©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Molecular Chaperonin cpn60 Displays Local Flexibility That Is Reduced after Binding with an Unfolded Protein (*)

Boris M. Gorovits , Paul M. Horowitz (§)

From the (1) Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Steady-state fluorescence polarization was used to examine the chaperonin cpn60 that was covalently labeled with pyrene. Two compounds, 1-pyrenesulfonyl chloride or N-(1-pyrene)maleimide, were used to incorporate up to 8 mol of pyrene per mol of cpn60 14-mer. The fluorescence lifetime of the cpn60-pyrenesulfonyl chloride conjugate exhibited a double exponential decay: 5.36 ns, with a fractional contribution to the intensity of 7%, and 48.77 ns, with a fractional contribution to the intensity of 93%. These yield a second-order average lifetime of 45.58 ns at 20 °C. Analysis of the fluorescence polarization data for the pyrene probe by the Perrin-Weber treatment revealed the existence of two components that account for the depolarization. The fast component accounted for 24% of the depolarization at 20 °C. The rotational relaxation time for the cpn60 14-mer derived from the low viscosity part of the Perrin-Weber plot which accentuates the slow motion gave = 1113 ± 55 ns. When this value of is compared with the calculated based on the Stokes radius of cpn60 from ultracentrifugation, , it leads to / = 0.4 which is considerably smaller than the value expected ( / = 1) or actually found in the cpn60-rhodanese complex ( / = 0.93). These considerations and the observed presence of the fast motion suggest that cpn60 is not a rigid protein. Analysis of the polarization data as a function of temperature, which is weighted more toward the fast motion, showed that the rotational relaxation time assessed by temperature variation is greatly increased (from 552.5 to 2591 ns) for the complex of cpn60 with partially folded rhodanese (34-kDa monomeric protein). No change in was observed upon formation of the cpn60ATP complex (= 556.9 ns). These data indicate that there is local motion in the cpn60 14-mer molecule that can be frozen by formation of a binary complex with partially folded proteins. This conclusion is in keeping with results showing that the structure of cpn60 is generally stabilized when it forms complexes with passenger proteins (Mendoza, J. A., and Horowitz, P. M.(1994) J. Biol. Chem. 269, 25963-25965).


INTRODUCTION

Molecular chaperonins are proteins that can, as one of their functions, mediate protein folding in an ATP-dependent manner (1) . One family of molecular chaperonins is represented by cpn60() (groEL) from Escherichia coli, which is homologous to Hsp60 in the mitochondrial matrix. cpn60 is a large multisubunit oligomeric protein consisting of two stacked rings. Each 7-fold rotationally symmetrical ring contains seven identical 60-kDa subunits. Each subunit consists of three domains: 1) a large equatorial domain at the interface between the two rings, 2) a large apical domain forming the ends of the cylinder, and 3) a small intermediate domain that connects the equatorial and apical domains (2). The cylindrical oligomer is 146 Å tall, 137 Å wide, with an inner channel that is approximately 45 Å in diameter (2) .

cpn60 has been reported to facilitate the in vitro folding of many proteins, including monomeric mitochondrial rhodanese, molecular mass = 33 kDa (3) ; ornithine transcarbamylase, a trimer of 36-kDa subunits (4) ; 6-hydroxy-D-nicotine oxidase, molecular mass = 48 kDa (5) ; tryptophanase, a tetramer of 52-kDa subunits (6) ; monomeric -glucosidase from yeast with molecular mass = 68 kDa (7) ; and malate dehydrogenase with a molecular mass = 70 kDa (8) . An initial step in refolding mediated by cpn60 is the association between the 14-mer and partially folded proteins. Subsequent interaction with the co-chaperonin cpn10 (groES) accompanied by ATPase activity of the cpn60 is required for the most efficient release of bound polypeptide leading to properly folded protein.

