(Received for publication, October 31, 1994; and in revised form, November 17, 1994)
From the
The molecular chaperone GroEL from Escherichia coli is
a member of the highly conserved Hsp60 family of proteins that
facilitates protein folding. A central question regarding the mechanism
of GroEL-assisted refolding of proteins concerns its broad substrate
specificity. The nature of GroEL-polypeptide chain interaction was
investigated by isothermal titration calorimetry using proteins that
maintain a non-native conformation in neutral buffer solutions. A
single molecule of an unfolded variant of subtilisin BPN` binds
non-cooperatively to GroEL with micromolar affinity and a positive
enthalpy change. Additional calorimetric titrations of this chain with
GroEL show that the positive enthalpy change decreases with increasing
temperature between 6 and 25 °C, yielding a C
of -0.85 kcal mol
degree
.
-Casein similarly binds to GroEL
with micromolar affinity and a positive enthalpy change in the range of
15-23 °C, yielding a
C
of
-0.44 kcal mol
degree
. The
negative heat capacity change provides strong evidence for the role of
hydrophobic interactions as the driving force for the association of
these substrates with the GroEL chaperonin.
Escherichia coli GroEL is a member of the highly
conserved Hsp60 family of molecular chaperones which facilitate protein
folding in vivo and in vitro(1) . A key issue
in the mechanism by which chaperonins facilitate protein folding is the
nature of their recognition of non-native structures. Although in
vivo studies have identified a potentially wide range of protein
substrates that bind to GroEL(2, 3) , and numerous in vitro studies have demonstrated that many chemically
unfolded proteins bind to GroEL(4, 5) , the nature of
this interaction remains unclear(6, 7) . Since
unfolded proteins possess a high degree of exposed hydrophobic surface,
it has been suggested that the association of nonnative chains with
GroEL is hydrophobically
driven(8, 9, 10, 11, 12, 13, 14) ,
but definitive experimental evidence in support of this view is
lacking. In this study, an unfolded mutant of subtilisin BPN` and
-casein were employed in calorimetric experiments to measure
directly the energetics of substrate binding to GroEL. These results
demonstrate unequivocally that the association of substrates with GroEL
is determined by hydrophobic interactions.
where Q is the amount of heat exchanged, X is
the ligand concentration (in this case, unfolded polypeptides), V is the volume of the cell (1.38 ml), P is the GroEL
concentration, 1/r = [X]K
and X
=
[P]
/[X]
(18) . The temperature dependence of
H was
analyzed to
where H is the observed enthalpy change for the
association of unfolded chains with GroEL at various temperatures, and
H
is the enthalpy change at a reference
temperature, T
. The temperature dependence of the
association of BPN` PJ9 with GroEL was estimated
from
where H
and K
at a reference (14.3 °C) temperature, and the determined
value of
C
, -0.85 kcal mol
deg
, were used(19) .
Since a quantitative study of the interaction of molecular
chaperones with nonnative polypeptides is potentially complicated by
the tendency of these chains to spontaneously refold or aggregate in
the absence of denaturants, an unfolded variant of subtilisin BPN` was
used to measure the energetics of substrate interaction with the GroEL
chaperonin from E. coli. Subtilisin BPN` PJ9 is unable to
adopt a native-like, folded conformation and remains unfolded in
non-denaturing aqueous solution. The nonnative character of subtilisin
BPN` PJ9 was established by circular dichroism (CD) and bisANS binding.
As can be seen in Fig. 1, the far-UV CD spectrum of subtilisin
BPN` PJ9 indicates that the protein is much less structured relative to
native BPN`, and the near-UV spectrum shows little indication of
native-like tertiary structure(20, 21) . On the other
hand, subtilisin BPN` PJ9 is not in a completely random conformation.
Residual secondary structure is also evident from the CD spectrum
presented in Fig. 1A, which differs from that for
subtilisin BPN` PJ9 in the presence of 8 M urea. Additionally,
as can be seen in Fig. 2, neither fully folded native BPN` nor
the BPN` PJ9 variant that was completely unfolded in the presence of 8 M urea showed measurable affinity for bisANS, which is a probe
for solvent-accessible hydrophobic areas. In contrast, about 1 mol of
bisANS/mol of BPN` PJ9 is bound with a K
5
µM, indicating the existence of exposed hydrophobic
clusters(22) . Subtilisin BPN` PJ9 therefore provides a useful,
stable model of an ``unfolded protein substrate'' for GroEL
under neutral buffer conditions(23, 24) .
