From the Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284-7760
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ABSTRACT |
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Fluorescent and non-fluorescent probes have been
used to show that divalent cations (Ca2+,
Mg2+, Mn2+, and Zn2+) significantly
increase hydrophobic exposure on GroEL, whereas monovalent cations
(K+ and Na+) have little effect.
Zn2+ always induced the largest amount of hydrophobic
exposure on GroEL. By using a new method based on interactions of GroEL
with octyl-Sepharose, it was demonstrated that Zn2+ binding
strengthens GroEL hydrophobic binding interactions and increases the
efficiency of substrate release upon the addition of MgATP and GroES.
The binding of 4,4-bis(1-anilino-8-naphthalenesulfonic acid) to GroEL
in the presence of Zn2+ has a Kd
1 µM, which is similar to that observed previously for the
GroEL 4,4
-bis(1-anilino-8-naphthalenesulfonic acid) complex. Urea
denaturation, sedimentation velocity ultracentrifugation, and electron
microscopy revealed that the quaternary structure of GroEL in the
presence of Zn2+ had a stability and morphology equivalent
to unliganded GroEL. In contrast, circular dichroism suggested some
loss in both
-helical and
-sheet secondary structure in the
presence of Zn2+. These data suggest that divalent cations
can modulate the amount of hydrophobic surface presented by GroEL.
Furthermore, the influence of Zn2+ on GroEL hydrophobic
surface exposure as well as substrate binding and release appears to be
distinct from the stabilizing effects of Mg2+ on GroEL
quaternary structure.
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INTRODUCTION |
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In vitro studies have demonstrated that the information necessary for a polypeptide to attain its native conformation is contained within its amino acid sequence (1). However, the yield of folded protein in vitro is often quite low, in part because aggregation competes with folding. Recently, a highly conserved family of proteins, the Hsp60 molecular chaperones, has been suggested to be able to facilitate protein folding both in vivo and in vitro (2). The primary function of the molecular chaperones appears to be preventing aggregation of partially folded intermediates (3, 4).
The Escherichia coli protein GroEL is homologous to the eukaryotic mitochondrial protein Hsp60, and it is among the best characterized molecular chaperones. GroEL is composed of 14 57-kDa subunits arranged in two stacked, seven-membered rings with 7-fold symmetry to form a cylinder with a central cavity (14-mer) (5). GroEL assists in vitro refolding of a number of proteins, and it has been shown to be able to interact with over half of the proteins from E. coli (6). The mechanistic features for the binding and release of substrate polypeptides by GroEL are not completely understood. The promiscuous nature of GroEL-polypeptide interactions suggests that a GroEL recognition motif is not specific for polypeptide sequence. Thus, a GroEL substrate protein recognition motif must include characteristics that are common in a variety of incompletely folded polypeptides that are not present in their native forms which interact weakly or not at all with the chaperonin.
A key feature of protein folding intermediates is the easy accessibility of hydrophobic residues that would typically be buried in the native conformation of the protein. It has been suggested that GroEL binds the compact folding intermediates (molten globules) of proteins produced in the course of folding (7), and recent calorimetric studies have demonstrated that hydrophobic interactions are an important driving force for the association of substrate proteins with GroEL (8). Further evidence for the importance of hydrophobic interactions came from mutational analysis (9). That study identified polypeptide binding sites on the inside surfaces of the GroEL apical domains (one of the three domains into which the monomer is folded), facing the central channel. Interestingly, all of the apical domain residues determined to be essential for substrate polypeptide binding were hydrophobic residues.
Paradoxically, when the accessibility of hydrophobic surfaces on GroEL
was assayed with the hydrophobic probe
bis-ANS,1 only 1.4 molecules
of bis-ANS were bound per GroEL 14-mer (10). This implies that most of
the hydrophobic residues used for polypeptide binding are either buried
or that the negatively charged bis-ANS molecule does not have access to
the hydrophobic residues, due to the high net negative charge on GroEL
(266 per 14-mer).