It has been suggested that during the cpn60-cpn10 refolding cycle the cpn60 molecule undergoes several conformational changes (9, 10, 11, 12, 13, 14, 15) . For example, the affinity of cpn60 for the unfolded polypeptide is decreased upon binding of cpn10 and MgATP (11, 12, 13, 14) . The ATPase activity of cpn60 is 50% inhibited by cpn10 binding, and it can be stimulated 20-fold by binding an unfolded polypeptide (15) . The binding of ATP leads to conformational changes in the cpn60ATP complex such that the ATP initially forms a weak collision complex with cpn60 (K= 4 mM) which isomerizes to a strongly binding state at a rate of 180 s(15

The large number of conformational changes proposed to be involved in the mechanism of cpn60 function and its ability to bind such a broad range of polypeptides (16) suggest that some degree of flexibility is exhibited by the polypeptide binding sites of cpn60. It has been suggested, based on recent x-ray and mutation studies, that the region of the apical domain facing the central cavity is involved in peptide binding and could be inherently flexible and poorly ordered (2, 17) .

Fluorescence spectroscopy is a sensitive method for obtaining information about shape, size, and segmental motion of macromolecules (18). Pyrene has been used as a sensitive fluorescent probe exhibiting a long lifetime to investigate the rotational diffusion behavior of several proteins, including bovine serum albumin (19) , human plasma fibronectin (20, 21) , and Torpedo acetylcholinesterase (22) . The long lifetime of pyrene is particularly suitable for measuring rotational motions in large proteins.

In the present work, we have studied the hydrodynamic properties of the chaperonin cpn60 by using pyrene-labeled derivatives. We report that portions of the cpn60 14-mer exhibit a high degree of flexibility as determined by steady-state fluorescence measurements coupled with lifetime determinations. The flexibility of the protein is not affected by formation of a cpn60ATP complex, but the flexibility is greatly reduced by interaction with partially folded conformers of the enzyme rhodanese.


MATERIALS AND METHODS

Reagents and Proteins

1-Pyrenesulfonyl chloride and N-(1-pyrene)maleimide were purchased from Molecular Probes (Eugene, OR). All other reagents were analytical grade. The chaperonin cpn60 was purified from lysates of E. coli cells bearing the multicopy plasmid pGroESL (23) . After purification, cpn60 was dialyzed against 50 mM Tris-HCl, pH 7.6, containing 1 mM dithiothreitol, then made 10% (v/v) in glycerol, rapidly frozen in liquid nitrogen, and stored at -70 °C. The protomer concentration of cpn60 was measured by the BCA method (Pierce), and assuming a molecular mass of 60 kDa. Bovine liver rhodanese was purified as described previously (24) . The purified enzyme was stored at -70 °C as a crystalline suspension in 1.8 M ammonium sulfate. Protein concentration was determined using A = 1.75 for 0.1% solution of purified rhodanese (25) and a molecular mass of 34 kDa (26).

Preparation and Characterization of cpn60-Pyrene Conjugates

In order to obtain cpn60-pyrene conjugates two reagents were employed: 1) the amine-directed derivative of pyrene, 1-pyrenesulfonyl chloride (PSC); and 2) the sulfhydryl group directed derivative, N-(1-pyrene)maleimide (PM). In the first case, cpn60 (20-30 mg/ml) was dialyzed against 0.2 M carbonate buffer, pH 9.0, and then incubated with various amounts (final concentration in the reaction mixture was 26-320 µM) of 1-pyrenesulfonyl chloride dissolved in dimethylformamide. The reaction mixture was incubated for 2 h at 25 °C followed by addition of concentrated Tris-HCl, pH 7.8 (final concentration 0.1 M). To conjugate cpn60 with N-(1-pyrene)maleimide, 1.1 mg of protein was dissolved in Tris buffer, pH 7.8, containing 50 mM KCl, 20 mM MgCl. Then 9 µl of N-(1-pyrene)maleimide solution (83 µg/ml) in dimethylformamide were added, followed by 2 h of incubation at 25 °C. In each case, the reaction mixture was finally dialyzed against 50 mM Tris, pH 7.8. Conjugates were stored at 4 °C. Protein concentrations were determined by the BCA method. The number of dye molecules bound per cpn60 molecule was determined by absorption spectroscopy using a molar extinction coefficient of 32,000 M cm at 352 nm for 1-pyrenesulfonyl chloride (27) and 38,000 M cm at 343 nm for N-(1-pyrene)maleimide (27) .

