Chaperonin-mediated protein folding

The mechanisms and components required for chaperonin-dependent folding of proteins has been elucidated using the groEL and groES proteins of E. coli to reconstitute enzymatic activity of two monomeric enzymes, dihydrofolate reductase (DHFR) and rhodanese following dilution from denaturant. The essential elements for properly folding any protein are Mg-ATP (provided in the preferred embodiment as 5 mM Mg acetate and 1 mM ATP), groEL or hsp60, and groES or eukaryotic equivalent. These can be provided in purified form or as a semi-purified cell extract. The groES eukaryotic equivalent, encoded by a gene which does not hybridize to the groES gene, can be isolated using the same technique as was described to isolate Hsp60: isolation of a temperature sensitive lethal yeast mutant (petite at permissive temperature) defective in folding and assembly of imported proteolytically processed human ornithine transcarbamylase (OTC). The yeast mutant is used to identify a yeast genomic DNA sequence that rescues the mutant following library transformation. The rescuing DNA is isolated, characterized, and expressed. The expressed protein is used to make an antibody which is in turn used to identify the protein in yeast mitochondrial extracts and facilitate biochemical isolation of the protein.

Little is known about the mechanisms by which newly-synthesized proteins 
fold inside cells. Recent findings suggest that, for many proteins, 
folding in vivo may not be a spontaneous process. For example, while in 
vitro folding reactions are carried out on completed polypeptide chains, 
these studies fail to address the situation faced with proteins 
synthesized in intact cells. In the cell, the NH.sub.2 -terminal epitopes 
of a nascent protein, which are required for the protein folding, may 
already have emerged from a ribosome, before the remaining COOH-terminal 
portion of the protein has been synthesized. Similarly, the NH.sub.2 
-terminal portion of a translocated polypeptide may emerge from the 
trans-side of a membrane before the COOH-terminal portion has 
translocated. In such instances, a "chaperoning" function is required to 
prevent illegitimate intra- and inter-molecular interactions of the 
nascent polypeptides. 
A number of components have been identified which are involved in mediating 
protein folding in a variety of cell types and compartments, as reported 
by Fischer, G. & Schmid, F. X. Biochemistry 29, 2206-2212 (1990); 
Freedman, R. B. Cell 57, 1069-1072 (1989); and Ellis and van der Vinn, 
Annu. Rev. Biochem. 60:337-347 (1991). They have been classified as 
"molecular chaperones" by Dingwall, C. K. & Laskey, R. A. Seminars in Cell 
Biol. 1, 11-17 (1990), or "polypeptide chain binding proteins" by Rothman, 
J. E. Cell 59, 591-601 (1989), based on their ability to prevent the 
formation of wrong protein aggregates by binding to unfolded or partially 
denatured polypeptides. The heat-shock proteins of the hsp70 and hsp60 
families are typical representatives of this heterogeneous group of 
components, as reviewed by Langer, T. & Neupert W. in Curr. Topics in 
Microbiol. and Immun. 167, 3-30 (1991); Pelham, H. R. B. Nature 332, 
776-777 (1988); and Hartl, F.-U Seminars in Immunol. 3, in press (1991). 
U.S. Ser. No. 07/261,573 filed Oct. 24, 1988, first described the folding 
function of hsp60, isolated from yeast mitochondria, and related proteins 
such as groEL, isolated from E. coli. The essential function in protein 
folding of the members of the hsp60 family has since been demonstrated in 
vivo and in vitro. These so-called "chaperonin", described by Hemmingsen, 
S. M., et al., Nature 333, 330-334 (1988), include the groEL protein of E. 
coli and other bacteria, reviewed by Georgopoulos, C., et al., J. Molec. 
Biol. 76, 45-60 (1973); Stornborg, N. J. molec. Biol. 76, 25 44 (1973); 
Hendrix, R. W. J. molec. Biol. 129, 375-392 (1979); Bochkareva, E. S., et 
al., Nature 336, 254-257 (1988); Goloubinoff, P. et al., Nature 342, 
884-889 (1989); Van Dyk, T. K., et al., Nature 342, 451-453; Lecker, S., 
et al. EMBO J. 8, 2703-2709 (1989); Laminet, A. A., et al., EMBO J. 
9:2315-2319 (1990); Buchner J., et al. Biochemistry 30, 1586-1591 (1991), 
the rubisco binding-protein of chloroplasts, reviewed by Barraclough, R. & 
Ellis, R. J. Biochim. Biophys. Acta 608, 19-31 (1980); Musgrove, J. E., et 
al., Eur. J. Biochem. 163, 529-534 (1987); and Gatenby, A. A. & Ellis R. 
J. A. Rev. Cell Biol. 6, 125-149 (1990), and the mitochondrial hsp60, 
reviewed by McMullin, T. W. & Hallbert, R. L. Molec. Cell. Biol. 8, 
371-380 (1988); Reading, D. S., et al., Nature 337-655-659 (1989); Cheng, 
M. Y., et al. Nature 337, 620-625 (1989); Ostermann, J., et al., Nature 
341, 125-130 (1989); and Cheng, M. Y., et al., Nature 348, 455-458 (1990). 
They form tetradecameric complexes composed of two stacked 7mer rings of 
approximately 60,000 Dalton subunits that have ATPase activity. 
GroEL and the mitochondrial hsp60 functionally cooperate with an additional 
component, groES, described by Chandrasekhar, G. N., et al., J. Biol. 
Chem. 261, 12414-12419 (1986); Lubben, T. H., et al., Proc. Natl. Acad. 
Sci. U.S.A. 87, 7683-7687 (1990); and Viitanen, P. V. et al. Biochemistry 
29, 5665-5670 (1990), a ring-shaped complex of seven approximately 10,000 
Dalton subunits which has been reported by Chandrasekhar and Viitanen to 
inhibit the ATPase activity of groEL. The groE proteins are required for 
lambda phage head-assembly. The rubisco binding-protein mediates the 
assembly of hexadecameric ribulose bisphosphate carboxylase (rubisco) in 
chloroplasts. The assembly of dimeric prokaryotic rubisco has been 
successfully reconstituted in vitro using purified groEL and groES by 
Goloubinoff, et al., and Viitanen, et al. Recently, the mitochondrial 
hsp60 has been shown to be necessary not only for the oligomeric assembly 
of proteins but also for the chain folding of monomeric polypeptides. 
However, the molecular mechanism of this ATP-driven process remains to be 
elucidated, and, as a result, how to use and manipulate this mechanism on 
a practical basis. 