The importance of charge to GroEL interactions has been suggested by studies of polypeptide binding and the effects of ions on the quaternary structure of the chaperonin (11-13). In this report, we demonstrate the importance of divalent cations in altering the apparent amount of hydrophobic surface presented by GroEL. The binding of divalent cations (Ca2+, Mg2+, Mn2+, and Zn2+), but not monovalent cations (K+ and Na+), results in an increase in the amount of GroEL hydrophobic surface exposure, with zinc inducing the greatest effects.
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MATERIALS AND METHODS |
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Reagents and Proteins--
All reagents used were analytical
grade. The chaperonin, GroEL, was purified from lysates of cells
containing the multicopy plasmid pGroESL (14). Following purification,
GroEL was dialyzed against 1000 volumes of 50 mM Tris-HCl
(pH 7.5) containing 1 mM dithiothreitol. Glycerol was then
added to 10% (v/v), and aliquots were frozen in liquid nitrogen and
stored at 80 °C. The monomer concentration of GroEL was measured
at 280 nm with an extinction coefficient of 1.22 × 107 M
1 cm
1 as
determined by quantitative amino acid analysis, assuming a molar mass
of 60 kDa (15).
Fluorescence Spectroscopy-- The buffer used in all experiments is 50 mM Tris-HCl (pH 7.0); the GroEL concentrations refer to monomers, and the salt concentrations are 10 mM, unless stated otherwise. All of the concentrations given are final concentrations. Bis-ANS fluorescence experiments in the presence of salts were performed with an excitation wavelength of 397 nm and an emission wavelength of 500 nm on either SLM 500c or SLM 48000s fluorometer. Each sample contained 2 µM GroEL and 20 µM bis-ANS and one of the following salts: NaCl, KCl, CaCl2, MnCl2, MgCl2, or ZnCl2. To determine a dissociation constant for bis-ANS in the presence of GroEL (2 µM) and ZnCl2, a titration was performed over a bis-ANS concentration range of 0-25 µM.
To assess the effects of high ionic strength, fluorescence spectra were acquired for samples containing bis-ANS (20 µM), GroEL (2 µM), and the required concentrations of ZnCl2, with and without NaCl (100 mM). To assess the reversibility of the effect of zinc binding, the fluorescence spectrum of bis-ANS (20 µM) and GroEL 2 (µM) was acquired with and without ZnCl2 (5 mM), followed by the addition of EDTA (8 mM). To determine the competition of MgCl2 for ZnCl2 binding sites on GroEL, samples of GroEL (10 µM) and bis-ANS (10 µM) in the presence and absence of ZnCl2 (5 mM) were titrated with MgCl2 (0-20 mM) and the fluorescence spectra acquired. A titration of bis-ANS with ethanol was performed to measure the fluorescence intensity changes as a function of the shift of the fluorescence wavelength maximum. Specifically, samples of bis-ANS (1 µM) were prepared with increasing concentrations of ethanol (0-95%), decreasing the dielectric constant of the solvent. The wavelength maximum of the bis-ANS fluorescence at each ethanol concentration was converted to frequency and plotted against the fluorescence intensity at the respective frequency maximum. Nile Red fluorescence measurements were performed with an excitation wavelength of 550 nm and an emission wavelength of 660 nm. Three samples were prepared containing Nile Red (10 µM) and GroEL (2 µM). Either MgCl2 or ZnCl2 was added to two of these samples. A fourth sample was prepared containing only Nile Red (10 µM). TNS fluorescence measurements were performed with an excitation wavelength of 315 nm and an emission wavelength of 445 nm. Three samples were prepared containing TNS (10 µM) and GroEL (2 µM). Either MgCl2 or ZnCl2 was added to two of these samples. A fourth sample was prepared containing only TNS (10 µM).GroEL Octyl-Sepharose Binding-- A slurry of octyl-Sepharose was made in 25% ethanol, and 300-µl aliquots were added to 