Fluorescence Polarization Measurements of the Pyrene Conjugates of cpn60

Fluorescence polarization, P, was measured using the T-format on an SLM 48000 spectrofluorometer (28) . Fluorescence of pyrene-labeled cpn60 was monitored at 398 nm with 345 nm excitation. The concentrations of samples were 0.1-1 mg/ml, depending on the type of experiment. Fluorescence polarization, P, was calculated according to Equation 1:

On-line formulae not verified for accuracy

To analyze the contribution of the fast rotational motion component to the depolarization of the cpn60-pyrene fluorescence signal, the following modified version of the Perrin-Weber equation was used (30, 31) :

On-line formulae not verified for accuracy

Measurements of cpn60-Pyrene Fluorescence Lifetimes

Fluorescence lifetimes of pyrene labeled cpn60 were derived from phase-modulation measurements (32) on an SLM 48000 fluorometer (SLM Instruments, Inc.). The temperature of the sample was maintained as above. Glycogen solutions were used as zero lifetime standards. Samples containing 1 mg/ml of the labeled protein were excited at 345 nm. Emission was monitored at 400 nm by using an interference filter. For each sample, the phase shift and demodulation of cpn60-pyrene fluorescence were measured as functions of modulation frequency at 2, 4, 8, and 16 MHz. Fluorescence lifetimes were calculated from these data using software supplied by SLM. This computation is based on modified analyses described by Brent (33) .

In all cases, double exponential fluorescence decay was observed. Average lifetimes were calculated using the equation described by Brochon and Wahl (34) :

On-line formulae not verified for accuracy

This second order lifetime was used in the Perrin-Weber analysis, because it was previously shown that the relaxation times evaluated using this average approached closely the relaxation times derived with anisotropy decay (34) .

Theoretical Estimation of the Rotation Relaxation Time for cpn60

The rotational relaxation time for the rigid, hydrated sphere with molecular mass of 840 kDa was estimated using:

On-line formulae not verified for accuracy

The rotational relaxation time for the cpn60 molecule was also estimated by employing the known value of the s for the 14-mer which has been reported and confirmed here to be 23 S. Assuming the molecular mass of the cpn60 oligomer as 840 kDa the Stokes radius of the protein was estimated as:

On-line formulae not verified for accuracy

Unfolding of the Rhodanese

Rhodanese was denatured in 8 M urea, 200 mM sodium phosphate, pH 7.4 and 1 mM 2-mercaptoethanol at a protein concentration of 1.06 mg/ml.


RESULTS

Conjugation of cpn60 with PSC

Given the large size of the 14-mer of cpn60 (840 kDa), the determination of its rotational relaxation characteristics requires the use of a fluorescent probe with a long fluorescence lifetime. The lifetime of pyrene derivatives are in the range of 20-100 ns which makes these probes sensitive to the slow rotation of large proteins (19) .

There are several amino groups and three cysteines on the cpn60 molecule. The pyrene-based amino reactive compound, 1-pyrenesulfonyl chloride, and SH group reactive compound, N-(1-pyrene)maleimide, were used separately to introduce pyrene label to the different sites of the protein.

The fluorescence spectrum of the cpn60-pyrenesulfonyl chloride derivative (cpn60-PSC), excited at 345 nm, demonstrated two distinct emission maxima (at 378 and 398 nm), which is characteristic of pyrene (15, 20). We prepared conjugates containing 1-8 mol of pyrene residue per mol of cpn60 14-mer. Higher fluorescence intensities were observed with samples containing more than 8:1 mol label/mol of 14-mer, but the fluorescence signals in these cases were depolarized significantly. This suggests that there can be energy transfer among pyrene molecules bound on the same molecule of cpn60 (37) when the degree of labeling is high (more than 10 pyrene residues per each cpn60 14-mer).