It is therefore an object of the present invention to provide all of the 
components, in purified form, and the method of use thereof, for refolding 
proteins in vitro, whether synthesized from recombinant sequences or 
denatured by heat or chemical means. 
It is a further object of the present invention to provide the eukaryotic 
equivalent of groES. 
SUMMARY OF THE INVENTION 
The mechanisms and components required for chaperon-independent folding of 
proteins has been elucidated using the groEL and groES proteins of E. coli 
to reconstituted two monomeric enzymes, dihydrofolate reductase (DHFR) and 
rhodanese. While DHFR is able to fold spontaneously upon dilution from 
denaturant, this is not observed with rhodanese. Folding reactions were 
monitored by measuring endogenous tryptophan fluorescence, adsorption of 
the hydrophobic fluorescent dye anilino-naphthalene-sulfonic acid, 
protease sensitivity and enzyme activity. The results demonstrate that: i) 
groEL, or its eukaryotic equivalent hsp60, stabilizes an early 
intermediate on the folding pathway which appears to be the equivalent to 
the folding state described as "molten globule"; ii) ATP-dependent folding 
occurs at the surface of groEL via intermediate conformations which are 
progressively more compact but still enzymatically inactive; iii) by 
regulating the groEL ATPase, groES, or its eukaryotic equivalent, 
accomplishes a critical folding step(s) at groEL by modulating step-wise, 
ATP-dependent release of the protein substrate from the groEL scaffold. 
The essential elements for properly folding any protein are Mg-ATP 
(provided in the preferred embodiment as 5 mM Mg acetate and 1 mM ATP), 
groEL or hsp60 14mer, and groES or eukaryotic equivalent. These can be 
provided in purified form or as a semi-purified cell extract. The groES 
eukaryotic equivalent, encoded by a gene which does not hybridize to the 
groES gene, can be isolated using the same technique as was described to 
isolate Hsp60: isolation of a temperature sensitive lethal yeast mutant 
(petite at permissive temperature) defective in folding and assembly of 
imported proteolytically processed human ornithine transcarbamylase (OTC). 
The yeast mutant is used to identify a yeast genomic DNA sequence that 
rescues the mutant following library transformation. The rescuing DNA is 
isolated, characterized, and expressed. The expressed protein is used to 
make an antibody which is in turn used to identify the protein in yeast 
mitochondrial extracts and facilitate biochemical isolation of the protein 
.

DETAILED DESCRIPTION OF THE INVENTION 
GroE-mediated folding of two monomeric enzymes has been reconstituted in 
vitro. GroEL stabilizes the polypeptides in a molten globule-conformation. 
GroES and Mg-ATP then promote the acquisition of ordered tertiary 
structure at the surface of groEL. Folding requires the hydrolysis of 
approximately 100 ATP molecules per protein monomer, and a solution 
concentration of at least 1 mM ATP and 5 mM Mg.sup.++. This active process 
of surface-mediated chain folding represents a general mechanism for the 
formation of protein structure in vivo. 
For general purposes, the following reagents can be combined to promote 
proper folding of proteins: Mg-ATP, groEL or Hsp60 14mer, and groES 7mer 
or eukaryotic equivalent protein. As used herein, the proteins are 
collectively referred to as the "chaperoning" protein system. These can be 
obtained or prepared as follows: 
Magnesium and ATP 
Magnesium is preferably provided as a 5 mM solution of magnesium acetate, 
either in cell culture fluid or a buffered solution. ATP must be provided 
in a hydrolyzable form, preferably in a concentration of at least 1 mM 
ATP. 
GroEL and GroEs proteins 
GroEL and groES proteins can be purified as described by Lecker, S., et al. 
EMBO J. 8, 2703-2709 (1989) or Viitanen, P. V. et al. Biochemistry 29, 
5665-5670 (1990), the teachings of which are specifically incorporated 
herein, using a groE overproducing strain of E. coli harboring the plasmid 
pOF39, described by Fayet, O., et al., Molec. Gen. Genet. 202, 35-445 
(1986). This strain can be obtained from Costageorgopoulos, at the 
University of Geneva. 
For purification of groEL, the cells are first sonicated at 4.degree. C., 
debri removed by centrifugation for 5 min.times.1000 g, then the 
supernatant placed on a sucrose gradient. The 10 to 30% fraction is 
centrifuged overnight at 4.degree. C., the 20 S fraction collected and 
precipitatedd with ammonium sulfate. The 30 to 60% ammonium sulfate 
fraction is collected, resuspended in buffer, and subjected to DEAE 
chromatography using a salt gradient equivalent to 50 to 250 mM NaCl. 
GroEL elutes at 170 mM NaCl. This fraction is then subjected to 
chromatography on a P100.TM. column (Pharmacia Fine Chemicals, New 
Jersey), and the fraction containing groEL subjected to chromatography on 
a Superose.TM. 6 (Pharmacia) column. The groEL elutes from this column at 
near homogeneity. 
GroES is isolated from the same overproducing strain of E. coli. The cells 
are sonicated, centrifuged, and the supernatant appliced to a sucrose 
gradient. GroES is found near the top of the gradient, as monitored by gel 
electrophoresis. The groES fraction is precipitated with ammonium sulfate. 
The groES is found in the 30 to 60% fraction. This is centrifuged, 
resuspended, and chromatographed on DEAE chromatography using a 50 to 250 
mM NaCl salt gradient. The groES is found in the 120 mM NaCl fraction. 
This faction is purified to near homogeneity by chromatography first on a 
Superose 6.TM. column, then on a Superose 12.TM. column. 
Hsp60 and eukaryotic equivalent of GroES proteins 
The hsp60 proteins can be provided in purified form or in the form of a 
partially purified cell extract (free of whole cells and nuclear materials 
such as DNA, cellular membrane proteins and lipids). The cell extract is 
normally a yeast mitochondrial matrix preparation containing hsp60. 
Mitochondrial matrix preparations are described by Cheng, et al., Proc. 
Nat. Acad. Sci. USA 84:4063-4067 (1987) and McMullin, et al., Mol. Cell. 
Biol. 8:371-380 (1988). Briefly, cells are lysed by sonication, osmotic 
shock, or a phospholipase, followed by centrifugation in a density 
gradient to remove membranes and associated proteins, which are 
solubilized with detergent to release the components of the inner 
membrane. 
