL1–Zn upon addition of Zn(II).



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  		Figure 3. CD spectra of L1 samples. The CD spectra were collected on 75 µg ml–1 enzyme samples (monomer) in 5 mM phosphate (pH 7.0) and at 25°C.

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 References
 
Biological protein folding of metalloproteins occurs via two limiting mechanisms (Hausinger 1990). The first mechanism involves protein folding in the absence of metal ion and the generation of a metal binding site. Metallation of this site can be under thermodynamic control, where metal ion selectivity and specificity is largely controlled by thermodynamic considerations (Lippard and Berg 1994), or under kinetic control, where accessory proteins are required to deliver the correct metal ion to the site, as in the case of the Cumetallochaperones (Rosenzweig 2001, 2002). The second mechanism requires the presence of metal ion(s) for the protein to fold properly and has been reported to be in operation for relatively few proteins, such as in azurin (Pozdnyakova and Wittung-Stafshede 2001) and in several zinc finger-containing proteins (reviewed in Cox and McLendon 2000). As in the first mechanism, metal ion binding could conceivably be under thermodynamic or kinetic control.

To evaluate which of the folding mechanisms is in operation with metallo-{beta}-lactamase L1, the enzyme was over-expressed in Escherichia coli using a minimal media and in the presence and absence of added Zn(II). At least five lines of evidence suggests that L1 overexpressed in minimal media and in the absence of added Zn(II) (mmL1–Zn) does not have the same structure as L1 overexpressed in rich media (LB-L1) or L1 overexpressed in minimal media supplemented with Zn(II) (mmL1+Zn). First, mmL1–Zn does not bind to the Q-Sepharose column in the same way that the other two enzymes do, suggesting different residues on the surfaces of the enzymes that interact with the resin. Second, gel filtration studies demonstrate that mmL1–Zn does not exist as a tetramer in solution as the other enzymes do. Previous studies showed that the different quaternary structure of mmL1–Zn cannot be used to explain the different Q-Sepharose binding characteristics of this enzyme as compared to the other enzymes (Simm et al. 2002). Third, mmL1–Zn does not bind significant amounts of Zn(II) nor does the enzyme exhibit steady-state kinetic properties like mmL1+Zn or LB-L1. Fourth, mmL1–Zn exhibits very different fluorescence properties than the other enzymes, suggesting different environments of the proteins’ tryptophan residues. Significantly, the fluorescence properties of mmL1–Zn do not change upon addition of Zn(II), suggesting that the structure of mmL1–Zn is not conducive to reversible Zn(II) binding. Fifth, CD spectra of the three enzymes demonstrate that mmL1–Zn does not have the same secondary structure as the other enzymes and that mmL1–Zn is made up of more random coil than LB-L1 and mmL1+Zn. As observed with the fluorescence studies, the addition of Zn(II) to mmL1–Zn does not result in a CD spectrum of the enzyme that resembles that of LB-L1. These characteristics indicate that mmL1–Zn has a different secondary, tertiary, and quaternary structure than the other enzymes, and suggest that proper folding of L1 requires the presence of Zn(II).

Recently, Wommer et al. have suggested that the metallo-{beta}-lactamases are apoenzymes in vivo and are mononuclear Zn(II) enzymes only in the presence of substrate (Wommer et al. 2002). In addition, the authors hypothesized that the existence of dinuclear Zn(II) metallo-{beta}-lactamases are the result of isolation artifacts. These hypotheses were extrapolated from thermodynamic binding/activity results on apoenzymes, which were generated by chelation of metal from four metallo-{beta}-lactamases (L1, BlaB, CphA, and BcII) overexpressed in rich media. A valid extrapolation of in vitro results to physiological relevance requires that the in vitro studies mimic how metal ions are incorporated into the proteins during protein synthesis/folding. The results presented herein demonstrate that the folding of L1 requires Zn(II) to be present for the enzyme to fold properly, suggesting that reversible metal binding to the properly-folded apo-L1 is not physiologically relevant.

While the results in this work do not directly address the issue of metal binding to L1 being under kinetic or thermodynamic control, previous studies by O’Halloran and coworkers (Hitomi et al. 2001; Finney and O’Halloran 2003) on two transcription regulators of Zn(II) influx/efflux pumps suggest that Zn(II) incorporation into E. coli proteins is under kinetic control. By using a cell assay and a Zn(II) regulatory protein, O’Halloran and coworkers (Outten and O’Halloran 2001) estimated that there is much less than one free Zn(II) ion in an E. coli cell despite E. coli cells having a Zn(II) quota of ~0.1 mM. Since there is no persistent pool of free Zn(II) in E. coli, O’Halloran and coworkers speculated that there must be Zn(II) metallochaperones that transport Zn(II) within the cell to various locations (Finney and O’Halloran 2003), including to the ribosomes during protein synthesis/folding. To date, no diffusible Zn(II) metal-lochaperones have been identified (Blencowe and Morby 2003; Finney and O’Halloran 2003; Luk et al. 2003); however, studies are on-going in our lab to identify these proteins in E. coli. Taken together, the data in this work clearly demonstrate that Zn(II) (or a Zn(II)-responsive protein) is required for the proper folding for L1, and we postulate that metal incorporation into L1 as it is being folded is under kinetic control.