Figure 1:
Circular dichroism
spectra of native BPN` and subtilisin BPN` PJ9. Molar ellipticity versus wavelength was measured as described under
``Experimental Procedures.'' A, in the far-UV
region, the CD spectrum of native subtilisin BPN` (thickline) yields a maximum at 192 nm and minima at about 220
and 210 nm, indicative of the high content of -helical structure
in the folded conformation. The CD spectrum of subtilisin BPN` PJ9 (thinline) shows a minimum at about 202 nm,
indicating a loss of helical content. The dashedline refers to the spectrum of subtilisin BPN` PJ9 in the presence of 8 M urea. B, in the near-UV region, native BPN` shows a
minimum at 278 nm, reflecting native tertiary structure, whereas the
region is featureless for BPN` PJ9 in the absence or presence of 8 M urea.
Figure 2:
Effect of native and non-native subtilisin
on the fluorescence of bisANS. The relative fluorescence emission at
484 nm of bisANS in the presence of various forms of subtilisin BPN`
was measured upon excitation at 394 nm. Native subtilisin BPN` in
buffer A (squares), and subtilisin BPN` PJ9 in buffer B in the
presence of 8 M urea (triangles) show no measurable
affinity for bisANS by the lack of fluorescence. Subtilisin BPN` PJ9 in
buffer B (circles) shows significant affinity for about 1 mol
of bisANS, with the theoretical curve corresponding to simple binding
with a K of about 5
µM.
The
titration calorimetry results in Fig. 3show that the
association of BPN` PJ9 to GroEL occurs with an uptake of heat, and an
analysis of the data seen in Fig. 3B yielded a positive
enthalpy change of H= +19.9 kcal/mol, an
equilibrium association constant of 5.91
10
M
at 14.3 °C, and a stoichiometry
of 1.10 polypeptides bound/GroEL. Thus, the binding of this unfolded
chain to GroEL is entropy-driven, which suggests a possible role for
hydrophobic interactions(25, 26, 27) . The
role of hydrophobic interactions was confirmed by performing additional
binding experiments as a function of temperature. As can be seen in Fig. 4A, the
H for subtilisin binding to
GroEL decreased with increasing temperature. These data were analyzed
to to yield a negative value for
C
of -0.85 kcal mol
deg
, which is the hallmark of the hydrophobic
interaction(28, 29, 30) .
Figure 3:
Exchange of heat on binding of subtilisin
BPN` PJ9 to GroEL. A, uptake of heat upon the addition of
15-µl aliquots of a 150 µM subtilisin BPN` PJ9
solution at 4-min intervals to a 12 µM solution of GroEL
in buffer A at 14.3 °C. The data correspond to heat absorbed versus time (injection number). B, integrated data
after correction for heat of ligand dilution determined in separate
experiments, presented as heat exchange versus the subtilisin
BPN` PJ9 to GroEL ratio (total concentrations). These data were
analyzed as described under ``Experimental Procedures,'' with
the theoretical curve corresponding to the parameter values of K = 5.91
10
M
,
H =
+19.9 kcal/mol, and a stoichiometry of 1.1 polypeptides
bound/GroEL.
Figure 4:
Energetics of subtilisin BPN` PJ9-GroEL
interaction. A, temperature dependence of the enthalpy change
for BPN` PJ9 binding to GroEL. The linear dependence of enthalpy versus temperature yields a heat capacity change,
C
, of -0.85 kcal mol
deg
. B, a stability curve for the
association of BPN` PJ9 with GroEL was determined using as
described under ``Experimental
Procedures.''
The micromolar binding constants determined calorimetrically for subtilisin BPN` JP9 differ significantly from the picomolar constants estimated for the affinity of chemically unfolded ribulose-1,5-bisphosphate carboxylase with GroEL by its rate of exchange with free chains(4) . This difference may be due to the nature of the unfolded form of the species used in these studies since BPN` PJ9, although largely unfolded under non-denaturing buffer conditions, contains residual structure.