Hsp60 can be isolated from yeast cell extracts using an antibody such as 
the antibody described by McMullin, et al., Mol. Cell. Biol. 7:4414-4423 
(1987) using known immunoaffinity methodology, or more preferably, 
expressed from an isolated nucleotide sequence encoding the protein in an 
appropriate host system. Such a sequence and methods for expression 
thereof are described in U.S. Ser. No. 07/261,573 filed Oct. 24, 1988, the 
teachings of which are incorporated herein. 
The isolation and identification of the gene encoding Hsp60 from yeast, as 
described in U.S. Ser. No. 07/261,573 filed Oct. 24, 1988, using a 
temperature sensitive OTC yeast mutant, can be used to isolate the gene 
for the eukaryotic equivalent of the groES protein. The following is a 
description of how the gene encoding hsp 60 was isolated. 
Conditional yeast mutants affecting the mitochondrial-import machinery were 
isolated using a strategy based on the presumption that (1) obstruction of 
import of essential metabolic enzymes that reside within mitochondria is 
lethal and (2) when the subunit precursor of the human mitochondrial 
matrix enzyme ornithine transcarbamylase (OTC) is expressed in yeast, the 
precursor follows the same pathway of import employed by endogenous yeast 
mitochondrial precursors. 
Temperature-sensitive (ts) lethal mutants were derived from a Saccharomyces 
cerevisiae yeast strain that is defective at the yeast OTC locus (Arg 3) 
but that contains in its URA 3 locus an integrated segment containing the 
human OTC coding sequence joined with an inducible yeast Gal 1 promoter 
(GALOTC strain). Expression of human OTC at non-permissive temperature was 
induced and production of OTC enzyme activity determined in order to 
identify temperature shock (ts) mutants affected in mitochondrial import. 
A group of mutants that failed to demonstrate activity was isolated. These 
mutants were further analyzed by immunoblot analysis of extracts with 
anti-OTC antiserum. Two classes of mutant were distinguished. In one 
class, precursors of both human OTC and endogenous yeast mitochondrial 
proteins accumulated at non-permissive temperature. Complementation 
analysis of this class revealed three groups, called mif 1, 2, and 3 
(mitochondrial import function mutants). mif 1 and 2 were shown to encode 
the two subunits of the mitochondrial processing protease, the processing 
enhancing protein (PEP) and the matrix processing peptidase (MPP), 
respectively. In the second class of ts lethal mutants, the mature-size 
OTC subunit was observed at non-permissive temperature (M.sup.+) but no 
OTC enzyme activity could be detected (A.sup.-). Three different molecular 
defects could explain such a phenotype: (1) A defect in translocation, 
leaving OTC subunits only partway translocated through the mitochondrial 
membranes, in a position where they were able to be cleaved by the matrix 
processing enzyme to a mature size but were not able to be further 
translocated through the membranes to a position where they could contact 
each other to assemble into the active homotrimeric enzyme; (2) A defect 
in the OTC subunit itself, allowing OTC subunits to be completely 
translocated to the matrix space but blocking their assembly into 
enzymatically active homotrimer; and (3) A defect in a mitochondrial 
component that normally plays a role in assembly of subunits reaching the 
matrix space. The second type of defect would not lead by itself to a 
lethal phenotype because OTC activity is not required for growth of cells 
as long as arginine is provided in the growth medium. However the first 
and third types of defect could produce a lethal phenotype because these 
defects could involve components likely to play a general role in import. 
To determine whether any of the mutants exhibited a defect of either 
translocation or assembly, each mutant was cured of the human OTC 
sequence, transformed with a new Gal-OTC segment, and tested at 37.degree. 
C. for both the size of OTC subunits and the presence of enzyme activity. 
More specifically, each mutant was first mated with an Arg 3 strain and 
sporulated. Tetrads were then examined for 2:2 ts lethal behavior to 
demonstrate that a single lethal mutation was present in the original 
mutant strain. Spores were also tested for the absence of the URA 3 gene 
in order to identify ts spores in which the ts mutation had segregated 
away from the original integrated Gal-OTC-URA 3 segment. 
Ts ura.sup.- spores were transformed with a new Gal-OTC-DRA 3 segment. The 
ura.sup.+ transformants were examined for OTC activity after galactose 
induction at 37.degree. C. Nine out of ten mutants exhibited normal levels 
of activity, suggesting that they had originally harbored two mutations, 
one a ts lethal mutation that does not directly affect the import pathway, 
and a second mutation affecting the human OTC sequence. 
One of the ten mutants, alpha-143, also referred to as ts143, behaved 
differently. As in the other mutants, mature size OTC subunits were 
detected following induction at 37.degree. C. However, no OTC activity was 
detected in this mutant. When examined at the permissive temperature, 
23.degree. C., this mutant behaved like wild-type. Both mature subunit and 
OTC activity were detected. It was determined that ts143 contained a 
single ts lethal mutation that affects the ability of mature-size 
wild-type human OTC subunits to produce enzyme activity. 
To determine whether OTC subunits in the ts143 cells had assembled into 
homotrimeric enzyme, an assay was carried out using the OTC substrate 
analogue,. delta-N phosphonoacetyl Lornithine (O). Total cell extracts 
were prepared from both ts143 and wild-type cells grown for 2 hours at 
37.degree. C. in galactose medium. The extracts were applied to columns 
containing O linked to epoxy-Sepharose.TM.. Column eluents were 
collected and immunoprecipitated with anti-OTC antiserum. The precipitates 
were solubilized, fractionated in SDS-PAGE, and the gel immunoblotted with 
anti-OTC antiserum. When wild-type extracts were applied to the column, no 
mature size subunit was observed in the flow-through fraction (CE and BK), 
consistent with the ability of the substrate analogue to quantitatively 
bind assembled OTC enzyme. In contrast, when an extract of ts143 cells was 
applied, the flow-through was found to contain both precursor and mature 
size OTC subunits, in an amount corresponding to that applied. 
Both wild-type and ts143 columns were next washed with buffer containing 40 
mM KCl (SW). No OTC submits eluted from either column, indicating absence 
of nonspecific binding. The columns were then washed with the OTC 
substrate carbamyl phosphate (Cp). An amount of mature-size OTC subunit 
corresponding approximately to that originally applied eluted from the 
wild-type column. In the case of the ts143 column, neither OTC precursor 
nor mature OTC subunit was detected, demonstrating the quantitative 
passage of subunits through the column during the initial application of 
extract. These results lead to the conclusion that mature-size wild-type 
human OTC subunits are produced in ts143 cells at 37.degree. C., but fail 
to assemble into homotrimeric, catalytically active, OTC enzyme. 