Several properties of the conjugates were similar to those observed with unlabeled cpn60: (a) The digestion patterns of the conjugates with chymotrypsin were identical to those derived from the native protein; (b) labeling did not affect the s value of the 14-mer which has been reported as 23 S for native cpn60 (38) ; (c) electrophoresis of the labeled protein on non-denaturing gels showed only 14-mers; and (d) labeled cpn60 was able to facilitate the refolding of urea unfolded rhodanese with an efficiency that was similar to the unlabeled protein (data not shown).

Fluorescence Lifetime Measurements of cpn60-PSC Conjugates

The phase-modulation data obtained were fitted to a double exponential decay, and for cpn60-PSC in Tris-HCl at 20 °C the following components were computed: 5.36 ns with a fractional contribution to the fluorescence intensity of 7%, and 48.77 ns with a contribution to the fluorescence intensity of 93%. The second-order average was used to calculate the lifetime at a particular temperature, since this was shown to be the most appropriate way to interpret the type of data acquired here in terms of rotational motion (34) . The data show the expected decrease in the average fluorescence lifetime of the cpn60-PSC conjugate when the temperature was raised from 0 to 40 °C (Fig. 1). Similar results were obtained for cpn60ATP and cpn60Ru complexes (data not shown).


Figure 1: Temperature dependence of the fluorescence lifetime of the cpn60-PSC conjugate. The sample contained 1 mg/ml labeled protein in 50 mM Tris buffer, pH 7.8. The sample was excited at 345 nm, and emission was monitored at 400 nm by using an interference filter (400 nm transmission maximum). Shown are second-order averages of the lifetimes for each temperature. The line is drawn only to help the eye.



Rotational Relaxation Time Determination for the cpn60-PSC

Fluorescence polarization data obtained for the cpn60-PSC conjugate as a function of sucrose concentration were analyzed by using Equation 2. The resulting Perrin-Weber plot is shown in Fig. 2. The first two points on the plot were obtained by utilizing high concentrations of sucrose, 54 and 45%, together with low temperature (0 °C) for the circle and triangle, respectively.


Figure 2: Perrin-Weber plot for the cpn60-PSC conjugate. The Perrin-Weber plot was generated by measuring the fluorescence polarization of the cpn60-PSC conjugate as a function of sucrose (0-45%) concentration. Temperature was kept constant at 25 °C. Protein (0.25 mg/ml) was dissolved in 50 mM Tris buffer, pH 7.8. The conjugate contained 3 pyrene residues per molecule of cpn60 oligomer. The first two points on the plot were obtained by at 0 °C and high concentrations of sucrose (circle, 54%; triangle, 48%).



The nonlinearity of this plot demonstrates the presence of at least two independent processes contributing to the depolarization of the cpn60-PSC fluorescence (39) . These can be: 1) rapid rotational motion due to local rotations of the probe at the point of attachment and/or in small, flexible sections of the protein; or 2) slow rotational motion that can include rotation of the entire protein molecule and/or rotation of some relatively large part of the molecule to which the fluorescent probe is attached. Highly viscous solutions freeze the slow rotational motion and make more apparent the contribution of the fast rotational motion to the observed depolarization. The contribution of the fast motion is predicted to increase at higher temperatures (39) . This rapid motion has been demonstrated for several proteins, including human IgG (31) , rabbit IgG (30) , human fibrinogen (40) , and human plasma fibronectin (20) . The contribution of the fast motion to the overall depolarization of the fluorescence signal can be determined, based on Equation 5, from the intercept of the isothermal and nonisothermal Perrin-Weber plots (30) . In order to measure this parameter, we combined the data obtained from isothermal experiments where viscosity was varied by using different sucrose concentrations (Fig. 3, closed squares), with data from nonisothermal experiments where the temperature was changed and the buffer contained either 0% sucrose (Fig. 3, diamonds), or 27% sucrose (Fig. 3, open squares). Assuming that the fluorescence lifetime () of the cpn60-pyrene conjugate is independent of the presence of sucrose in solution, Fig. 3was constructed as a Perrin-Weber plot using the coordinates (1/P - 1/3)/(1/P - 1/3) versusT/, where P was estimated as 0.296. The leftmost point on the plot (circle) was obtained at high sucrose concentration (54%) and low temperature (0 °C). The contribution of the fast motion at 20 °C for cpn60-PSC conjugate in solution containing 0% sucrose was estimated as 24%. We observed a significant increase in the slope of the Perrin-Weber plot when nonisothermal experiment was carried out in a solution containing 27% sucrose (Fig. 3, open squares). This decrease in value suggests that the slow rotational motion was frozen while the independent fast motion continued to produce depolarization of the fluorescence signal, although we cannot exclude the possibility of some conformational stabilization of cpn60 at the high concentrations of sucrose.


