From the
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
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
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
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 cpn60
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 cpn60
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
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) .
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
On-line formulae not verified for accuracy
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
The value
calculated for
The low value of the
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/
The
relatively low value for the measured
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 cpn60
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.
= 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 cpn60
ATP 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).
(
)
(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) .
-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.
ATP
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
ATP complex, but the
flexibility is greatly reduced by interaction with partially folded
conformers of the enzyme rhodanese.
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:
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) .
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:
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:
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.
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) .
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 cpn60
Ru 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 H
O 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) .
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.
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.
. 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.
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) .
PSC. 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.
ATP, noncovalent complex of chaperonin 60 and ATP.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.