To isolate a wild-type copy of the gene affected in ts143, the haploid 
strain was transformed with a CEN library substantially identical to that 
of ATCC Deposit No. 37415 prepared using shuttle vector YCp50 (ATCC 37419) 
described by Rose et al., Gene, 60:237-243 (1987), containing fragments of 
yeast genomic DNA inserted into a plasmid that contains both a centromere 
sequence and a URA3 marker. The particular library utilized was a retained 
library that was obtained directly from Dr. Peter Novick, a co-author of 
the above Rose et al. article. That library was therefore substantially 
the same as ATCC No. 37415. 
In one strategy of genetic rescue, URA.sup.+ transformants were selected 
at 23.degree. C., then replica plated to rich medium (YPD) at 37.degree. 
C. In a second strategy, yeast cells were directly plated on YPD medium at 
37.degree. C. following transformation. 
In both cases, DNA was prepared from colony-purified transformants and used 
to transform E. coli to ampicillin-resistance. Plasmid DNAs were then 
isolated and characterized by restriction analysis. Plasmids from the 
strategy involving initial URA selection at 23.degree. C. followed by 
replica plating to 37.degree. C. all shared several restriction fragments 
containing inserted yeast genomic DNA. Plasmids from the strategy 
involving direct plating at 37.degree. C. exhibited not only this pattern 
in several isolates but also other distinct restriction patterns each 
isolated independently more than once. 
The rescuing plasmids were then tested to see if they encoded a heat shock 
protein. Yeast DNA inserts from the plasmids were nick-translated and used 
to probe blots containing RNA prepared from wild-type yeast grown at 
23.degree. C. and either maintained at this temperature or exposed first 
to an incubation at 42.degree. C. for one hour. The results of this study 
with the insert from a plasmid designated p8 belonging to the unique group 
of plasmids obtained by both rescue strategies indicated an increase in 
the amount of assayed-for RNA in the cells incubated at the higher 
temperature than those incubated at the lower temperature. Plasmid P8 was 
determined to contain plasmid YCp50 plus the gene for the rescuing 
protein, hsp60. 
This gene was then isolated and sequenced using standard techniques, as 
described in U.S. Ser. No. 07/261,573 filed Oct. 24, 1988. 
Substantially the same technology has been used to isolate mutants 
defective in the eukaryotic equivalent of groES. The gene encoding this 
protein can be isolated using the temperature sensitive lethal yeast 
mutants (petite at permissive temperature) defective in folding and 
assembly of imported proteolytic processed human ornithine 
transcarbamylase (OTC). The yeast mutant is used to identify a yeast 
genomic DNA sequence that rescues the mutant following library 
transformation. The rescuing DNA is isolated, characterized, and 
expressed. The expressed protein is used to make an antibody which is in 
turn used to identify the protein in yeast mitochondrial extracts and 
facilitate biochemical isolation of the protein. The encoded protein is 
expected to have a molecular weight of approximatley 10,000 Daltons and to 
be encoded by a nucleotide sequence of approximately 300 base pairs. 
Folding of proteins using the proteins in combination with Mg-ATP 
The purified reagents can either be added to the system in which the 
protein to be properly folded (either as it is synthesized or after 
chemical or heat denaturation) is present, or the genes encoding the 
relevant proteins expressed in the cell culture system in which the 
protein to be properly folded is being expressed. In the preferred 
embodiment, the genes for the groE or hsp60 proteins are introduced into 
the cell culture system using an appropriate vector. Such vectors are 
commercially available, for example, from BioRad Laboratories, Richmond, 
Calif. or from International Biotechnologies, Inc., either for expression 
of proteins in procaryotic or eukaryotic cell culture. The gene to be 
expressed is inserted into the vector and the cells in which the gene is 
to be expressed as described by the supplier. Suitable cell culture 
systems include E. coli, yeast such as S. cerevisae, and mammalian cells 
such as CHO cells, all available from a variety of sources, including the 
American Type Culture Collection of Rockville, Md. 
The chaperonin protein system is added to the protein to be folded/refolded 
under conditions generally in the range of a temperature of between about 
1.degree. C. and 40.degree. C., preferably between 15.degree. and 
23.degree. C., at a pH of between about 7 and 8.0, preferably at about 
7.4. The chaperonin proteins, for example, the groEL 14mer and the groES 
7mer, are added in a preferred ratio of 1:1 to the protein substrate, at a 
ratio of chaperonin proteins to substrate of at least 1:1, preferably at a 
ratio of 1:3 or 4. The time required for folding is short; i.e., minutes 
or less for a mature protein, simultaneously with the production of a 
newly expressed protein. 
The method and reagents of the present invention will be further understood 
by reference to the following non-limiting examples of the refolding of 
two distinct enzymes using purified groEL and groES in the presence of 
Mg-ATP. 
Reconstitution of Chaperonin-Dependent Folding 
Chicken DHFR is a globular, single-domain protein of 189 residues. Upon 
dilution from denaturant, DHFR refolds to greater than 90% of the activity 
of the native enzyme. This property was used to establish the conditions 
allowing binding of DHFR to purified groEL. These experiments were 
performed at 15.degree. C. to obtain a better resolution of the 
time-course of reactivation. 
The following methods and reagents were used. The results are shown in 
FIGS. 1a, 1b, 1c, and 1d. 
GroEL and groES proteins were purified to greater than 95% purity using a 
groE overproducing strain of E. coli harboring the plasmid pOF39, as 
described above. 
As shown in FIG. 1a, chicken DHFR (greater than 95% purity, Sigma) was 
denatured at 100 .mu.g/ml in buffer A (6M guanidinium-HCl (GdmCl), 30 mM 
Tris pH 7.4, 1 mM dithiothreitol (DTT)) and diluted at 15.degree. C. 
200-fold into buffer B (30 mM Tris pH 7.2, 2 mM DTT, 50 mM KCl) containing 
50 .mu.M dihydrofolate and purified groEL 14mer as indicated in FIG. 1. 
Reactivation was measured photometrically by monitoring the oxidation of 
NADPH at 340 nm. Binding to groEL was analyzed by adding increasing 
amounts of denatured DHFR at 15.degree. C. to buffer B containing 50 nm 
groEL; the final concentration of GdmCl was adjusted to 100 nM. After 5 
min of incubation, the reactions were separated on 5 ml Sephacryl.TM. 