1.5-ml Eppendorf tubes and centrifuged for 30 s. The supernatants were removed, and the octyl-Sepharose in each tube was washed with 400 µl of 50 mM Tris-HCl (pH 7.0), containing 10 mM appropriate salt. The samples were vortexed and then centrifuged for 30 s. The supernatants were removed, and the procedure was repeated three times. Following octyl-Sepharose equilibration, 100-µl samples containing GroEL (10 µM), ± ATP (10 mM), and 10 mM of one of the following salts NaCl, KCl, MnCl2, MgCl2, CaCl2, or ZnCl2 were added to the appropriate octyl-Sepharose pellet. Each sample was mixed and incubated for 5 min. After incubation, the samples were centrifuged for 30 s, and 10 µl of the supernatant from each tube were removed. To assay the release of GroEL from the octyl-Sepharose resin, GroES (15 µM) was subsequently added to the appropriate incubation mixture of GroEL, MgATP, and divalent cation. To assay the competition between GroES and octyl-Sepharose for GroEL binding sites, increasing concentrations of GroES were incubated with GroEL (1:1, 2:1, 3:1, and 4:1; GroES 7-mer/GroEL 14-mer) before addition to the octyl-Sepharose resin. The GroES/EL incubation mixture also contained 10 mM MgCl2, 10 mM ZnCl2, 5 mM KCl, and 5 mM ATP in 50 mM Tris-HCl (pH 7.0). The protein content in the supernatants was analyzed by either 12% SDS-polyacrylamide gel electrophoresis or by BCA protein assay. Gel images were captured electronically, and the optical densities of the GroEL bands were quantified using the computer program, NIH image (version 1.6).
Urea Dissociation of GroEL Followed by Intrinsic Tyrosine Fluorescence and Bis-ANS Fluorescence Spectroscopy-- The urea dissociation of GroEL tetradecamers was detected by intrinsic tyrosine fluorescence and bis-ANS fluorescence. GroEL tyrosine fluorescence was excited at a wavelength of 274 nm, and the emission was detected at a wavelength of 310 nm. Bis-ANS fluorescence was excited at a wavelength of 397 nm, and the emission was detected at 500 nm. To follow the dissociation of GroEL tetradecamers by tyrosine fluorescence and bis-ANS fluorescence, an identical range of urea concentrations (0-6 M) was covered for three different GroEL conditions. The three GroEL conditions used for tyrosine fluorescence measurements were as follows: GroEL (15 µM), GroEL + Mg2+, and GroEL + Zn2+. The conditions for bis-ANS (10 µM) measurements were GroEL (1 µM), GroEL + MgCl2, and GroEL + ZnCl2.
GroEL Light Scattering-- Kinetics of GroEL aggregation were followed by 90° light scattering using an SLM 500c fluorometer by setting both the excitation and emission wavelengths at 400 nm. The protein samples were GroEL (1 µM), GroEL + ZnCl2, and BSA (15 µM) + ZnCl2. Buffer blanks were collected and subtracted from the light scattering data collected for each protein sample.
Circular Dichroism Spectroscopy--
Circular dichroism spectra
of GroEL (5 µM) at three different ZnCl2
concentrations (0, 1, and 10 mM) were collected on a Jasco J500c spectropolarimeter from 250 to 180 nm, at 25 °C. Buffer blanks
were collected and subtracted from the appropriate sample. A spectral
bandwidth of 2 nm, a digitizing interval of 0.1 nm and a 2-s time
constant were used for all samples. Ellipticity () is expressed as
degrees cm2 (dmol amino acid)
1. The spectra
were fit using the k2d program of Andrade et al. (16)
utilizing a neural network algorithm.
Sedimentation Analysis-- GroEL samples (5.0 µM) at three different ZnCl2 concentrations (2.5, 5.0, and 10 mM) were subjected to sedimentation velocity analysis at 25 °C using a Beckman XL-A analytical ultracentrifuge with 12-mm double sector cells in a four-hole Ti-60 rotor with a rotor speed of 18,000 rpm. The scans were analyzed by the method of van Holde and Weischet (17) using the Ultrascan ultracentrifuge data collection and analysis program (B. Demeler, Missoula, MT). All data were corrected to standard conditions.