Figure 3: The effect of temperature and sucrose on the fluorescence polarization of the cpn60-PSC conjugate. Curves are: (a) temperature variation (5-40 °C), in the presence of 27% (open squares) or absence of sucrose (open circles) and (b) sucrose concentration variation (0-45%) at 20 °C temperature (closed squares). Protein (0.1 mg/ml) was dissolved in 50 mM Tris buffer, pH 7.8. The P from these data was 0.296



In order to estimate for the labeled cpn60, we used the low viscosity part of the curve (Fig. 2) (less then 30% sucrose, i.e. at 25 °C T/ > 1.1 10 K P). The rotational relaxation time obtained from these data is 1113 ± 55 ns. The conjugate used for these measurements contained 3 mol of pyrene label per mol of cpn60 14-mer. A similar result, 1070 ns, was obtained for a conjugate with 8:1 molar ratio of the probe to the cpn60 oligomer. These results demonstrate that, within experimental error, did not depend greatly on the extent of labeling. To calculate the theoretical value of the , we assumed that the molecular mass of the cpn60 tetradecamer is 840 kDa and that the hydration is 0.23 g of HO per g of the protein (35) . The partial specific volume (v = 0.723 ml/g) of the protein was calculated by using standard values for the partial specific volumes of the amino acid residues (36) .

The value calculated for at 20 °C is 989.8 ns, which leads to the ratio of / = 1.13. The value of represents the shortest possible rotational relaxation time for the protein of given molecular weight considered as a sphere, and any corrections for the geometry of the cpn60 molecule would only increase the / ratio. Thus, for a rigid molecule, / must be >1, and it is typically >1.5 because of hydration and nonspherical shape. The value of / = 1.13 and the appearance of the fast motion suggest that cpn60 is not a rigid protein. The estimated as 2777 ns, was calculated as described under ``Materials and Methods'' by employing the s value of the 14-mer, which is 23 S for native cpn60. That leads to the very low value of / = 0.4. Since this ratio is based on the effective hydrodynamic size of the protein, the value of / should be close to 1.

Rotational Relaxation Measurements for the N-(1-Pyrene)maleimide Derivative of cpn60 (cpn60-PM)

The pyrene-based reagent, PM, selectively reacts with free SH groups. It has been demonstrated that cpn60 can be effectively labeled by PM (15) . This fluorescently labeled protein remains active in chaperonin functions, and it was used to study cpn60-ATP interactions (15) . We investigated the rotational relaxation of conjugates that contained 1 pyrene residue per 14-mer of cpn60. A Perrin-Weber plot was constructed based on the temperature dependence (between 0 and 40 °C) of the polarization. The leftmost point (Fig. 4, circle) corresponds to high sucrose (54%) and low temperature (0 °C) conditions. No significant change in the polarization of cpn60-PM was detected over this temperature range (Fig. 4), indicating that the cpn60-PM does not exhibit significant apparent rotational motion at the site of labeling during the pyrene fluorescence lifetime. This can be due to the fact that pyrene maleimide reacts with only one of the three sulfhydryl groups in the cpn60 monomer, and therefore is in a single, rigid site. There is an additional possibility that the transition moment of the incorporated pyrene in this derivative is oriented within the structure relative to the rotational axes so that it is less sensitive to rotations.


Figure 4: Perrin-Weber plot for the cpn60-PM conjugate. The Perrin-Weber plot was generated by measuring the fluorescence polarization of the cpn60-PM conjugate as a function of the temperature. Temperature was changed from 0 to 40 °C. Protein (0.1 mg/ml) was dissolved in 50 mM Tris buffer, pH 7.8. cpn60-PM contained 1 pyrene residues per molecule of cpn60 oligomer.