S300-columns (Pharmacia), allowing complete separation between groEL-bound 
and free DHFR. Fractions (120 .mu.l) containing the peak concentrations of 
groEL were pooled and analyzed by SDS-polyacrylamide electrophoresis 
(SDS-PAGE) and immunoblotting using anti-DHFR and anti-groEL antibodies 
were visualized by the luminescence-based detection system ECL (Amersham). 
Fluorographs were quantified by laser-densitometry using known amounts of 
groEL and DHFR as standards. 
As shown in FIG. 1b, unfolded DHFR (25 nM, final concentration) was diluted 
into buffer B in the absence or presence of 50 nM groEL. Reactivation was 
started by adding 5 mM Mg-acetate/1 mM ATP after 3 min when spontaneous 
refolding of free DHFR was complete. When indicated, groES 7mer (50 nM, 
final concentration) was added prior to MG-ATP. Progress curves of 
activity were recorded by plotting the changes in absorption at 340 mn ( 
A/17 sec) versus time. 
As shown in FIG. 1c, the DHFR-groEL complex was generated as in (a) (final 
concentrations of groEL and DHFR 50 nM and 25 nM, respectively). After 3 
min of incubation, increasing amounts of groEL were added in the presence 
or absence of equimolar amounts of groES. Total concentrations of groEL 
are indicated. Reactivation was initiated by adding Mg-ATP as above. Based 
on progress curves, t.sub.K is defined as the time required for 60% 
reactivation considering that approximately 20% of total DHFR activity was 
due to spontaneous reactivation. 
As shown in FIG. 1d, unfolded DHFR (25 nM) was diluted into buffer B 
containing 100 nM groEL in the presence or absence of groES (100 nM). 
After 3 min, .alpha..sub.51 -casein (Sigma) was added at the indicated 
concentrations. After continuing incubation for 3 min to confirm stability 
of the DHFR-groEL complex, MG-ATP was added and t.sub.K of reactivation 
was determined as in (c). 
DHFR was completely unfolded in 6M guanidinum-HCl (GdmCl) as based on 
circular dichroism-spectroscopy and then diluted 200-fold into 
physiological buffer containing increasing concentrations of groEL (FIG. 
1a). An equimolar concentration of groEL (25 nM with respect to groEL 
14mer) reduced the yield of reactivated DHFR by about 80%. Unfolded DHFR 
bound to groEL with high affinity which was not influenced significantly 
by the present of ATP (in the absence of Mg.sup.2 +) or the 
non-hydrolyzable ATP analogue AMP-PNP. The groEL 14mer appears to bind 
only 1-2 molecules of DHFR (FIG. 1a, insert). Addition of Mg-ATP to the 
DHFR-groEL complex allowed refolding to occur with a half-time of 
approximately three min (FIG. 1b). This was considerably slower than the 
spontaneous folding of DHFR ('1/2 approximately 1 min). In the presence of 
groEL, reactivation curves were sigmoidal, suggesting the existence of 
additional species of DHFR during refolding other than the fully unfolded 
and native forms. GroES 7mer added at a 1:1 molar ratio to groEL 
accelerated the groEL-dependent folding. Higher amounts of groES were 
without further effect. 
Starting with the preformed DHFR-groEL complex, increasing the 
concentration of groEL was found to retard the Mg-ATP-dependent 
reactivation of DHFR (FIG. 1c). This suggested that, following release, 
DHFR could re-bind to groEL before completing folding. This was 
demonstrated using .alpha..sub.51 -casein as a competitor of protein 
binding to groEL (FIG. 1d). Casein was chosen because it has certain 
properties of a partially denatured protein. Although being soluble, the 
native form exposes a considerable part of its hydrophobic residues to 
solvent and contains a high amount of disordered structure. Casein bound 
to groEL with high affinity and thereby prevented the re-binding of DHFR 
to groEL. Displacement of DHFR by casein apparently required 
ATP-hydrolysis since it was only detected in the presence of Mg-ATP, at 
least with the concentrations of casein employed. Notably, the decreased 
rate of reactivation due to the re-binding of DHFR was not observed when 
groES was present in amounts stoichiometric to groEL (FIG. 1c, d). It thus 
appears likely that only when groES is absent does DHFR reach its native 
state via cycles of repeated binding to groEL and Mg-ATP-dependent 
release. 
In contrast to DHFR, a number of proteins do not refold spontaneously upon 
dilution from denaturant but rather form misfolded aggregates. GroEL and 
groES allowed the efficient refolding of one such monomeric enzyme, 
rhodanese of bovine liver. Rhodanese (293 residues) is considerably larger 
than DHFR and, in contrast to the former, is composed of two equal-sized 
domains which are stabilized by hydrophobic interactions. 
The following methods and materials were used to demonstrate refolding of 
rhodanese. Rhodanese from bovine liver (greater than 95% purity, Sigma) 
was denatured in buffer A. 
As shown in FIG. 2a, unfolded rhodanese was diluted 100-fold (0.46 .mu.M, 
final concentration) into buffer C (30 mM Tris pH 7.2, 50 mM KCl) 
containing 0, 0.12, 0.23 and 0.46 .mu.M groEL 14mer, respectively. 
Aggregation (turbidity) was measured as absorbance at 320 nm. 
As shown in FIG. 2b, rhodanese-groEL complex was generated as above (final 
concentrations of rhodanese and groEL 0.46 .mu.M and 0.69 .mu.M, 
respectively). Aggregation of rhodanese was measured as in (a) upon 
addition of 1 mM ATP and 5 mM Mg-acetate in the absence or presence of 4.4 
.mu.M casein. Aggregates were sedimented in an Eppendorf centrifuge while 
groEL remained in the supernatant fraction. 
As shown in FIG. 2c, rhodanese-groEL complex was formed as in (b). When 
indicated, groES was present at 0.69 .mu.M. At various times after adding 
Mg-ATP, enzyme activity was determined (0.14 .mu.M rhodanese, final 
concentration) in the presence of 10 mM 
trans-1,2-cyclohexanediaminetetraacetic acid (CDTA) to stop groE-dependent 
reactivation. 
As expected, dilution of rhodanese from 6M GdmCl resulted in extensive 
aggregation (FIG. 2a). GroEL prevented this aggregation and formed a 
stable complex with rhodanese. Unlike DHFR, only a small amount of 
rhodanese was released from groEL upon ATP-hydrolysis; this portion of 
rhodanese did not reactivate but formed inactive aggregates (FIG. 2b). 
Complete release of rhodanese was achieved by the competitor casein. 