Electron Microscopy-- Two 5 µM GroEL (50 µl) samples were prepared ± ZnCl2 and incubated for 30 min. The samples (8 µl) were then placed on carbon-coated grids and immediately washed (three times) with 15 µl of H2O. The samples were then negatively stained with 8 µl of 1% (w/v) uranyl acetate and dried by touching the edge of each grid with a piece of filter paper. The micrographs were examined and photographed in a Philips 301 transmission electron microscope.
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RESULTS |
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GroEL Hydrophobic Exposure in the Presence of Divalent Cations-- The influence of cations on the exposure of hydrophobic surfaces on GroEL was probed using the fluorescence associated with bis-ANS binding. Fig. 1 shows the effects on the bis-ANS fluorescence when salts were added to separate solutions of GroEL (2 µM) and bis-ANS (20 µM). The bottom three spectra show there is little influence of the monovalent cations (K+ and Na+). However, the addition of MgCl2, CaCl2, MnCl2, or ZnCl2 resulted in 2.8-, 3.5-, 3.5-, and 15.9-fold increases in bis-ANS fluorescence, respectively. ZnCl2 also caused a 15-nm shift of the bis-ANS emission maximum to a shorter wavelength (505 nm).
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Zn2+ Strengthens GroEL Hydrophobic Binding Interactions and Improves the Efficiency of GroEL Release upon the Addition of MgATP and GroES-- The use of octyl-Sepharose as a GroEL substrate permits the assessment of substrate (octyl-Sepharose) binding and release by monitoring the GroEL protein concentration. Less than 50% of the GroEL is bound in the absence of any ligands (Fig. 3). The addition of either MgCl2 (10 mM) or MgCl2 (10 mM) + ZnCl2 (10 mM) resulted in a 2.5% and 25% increase in GroEL octyl-Sepharose binding, respectively. The addition of ATP increased GroEL binding in the presence of MgCl2 (10 mM) and decreased GroEL binding in the presence of MgCl2 (10 mM) + ZnCl2 (10 mM). Further addition of GroES to these samples resulted in the partial release of GroEL (6% of the GroEL bound) in the presence of MgCl2 (10 mM) and complete release of bound GroEL in the presence of MgCl2 (10 mM) + ZnCl2 (10 mM). This demonstrated that Zn2+ not only supports GroEL function by strengthening hydrophobic interactions with substrate, but it also improves the efficiency of substrate release in the presence of MgATP and GroES.
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Structural Characterization GroEL-Zn2+-- Functionally active GroEL is a homotetradecameric protein made of two stacked 7-subunit rings. In the absence of ligands, the GroEL tetradecamer must be perturbed, e.g. with 2.5 M urea, to observe a large increase in hydrophobic exposure when probed with bis-ANS. The ability of Zn2+ to modulate large changes in GroEL hydrophobic surface exposure was characterized further.
Intrinsic tyrosine fluorescence, bis-ANS fluorescence, and sedimentation velocity ultracentrifugation were used to follow the urea dissociation of GroEL 14-mers into monomers. It has been demonstrated in urea denaturation experiments, which follow intrinsic GroEL tyrosine fluorescence (Fig. 4), that Mg2+ (filled circles) stabilizes the GroEL 14-mer (U1/2
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DISCUSSION |
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GroEL binds a variety of polypeptides in their non-native states (3, 20-21). This implies that the primary sequence of a substrate polypeptide does not supply the information required for recognition. Thus, binding must occur by a more general mechanism of molecular recognition. A common feature of unfolded and misfolded polypeptides is the solvent exposure of hydrophobic residues that would be buried in the native state. It is generally accepted that GroEL polypeptide interactions occur by hydrophobic association. Several studies have described these interactions and stated the importance of GroEL hydrophobic binding of substrate polypeptides (8, 22-23). The GroEL crystal structure (24), complemented by GroEL mutational analysis (9), has made it possible to suggest the substrate polypeptide binding sites on GroEL. These sites are within flexible segments containing hydrophobic residues in the apical domains of the GroEL 14-mer. However, when hydrophobic surfaces on unliganded GroEL are probed with bis-ANS, only a few molecules of bis-ANS bind per GroEL 14-mer (10). This result raises an interesting question. If GroEL substrate polypeptide binding occurs by hydrophobic association in the apical domains of GroEL, where are all the hydrophobic sites on GroEL?