Binding of Unfolded Protein Alters the Rotational Characteristics of cpn60-PSC

To elucidate the possible modulation of segmental motion within cpn60-PSC, the fluorescence polarization of the derivative was studied over the temperature range from 0 to 40 °C (no sucrose was added) in the presence and absence of MgATP and unfolded rhodanese. The resulting polarization data are plotted in Fig. 5as a function of T/ to correct for the temperature dependence of the pyrene fluorescence lifetime.


Figure 5: Binding of unfolded rhodanese alters hydrodynamic properties of cpn60. A Perrin-Weber plot was constructed by measuring fluorescence polarization of the cpn60-PSC conjugate as a function of temperature in the: (a) absence (closed squares) or (b) presence of unfolded rhodanese (opened circles), or (c) presence of MgATP (open squares). Temperature was varied from 0 to 40 °C. The P from these data was 0.300. Protein (1 mg/ml or 2 µM) was dissolved in 50 mM Tris, pH 7.8. Final concentrations of the unfolded rhodanese, Mg, and ATP were 2.07 µM, 6 mM, and 5 mM, respectively. The cpn60-PSC conjugate contained 1 pyrene residue per molecule of cpn60 oligomer.



As shown in Fig. 5(open squares), formation of the cpn60-PSC complex with MgATP does not change the temperature dependence of the fluorescence polarization. The rotational relaxation times derived from the data presented in the Fig. 5 are 552.5 and 556.9 ns for free molecule of cpn60 and cpn60ATP complex, respectively. P was estimated to be 0.300.

The low value of the obtained here demonstrates that the fast motion is accentuated in the depolarization of the fluorescence signal of pyrene when T/ ratio was varied by temperature. In contrast with the results using ATP, the polarization was greatly increased when labeled cpn60 was complexed with partially folded rhodanese (Fig. 5, open circles). As a consequence, the slope of the Perrin-Weber plot is decreased (the leftmost point on the plot (closed circle) was obtained at 0 °C by utilizing high concentration of sucrose (54%)). The rotational relaxation time calculated for the cpn60-rhodanese complex was estimated as 2591 ns which is 4.69 times larger than the corresponding number for the unbound cpn60 molecule (552.5 nanoseconds). Thus, binding of rhodanese leads to / = 2.62 and / = 0.93. This apparent increase in of the labeled cpn60 molecule can be explained by ``freezing'' of its structure upon binding to the unfolded state of the rhodanese.


DISCUSSION

Flexibility in the cpn60 structure is suggested by its ability to bind polypeptides in a broad range of molecular masses and by proposed refolding mechanisms that include conformational changes in the cpn60 molecule (9, 10, 13, 15, 16, 41) . Recent studies of the crystal structure show poor side-chain density in the apical domain facing the central channel (2) , and it was not possible to define the conformation of about 50 residues located on the surface of the apical domain at the current resolution of 2.8 Å (2) . This part of the protein has been implicated by mutagenesis as containing part of the binding site for unfolded polypeptide (17) .

The amine-directed fluorescent probe, 1-pyrenesulfonyl chloride, enabled us to study the hydrodynamic properties of the cpn60 oligomer. cpn60-PSC fluorescence polarization data measured as a function of solvent viscosity at a constant temperature and analyzed by the Perrin-Weber method demonstrated a nonlinear dependence of (1/P - 1/3) on T/. This result indicates that at least two independent rotational motions contribute to the depolarization of the probe fluorescence (18) . The fast rotational motion was estimated to produce 24% of the signal depolarization at 20 °C. The part of the Perrin-Weber plot where T/ exceeds 1.1 10 K P represents slow rotational motion of the protein. This linear part of the plot was used to estimate the value of = 1113 ± 55 ns for the cpn60 14-mer which is similar to that expected for a rigid sphere with the same molecular mass. The ratios / and / were estimated to be 1.13 and 0.4, respectively. In this study, we examined cpn60-PSC conjugates containing from 1 to 8 probe molecules per molecule of the cpn60 oligomer. The results show no difference, within experimental error, in the estimated values.