Again, this displacement was only observed in the presence of Mg-ATP and, 
in contrast to the findings with DHFR, resulted in the aggregation of the 
released protein. Only in the presence of groEs does efficient 
reactivation of rhodanese (t.sup.K .about.10 min) occur (FIG. 2c), 
suggesting a specific function of groES for folding beyond just promoting 
the release of a groEL-stabilized intermediate. 
GroEL stabilizes a molten globule-like state 
The endogenous tryptophan fluorescence of groEL-bound DHFR and rhodanese 
was determined as a measure of tertiary structure. GroEL and groES 
themselves interfere only minimally with this analysis since both 
components are devoid of Trp residues. 
The conformations of groEL-associated proteins were compared by Trp 
fluorescence of DHFR: (1), in 6M GdmCl; (2), native; (3), groEL-bound 
DHFR-11; (4), groEL-bound DHFR-12; (5) DHFR-12 after incubation in the 
presence of DCTA; (6), DHFR after complete reactivation in the presence of 
groEL and Mg-ATP, (ii), Trp fluorescence of rhodanese: (1), in 6M GdmCl; 
(2), native; (3), groEL-bound RHO-11; (4), RHO-11 upon incubation with 
Mg-ATP in the absence of groES; (5), RHO-11 after reactivation in the 
presence of groES and Mg-ATP; by 1,8 ANS fluorescence of free and 
groEL-bound forms of DHFR and rhodanese, and of groEL; and by proteinase K 
resistance of DHFR and rhodanese: (i, iv), native; (ii, v), DHFR-11 and 
RHO-11, respectively; (iii, vi), DHFR-12 and RHO-12, respectively. 
The following materials and methods were used. Fluorescence spectra were 
recorded on a SPEX spectrofluorimeter. Absorbance of the samples was lower 
than 0.05 at the excitation wavelength. The substrate-groEL complexes were 
formed as described above at a 2.5-fold molar excess of groEL 14mer over 
substrate protein (final concentrations of DHFR and rhodanese, 0.65 .mu.M, 
respectively). Under these conditions, greater than 90% of DHFR and 
greater than 95% of rhodanese were bound to groEL. Where indicated, groES 
7mer was added at an equimolar concentration to groEL. Trp fluorescence 
was excited at 295 nm. Spectra were obtained by subtracting the background 
fluorescence of the respective chemically identical reactions from which 
DHFR or rhodanese were omitted. Background fluorescence of groEL and groES 
due to minor impurities was 15-30% of the total fluorescence measured in 
the presence of substrate proteins. The small amount of DHFR which had 
escaped binding to groEL was corrected for. Analysis of DHFR-12 in 
reaction (i) 4 was generated by adding Mg-ATP (1 mM Mg-acetate, 2 mM ATP) 
to groEL-bound DHFR-11. (Neither ATP nor Mg.sup.2+ affected the 
fluorescence properties of DHFR-11) Spectrum (i) 4 was recorded 30 sec and 
spectrum (i) 6 10 min after addition of Mg-ATP. Unfolding of DHFR-12 (i) 5 
was observed upon incubation of reaction (i) 4 in the presence of 10 mM 
CDTA for 5 min. In reaction (ii) 4, groEL-bound RHO-11 was incubated with 
Mg-ATP for 30 min. Reactivated rhodanese was analyzed after 40 min 
incubation of RHO-11 in the presence of groES and Mg-ATP. 
Various forms of DHFR and rhodanese prepared as above were incubated for 5 
min at 15.degree. C. or 20.degree. C. in the presence of a 20-fold molar 
excess of 1-anilino-naphthalene-8-sulfonic acid (ANS) for ANS analysis. 
(GroEL-substrate complexes were stable in the presence of ANS for at least 
30 min.) Excitation was at 390 nm and emission was monitored at 470 nm. 
Emission spectra were corrected for background fluorescence caused by ANS 
in the presence or absence of groEL. DHFR-12 and reactivated DHFR were 
formed as above in the presence of ANS and the loss of fluorescence at 470 
nm was followed. RHO-12 was generated in the presence of ANS by incubation 
of RHO-11 with Mg-ATP and groES for 3 min at 25.degree. C. Fluorescence 
intensity was calculated in arbitrary units per mole of protein (for the 
14mer in case of groEL). 
GroEL-bound DHFR-11 and DHFR-12 were separated from free DHFR by sizing 
chromatography as in FIG. 1. GroEL-bound rhodanese was isolated by 
sedimenting aggregates for 30 min at 25.000 xg. RHO-12 was generated as 
above (but in the absence of ANS). The reaction was stopped by the 
addition of 10 mM CDTA and cooling to 0.degree. C. Protease treatment was 
performed in buffer B (final concentration of DHFR and rhodanese 
approximately 0.25 .mu.M and 0.95 .mu.M, respectively) for 10 min at 
0.degree. C. at concentrations of proteinase K (PK) of 0, 0.5, 1, 5, 10, 
and 20 .mu.g/ml. Protease action was stopped by adding 
phenylmethylsulfonylfluoride (PMSF) to 2 mM. TCA precipitates were 
analyzed by SDS-PAGE and immunoblotting. Fluorographs were exposed for 
either 15 sec or 90 sec. 
DHFR from chicken contains three tryptophans, at positions 24, 57 and 113. 
Their fluorescence upon excitation at 295 nm changes markedly when the 
protein is unfolded: the emission maximum (lambda max. em) shifts from 331 
nm to 356 nm accompanied by an approximately 2-fold increase in the 
fluorescence intensity. The red shift of fluorescence generally reflects 
transfer of Trp residues to a more polar environment. The increase in 
fluorescence intensity upon unfolding has been shown to be largely due to 
the removal of quenching amino acids from the neighborhood of the active 
site residue trp 24. The fluorescence spectrum of DHFR was stabilized by 
groEL in the absence of ATP-hydrolysis (termed DHFR-11). The lambda max, 
em was at 345 nm, half-way between the lambda max, em values of the native 
and unfolded proteins (Table 1). Strikingly, the relative fluorescence 
intensity of DHFR-11 was almost as high as that of the completely unfolded 
protein. Apparently, groEL-bound DHFR lacks an ordered tertiary structure. 
This is consistent with the very high protease sensitivity of the protein. 
As indicated by the 50% blue shift of emission, the Trp residues in 
DHFR-11 are already in a more hydrophobic environment relative to the 
completely unfolded protein. Using acrylamide as quencher, their 
accessibility to solvent was found to be significantly increased as 
compared to the native structure. Acrylamide is a polar but non-ionic 
collisional quencher that may be expected to partition into the 
loosely-folded interior of folding intermediates. 