   Materials and methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
References
 
Overexpression and purification of L1 samples
Metallo-{beta}-lactamase L1 was overexpressed in the minimal media (Rajagopalan et al. 2000) using three different preparation conditions: (1) in minimal media containing 100 µM ZnCl2 (mmL1+Zn), (2) in minimal media that was chelexed and to which no Zn(II) was added (mmL1–Zn), and (3) in LB media (LB-L1), as previously described (Crowder et al. 1998). ICP-AES analyses of the resulting media demonstrated that LB contained 9.2 µM Zn(II) and minimal media with added metal mix contained 2.4 µM Zn(II). In all cases, L1 was overexpressed and purified using the same procedure as was previously reported (Crowder et al. 1998). SDS-PAGE gels of boiled cell fractions (Crowder et al. 1998) demonstrated that L1 overexpressed at similar levels in all three cultures. The amounts (mg of L1 per liter of culture) of isolatable L1, after Q-Sepharose chromatography, was 10–12 mg/L of LB-L1, 10 mg/L of mmL1+Zn, and 3 mg/L of mmL1–Zn. Protein purity was ascertained by using SDS-PAGE. Size exclusion chromatography was performed on a Sephacryl S-200 column using 50 mM Na3PO4 (pH 7.5) containing 150 mM NaCl. Extinction coefficients for L1 samples were determined utilizing the BCA protein assay kit purchased from Fisher, with L1-LB used to create the calibration curve.

Protein identity of mmL1–Zn was accomplished using trypsin digestions and MALDI-TOF mass spectrometry. Samples of mmL1–Zn and mmL1+Zn were obtained from the FPLC and dialyzed in 30 mM TRIS (pH 7.5) overnight. The sample proteins then were quantitated, and 5 µg of each protein was digested overnight with proteomics-grade trypsin (Sigma) at 37°C. Aliquots (2 µL) of the trypsin-digested peptides were mixed with same volume of {alpha}-cyano-4-hydroxycinnamic acid (10 mg/mL in aceto-nitrile:trifluoroacetic acid, 50%: 0.05%). A 2 µL sample of the mixture was loaded onto a target for a Bruker Reflex III MALDI-TOF mass spectrometer. Analyses were performed in reflectron mode with an acceleration voltage of 20 kV and delayed extraction of 40,000 ns. Peptides masses ranging from 1000–2600 Da were collected to avoid any lower molecular mass matrix molecules, and these conditions also helped to enhance the signals from the peptides. The instrument was calibrated by using standard peptides angiotensin II [(M+H+)+ = 1046.54] and ACTH, fragment 18–39 [(M+H+)+ = 2465.20] as external calibrants. The spectra were analyzed by using the Bruker XTOF software.

Metal analyses and steady-state kinetics
Protein concentration was determined by measuring the absorbance at 280 nm and using the extinction coefficients of L1 [L1-LB and mmL1+Zn: {varepsilon}280 = 54,600 M–1cm–1 (Crowder et al. 1998); mmL1–Zn {varepsilon}280 = 78,600 M–1cm–1]. The metal content of the L1 samples (typically 10 µM) was determined by using a Perkin-Elmer Inductively Coupled Plasma spectrometer with atomic emission detection (ICP-AES). The detection limit of our ICP-AES for Zn(II) is 2–5 µM. All kinetic studies were conducted on a Hewlett-Packard 8453A UV-Vis diode array spectrophotometer at 25°C. Steady-state kinetic parameters, the Michaelis constant Km and the turnover number kcat, were determined using nitrocefin as substrate in 50 mM cacodylate (pH 7.0) containing 100 µM ZnCl2, as previously described (Crowder et al. 1998).

Circular dichroism spectroscopy
Samples were prepared by dialyzing L1 samples versus 3 x 2L of 5 mM phosphate buffer (pH 7.0) over 6 hours. The samples were diluted to a final concentration of 75 µg/mL. A JASCO J-810 CD spectrometer operating at 25°C and a 0.1 mm quartz cuvette were used to collect CD spectra. CD spectra were analyzed for secondary structural content using the CDSSTR, CONTINLL, SELCON3, VARSLC, and K2D simulation programs at DI-CHROWEB (http://www.cryst.bbk.ac.uk/cdweb/html/home.html) (Lobley and Wallace 2001; Lobley et al. 2002). The standard NRMSD goodness-of-fit parameter was used to determine which program best fitted the data (Mao et al. 1982).

Fluorescence spectroscopy
A Perkin-Elmer LS55 Luminescence Spectrometer, tuned to an excitation wavelength of 295 nm and emission wavelength of 340 nm with a slit width of 5 nm, was used to monitor fluorescence emission spectra of the L1 samples. A 4-mm quartz cuvette was used. Protein concentrations were 250 nM, and chelexed, 50 mM HEPES buffer was used as blank.


   Acknowledgments
 
The authors thank James Garrity for carefully reading this manuscript. This work was supported by the NIH (AI40052 and GM40052). Funds to purchase the CD spectrapolarimeter (DBI-0070169) were provided by the NSF. Patrick Shaw was a 2003 Hughes summer scholar.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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Carenbauer, A.L., Garrity, J.A., Periyannan, G., Yates, R.B., and Crowder, M.W. 2002. Probing substrate binding to metallo-{beta}-lactamase L1 from Stenotrophomonas maltophilia by using site-directed mutagenesis. BMC Biochem. 3: 4–10.[CrossRef][Medline]

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