The relatively low value for the measured and appearance of the fast motion suggest that the molecular unit that causes the observed slow motion depolarization is smaller than the entire cpn60 14-mer molecule. This result appears to demonstrate the presence of segmental motion in the protein structure (18, 22, 42) .

The apical domain which has been suggested to contain the polypeptide binding region of cpn60 (2, 17) contains a considerable number of lysines that can be labeled by the amine reactive pyrene derivative employed in this study. The x-ray studies suggested that the apical domain might exhibit two forms of flexibility: (a) en bloc movement generated by hingelike motion at its junction with the intermediate domain and/or (b) a local or segmental flexibility within the apical domain (2) . The movement of a large part of the protein, the en bloc movement, can introduce depolarization as a relatively slow motion, while the local or segmental flexibility can be considered as an element of a faster motion.

In our study, the PM label was also introduced into the cpn60 molecule by modifying one of its cysteines. It is important to note that all three cysteines in each monomer are located within the intermediate and equatorial domains of the protein. Unlike the apical domain, the equatorial domain of the cpn60 structure appears to be well defined in the crystal structure (2) . Therefore, it is not surprising that the fluorescence polarization of the cpn60 conjugate was not sensitive to the change in solution temperature over the range studied. In addition, the unique labeling at sulfhydryl groups apparently orients the transition moments within the fluorophore relative to the rotational axes of the cpn60 14-mer such that it does not show strongly depolarizing rotations with respect to the axes of rotation.

One might expect that if the flexibility of the cpn60 apical domain is necessary to facilitate conformational adjustments of the protein in order to provide the best binding affinity for an unfolded polypeptide, then the flexibility should be greatly diminished when cpn60 forms a complex with a partially folded polypeptide. Here we have shown that the rotational relaxation parameters of the PSC-labeled cpn60 change considerably upon its binding to unfolded rhodanese. In the case where the fluorescence polarization was measured as a function of the solution temperature (Fig. 5), the cpn60-rhodanese complex exhibited less dependence of the fluorescence polarization signal on temperature then uncomplexed cpn60PSC. The decrease in the slope of the thermally derived Perrin-Weber plot is evidence of an increase in . This large change cannot be explained by the small increase in the molecular mass of the complex (from 840 to 874 kDa). Similar effects have been observed for Ca-induced conformational changes in porcine intestinal brush border membrane (43), acid-induced isomerization and expansion of bovine plasma albumin (44), and changes in the conformation of 4-aminobutyrate aminotransferase induced by tryptic digestion (45) . Interestingly the ratio, /, for the cpn60-unfolded rhodanese complex is close to 1, showing that, under these conditions, cpn60 structure is closer to the rigid hydrodynamic unit observed in the ultracentrifuge.

Unlike the case with unfolded polypeptide, binding of ATP did not change the hydrodynamic parameters of cpn60. Although it has been shown that binding of ATP leads to changes in the cpn60 conformation, it appears that these changes do not affect the flexibility cpn60 that can be detected by the methods used here.

The detailed analysis of the steady state polarization data is complex, and, in general, it is not straightforward to parse the information into unique rotational contributions (34) . Overall, the results make it tempting to speculate that conformational adjustments within cpn60 can be involved in accommodating a range of protein substrates, and modulation of the structure may be an element of the mechanism by which this chaperonin can influence protein folding.


FOOTNOTES

*
This research was supported by National Institutes of Health Research Grants GM25177 and ESO5729 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. This article must therefore by 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 at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78284. Tel: 210-567-3737; Fax: 210-567-6595.

The abbreviations used are: cpn60, chaperonin 60 or groEL; PSC, 1-pyrenesulfonyl chloride; PM, N-(1-pyrene)maleimide; cpn60-PSC, chaperonin 60 conjugated with pyrene by using 1-pyrenesulfonyl chloride; cpn60-PM, chaperonin 60 conjugated with pyrene by using N-(1-pyrene)maleimide; cpn60ATP, noncovalent complex of chaperonin 60 and ATP.


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