The properties of groEL-bound rhodanese (RHO-11) were similar to that of 
DHFR-11. Rhodanese contains eight tryptophans which are distributed 
throughout the sequence. Upon complete unfolding, their fluorescence 
maximum shifts from 330 nm to 355 nm without major change in intensity. 
The lambda max, em of RHO-11 was at 342 nm, again corresponding to an 
approximately 50% shift towards the unfolded form. The fluorescence 
intensity of RHO-11 was increased and was characterized by the presence of 
partially accessible Trp residues. Like DHFR-11, groEL-bound rhodanese 
exhibited very high protease-sensitivity. 
DHFR-11 and RHO-11 were tested to determine if they contained hydrophobic 
regions that could be detected by 1-anilino-naphthalene-8-sulfonic acid 
(ANS), a probe for apolar binding sites whose fluorescence is strongly 
dependent on the hydrophobicity of the environment. ANS is known to 
accumulate within the solvated hydrophobic core of early folding 
intermediates generally termed "molten globule" or "compact intermediate". 
Table 1 shows the florescence parameters of DHFR and rhodanese species. Trp 
fluorescence spectra were recorded as described above. Fluorescence 
quenching using acrylamide (0-400 mM) was measured at the maximum of 
emission. Quenching data were analyzed according to the Storn Volmer 
relationship Fo/F=1+K.sub.Q X!, where Fo and F are the fluorescence 
intensities in the absence and presence of the quencher, respectively, and 
X! is the quencher concentration. K.sub.Q is the apparent quenching 
constant. In the case of heterogeneous emission (i.e. presence of more 
than one population of Trp residues with different microenvironments) a 
modified Stern-Volmer equation was applied which allows the calculation of 
the effective quenching constant. (K.sub.Q)eff and the fraction of Trp 
residues (f.sub.a)eff with the highest accessibility to the quencher. 
Quenching data were corrected for background fluorescence caused by minor 
impurities of the groEL preparation. The inner-filter effect due to 
acrylamide was corrected for by the factor Y=antilog (d.sub.4 
+d.sub..epsilon.)/2, where d.sub.4 and d.sub..epsilon. are the absorbances 
at the excitation and emission wavelengths, respectively. 
Forster-distances r were calculated on the basis of singlet--singlet 
energy transfer from Trp residues to 1,8 ANS. Both groEL-stabilized 
proteins showed strong ANS fluorescence. 
TABLE 1 
______________________________________ 
Fluorescence data for groEL-stabilized proteins: 
Maximum of 
Fluorescence at 
Tryptophan 
Emission 
Fluorescence 
Maximum K.sub.Q r (Trp. ANS) 
nm! Arb. Units! 
1.M! f.sub.a 
.ANG.! 
______________________________________ 
DHFR 
Native 331 6.7 2.5 1.0 -- 
6M GdmCl 356 15.5 8.5 1.0 -- 
GroEL-bound 
345 14.1 5.5 0.8 2.9 
(DHFR-11) 
Rhodanese 
Native 333 13.1 1.5 1.0 -- 
6M GdmCl 356 12.2 9.0 1.0 -- 
GroEL-bound 
342 17.8 4.1 1.0 3.0 
(RHO-11) 
______________________________________ 
Notably, the interaction with ANS did not affect the stability of the 
complex between groEL and the substrate proteins. Little fluorescence was 
measurable with the native or guanidine-unfolded DHFR and rhodanese, or 
with groEL 14mer alone, indicating that groEL does not expose domains that 
could provide a hydrophobic environment for ANS. This does not exclude 
that groEL may shield hydrophobic regions of the associated proteins, 
thereby increasing the hydrophobic space accessible to ANS. The near 
spatial relationship of the tryptophan and ANS chromophores in DHFR-11 and 
RHO-11 is reflected in fluorescence energy transfer from the Trp residues 
(excitation at 295 nm) to ANS occurring with an efficiency of 
approximately 30% (Table 1). The Forster-distances between tryptophans and 
ANS were calculated as a relative parameter. In both cases a mean distance 
of approximately 30 .ANG. was found, further suggesting a similar global 
shape of the two groEL-bound intermediates. 
Altogether, these results indicate that groEL stabilizes unfolded substrate 
proteins in a loose conformation devoid of ordered tertiary structure. The 
fluorescence properties and the increased solvent accessibility of the Trp 
residues, as well as the incorporation of ANS, point to the presence of 
flexible tertiary structure internalizing a fluctuating, hydrophobic core, 
as described for the molten globule state. 
Folding of DHFR Via a More Compact Intermediate 
The changes in Trp fluorescence during groEL-dependent folding of DHFR-11 
were monitored. Within 15-30 sec of the addition of Mg-ATP to the 
DHFR-groEL complex, the relative fluorescence intensity of DHFR-11 dropped 
markedly, approaching that of the native protein, while lambda max, em 
remained unaltered, defining the folding intermediate DHFR-12. In a 
subsequent phase extending over 3-7 min. Lambda max, em shifted from 345 
nm to 331 nm accompanied by the appearance of enzyme activity. Apparently, 
a partial folding reaction including Trp 24 occurs early in 
groEL-dependent folding of DHFR, exposing this residue to the native-like 
fluorescence quenching environment. 
In parallel with the loss of Trp fluorescence, the conformational change to 
DHFR-12 resulted in a loss of 60% of the ANS fluorescence detected with 
DHFR-11. This phenomenon is typically observed with certain early folding 
intermediates, resembling the molten globule, upon their folding to a more 
compact structure. This could be confirmed by analyzing the endogenous 
resistance of DHFR-12 towards protease. DHFR-12 was generated by 
initiating ATP-hydrolysis as above. After 30 sec at 15.degree. C., the 
folding reaction was stopped by adding the Mg.sup.2+ -chelator CDTA and 
cooling to 0.degree. C. DHFR-12, in contrast to DHFR-11, was resistant to 
intermediate concentrations of proteinase K. A small amount of an 
approximately 10,000 Dalton fragment was detectable which was digested at 
higher concentrations of protease. In contrast to the protease-stable, 
native enzyme, DHFR-12 was still associated with groEL. When the reaction 
containing DHFR-12 and CDTA was maintained at 15.degree. C. (instead of 
0.degree. C.), partial unfolding, as visualized by an increase in 
fluorescence intensity, was observed. Addition of sufficient Mg.sup.2+ to 
compensate for the CDTA present re-initiated the folding reaction, leading 
to the conclusion that the folding of groEL-bound DHFR-11 involves the 
transient formation of an intermediate with partially native tertiary 
structure, DHFR-12. This compactization occurs in close association with 
the groEL complex. 