The answer to this question may be that hydrophobic sites on GroEL are buried until the appropriate ligands signal their exposure. For example, several studies have demonstrated that GroEL quaternary structure changes after the addition of adenine nucleotides and divalent cations (12, 25). Thus, it could be conjectured that subsequent to these quaternary changes, there is an exposure of GroEL hydrophobic surfaces. It is also possible that the substrate polypeptide itself might be able to trigger GroEL hydrophobic surface(s). It was shown in an experiment using a positively charged amphipathic peptide that the binding of the peptide to GroEL induced hydrophobic surfaces on GroEL (26). Thus, ligand-induced exposure of hydrophobic surfaces on GroEL may be a mechanism to present hydrophobic binding surfaces to substrate polypeptide. Furthermore, the interaction of a positively charged protein with GroEL has also been described as an effector of GroEL substrate polypeptide binding (11). In that study, it was demonstrated by point mutations within the protein chymotrypsin inhibitor 2 that positive charges on the protein confer tighter binding to GroEL, whereas negative charges on chymotrypsin inhibitor 2 diminish binding (11).
It has been demonstrated in this study that hydrophobic surface(s) on GroEL can be significantly increased upon the addition of divalent cations. The fluorescent probes bis-ANS, Nile Red, and TNS were used to demonstrate that additions of divalent cations (Ca2+, Mg2+, Mn2+, and Zn2+) but not monovalent cations (K+ and Na+) result in an increase in the fluorescence intensity of each probe. The presence of hydrophobic surfaces on GroEL was also measured by the non-fluorescent method using octyl-Sepharose (Fig. 3). The results of that experiment also demonstrate an increase in GroEL hydrophobic surfaces in the presence of divalent cations. This indicates that, independent of the method of hydrophobic measurements, the effects of divalent cations on GroEL hydrophobic surface exposure still occur. One of the most interesting findings in this study is the contribution of Zn2+ in strengthening GroEL substrate binding and increasing the efficiency of substrate release upon the addition of MgATP and GroES.
Magnesium has been previously shown to stabilize the quaternary structure of GroEL (12, 13). The present work shows that zinc does not stabilize the GroEL quaternary structure (Fig. 4), but it does appear to influence the secondary structure of GroEL (Fig. 5). In contrast, there was no detectable change in the GroEL secondary structure in the presence of magnesium (data not shown). These results suggest that Zn2+ binding can affect the secondary structure in a region that does not significantly contribute to the global stability of GroEL as reported by the methods used in this study.
Thus, it appears that interactions with divalent cations can modulate
the exposure of hydrophobic surfaces presented by GroEL, with
Zn2+ being the most effective. It is possible that GroEL
has a low affinity metal binding site (12) or that the divalent cations act in a more general fashion by shielding the negative charge on
GroEL. For example, due to the high net negative charge on GroEL (266
per 14-mer), it may be that cationic interactions of salts and/or
cationic amphipathic secondary structure on substrate polypeptides are
required for a coordinated exposure of buried hydrophobic binding sites
on GroEL. Thus, the binding of divalent cations to GroEL may be a way
of fine-tuning the exposure of hydrophobic residues for the binding of
unfolded substrate polypeptides.
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FOOTNOTES |
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* This research was supported by Research Grants GM25177 and ES05729 from the National Institutes of Health and Welch Grant AQ 723 (to P. M. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry,
University of Texas Health Science Center, San Antonio, TX 78284-7760. Tel.: 210-567-3737; Fax: 210-567-6595; E-mail: Horowitz{at}bioc09.uthscsa.edu.
1
The abbreviations used are: bis-ANS,
4,4-bis(1-anilino-8-naphthalenesulfonic acid); TNS, 6-(p-
toluidinyl)naphthalene-2-sulfonic acid; BSA, bovine serum
albumin.
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REFERENCES |
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