Both
the solubility of BPN` PJ9 and instrumental sensitivity limited the
binding measurements to the temperature range of 5-25 °C. The
stability curve presented in Fig. 4B was constructed
using the binding constants determined at three temperatures along with
the heat capacity change, C
(19) .
This analysis indicates that the maximum association of BPN` PJ9 with
GroEL occurs at about 37 °C where, since the enthalpy change is
approximately zero, the association of BPN` PJ9 with GroEL is entirely
entropically driven. The dissociation constant at this temperature was
estimated to be approximately 0.4 µM. The
temperature-dependent fashion of GroEL binding suggested by the
stability curve provides a possible explanation for the reduction in
affinity GroEL shows for various unfolded substrates at lower
temperatures(11, 31) . Furthermore, a single chain of
subtilisin BPN` PJ9 was bound to GroEL throughout the temperature range
investigated, with no evidence for cooperative polypeptide binding to
the chaperonin(32) .
It is of interest to note that
negligible heat exchange was seen for the association of GroEL with
GroES, a cofactor in many refolding
reactions(33, 34) . However, similar results to
subtilisin BPN` PJ9 were seen for the association of GroEL with
-casein, a disordered protein that has been shown to compete with
unfolded substrates for binding to GroEL(33, 35) .
Titration calorimetry experiments were performed using
-casein
under similar solution conditions at 15.0 and 22.5 °C. Analysis of
the binding isotherms yielded enthalpies of +9.37 and +6.08
kcal/mol, binding constants of 6.2
10
M
and 1.9
10
M
, respectively, providing an
estimate for
C
of -0.44 kcal
mol
deg
. Thus it appears that the
association of
-casein with GroEL is also driven by hydrophobic
interactions. However, a more detailed analysis of the calorimetric
data for casein was complicated by the unusual binding stoichiometry of
approximately 0.5 casein molecule/GroEL oligomer. Additional
calorimetric experiments were also performed using RCM-lactalbumin and
melittin. No detectable enthalpy change was seen for RCM-lactalbumin
addition to GroEL. This indicates RCM-lactalbumin has negligible
affinity for GroEL, in agreement with recent results, which indicate
that only specific conformers of lactalbumin are able to interact with
this chaperonin (14) . On the other hand, melittin, a small,
hydrophobic protein, showed a small positive enthalpy change on
addition to GroEL, but its association was too weak (K
mM) for a quantitative analysis.
The substrate
promiscuity of GroEL is an intriguing matter(6) . Previous NMR
studies have identified two peptides, STKWLAESVRAGK and KLIGVLSSLFRDK,
that bind to GroEL in an -helical conformation, possibly through
an apolar face(8, 36) . However, GroEL can facilitate
the refolding of an F
fragment of an antibody, which
contains no
-helical structure(9) , and it has been shown
recently that the secondary structure of cyclophilin is completely
eliminated upon binding to GroEL(11) . Thus, rather than a
cognate structural element responsible for GroEL
binding(8, 9, 10, 11) , it seems
more probable that GroEL recognizes unfolded proteins through their
common characteristic of exposed hydrophobic surface, which is not
generally present in their folded conformations, and binds these
segments through hydrophobic interactions. Not only have hydrophobic
amino acids been shown to stimulate preferentially the ATPase activity
of GroEL(37) , a detailed mutational study of GroEL also maps a
number of polypeptide binding deficient mutants to hydrophobic residues
within the central cavity of the two-stacked, seven-membered ringed
structure(38, 39) . Independent of whether the
association of polypeptides with GroEL are mediated through particular
structural elements, however, the energetics of subtilisin BPN` PJ9 and
casein binding to GroEL strongly indicate that the major determinant of
substrate association with GroEL is governed by the ability to form
hydrophobic interactions. The heat capacity changes seen for the
interaction of GroEL with subtilisin BPN` PJ9 and casein suggest that a
relatively large hydrophobic accessible surface area is buried upon
association with the chaperonin. If the correlation between
C
and the water-accessible nonpolar surface
area is adopted(29) , then roughly 3,040 Å
would be buried when subtilisin BPN` JP9 binds to GroEL, whereas
1,570 Å
should be buried upon casein binding. The
difference in accessible surface buried may be explained by a collapse
of BPN` PJ9 when bound to GroEL relative to casein, which is less
flexible than unfolded proteins(40) .