GroES Commits Proteins to Folding at the GroEL Surface 
The interaction of groES with groEL was found to accelerate the conversion 
of DHFR-11 (FIG. 1b) to the native protein, perhaps simply by facilitating 
its Mg-ATP-dependent release. On the other hand, based on the observation 
that folding of rhodanese is essentially groES-dependent (FIG. 2c), one 
would predict that the action of groES allows a critical folding step(s) 
to proceed at the groEL surface. To test this hypothesis, rhodanese 
folding was dissected into intermediate steps. 
The following materials and methods were used. The results are shown in 
FIG. 3a for unfolded rhodanese diluted as above from 6M GdmCl into buffer 
C containing 2.34 .mu.M groEL 14mer. The final concentration of rhodanese 
was 1.17 .mu.M. After centrifugation to exclude the presence of aggregated 
rhodanese, 2.34 .mu.M groES was added to the supernatant fraction and 
refolding was started at 25.degree. C. by addition of ATP and Mg-acetate 
to 1 and 2 mM, respectively. At the times indicated, folding was stopped 
in aliquots of the reaction by adding CDTA to 10 mM and cooling to 
0.degree. C. Enzyme activity and endogenous protease resistance of 
rhodanese were determined as in FIG. 2. Rho-11 was defined as the amount 
of rhodanese resistant to 2 .mu.g/ml proteinase K as quantified by 
densitometry of fluorographs. 
Association with groEL was determined and the results shown in FIG. 3b. 
Rhodanese-groEL complex (0.46 .mu.M/0.69 .mu.M, respectively) was formed 
as above. Refolding reactions received 4.4 .mu.M casein, 0.69 .mu.M groES, 
1 mM ATP and 5 mM Mg-acetate according to the order of addition indicated. 
In the absence of groES, the addition of Mg-ATP had no effect on the 
fluorescence properties or the protease-sensitivity of groEL-bound RHO-11. 
The folding reaction in the presence of groES was stopped after various 
times by adding CDTA to inhibit ATP hydrolysis. The amount of groEL-bound 
rhodanese, its endogenous protease resistance, and enzyme activity were 
analyzed during the time-course of the reaction, as shown in FIG. 3a. 
During the first 5 min of incubation in the presence of Mg-ATP, a form of 
rhodanese accumulated, defined as RHO-12, which had lost 50% of the ANS 
fluorescence of RHO-11 and had acquired an increased resistance to 
intermediate concentrations of proteinase K. The formation of this 
enzymatically inactive intermediate preceded that of the 
protease-resistant, active rhodanese which showed only little ANS 
fluorescence. In contrast to the native enzyme, RHO-12 was still bound to 
groEL as revealed by gel chromatography (FIG. 3a). Altogether, RHO-12 has 
properties resembling DHFR-12 but, in contrast to DHFR-12, its formation 
is critically dependent on groES. 
Competition by casein for the groES-dependent conversion to RHO-12 and to 
the native enzyme would be expected if folding involved cycles of complete 
release and rebinding to groEL. As shown in FIG. 2b, a 6-fold molar excess 
of casein over groEL efficiently displaced RHO-11 in the presence of 
Mg-ATP, resulting in aggregation. When added prior to rhodanese, this 
amount of casein was sufficient to almost completely prevent the 
reactivation of rhodanese by groEL and groES. Strikingly, when both casein 
and groES were added to preformed rhodanese-groEL complex before 
initiating folding by Mg-ATP, reactivation was as efficient as in the 
absence of casein (FIG. 3b). Thus, the competitor was unable to interrupt 
the reactivation once the groEL-rhodanese complex had been formed and 
groES was present. This indicates that a single round of interaction 
between rhodanese and groEL is sufficient for folding. On the other hand, 
when groES was added to a reaction containing rhodanese-groEL complex and 
casein 15-120 sec after starting ATP hydrolysis, the yield of active 
enzyme was decreased by 50-80% (FIG. 3c). This corresponded to the amount 
of rhodanese displaced from groEL during that incubation (see FIG. 2). 
This lead to the conclusion that during folding to RHO-12, rhodanese 
maintains contact with the groEL 14mer. In the presence of groES, the 
protein is committed for folding. The underlying mechanism of this 
surface-mediated reaction could be a progressive, Mg-ATP-dependent release 
of the folding polypeptide. Achievement of the native conformation appears 
to be closely coupled with final release from groEL. 
ATP Requirement of Folding 
The following materials and methods were used to determine the ATP 
requirement for folding. 
ATPase-activity was measured at 25.degree. C. by colorimetric determination 
of free phosphate produced by ATP-hydrolysis using malachite green as 
color reagent. Spontaneous hydrolysis of ATP as well as free phosphate in 
reagents were corrected for. One unit of absorbance at 640 nm corresponded 
to 9.45 nmole phosphate. As shown in FIG. 4a, after addition of groEL 
(0.33 .mu.M in buffer C) of groES (0.33 .mu.M), casein (4.4 .mu.M) or 
denatured rhodanese (1.0 .mu.M), ATP-hydrolysis was started with 1 mM ATP 
and 5 mM Mg-acetate. GroES was added to the reaction containing casein 
after 20 min of incubation. The final concentration of GdmCl in the assay 
of greater than 30 mM did not detectably affect the groEL ATPase. 
Spontaneously aggregated rhodanese (up to 1.6 .mu.M final concentration) 
did not stimulate the ATPase activity of groEL. 
As shown in FIG. 4b, GroEL (0.33 .mu.M) and groES (0.66 .mu.M) were 
preincubated for 30 min at 25.degree. C. in the presence of Mg-ATP to 
ensure maximal inhibition of the groEL ATPase. Denatured rhodanese was 
then added to a final concentration of 1.0 .mu.M (t=0 min). Enzyme 
activity and ATP-hydrolysis were measured in parallel aliquots of the same 
reaction. The concentration of reactivated rhodanese was calculated based 
on the specific activity of the native enzyme. (One out of five 
experiments is shown.) 
The ATPase activity of groEL, measurable in the absence of protein 
substrate, is known to be inhibited by groES. Under the assay conditions 
described herein, groEL 14mer hydrolyzed ATP at a rate of approximately 7 
min.sup.-1, as shown in FIG. 4a. GroES 7mer, at a 1:1 molar ratio to groEL 
14mer, reduced this rate to approximately 0.25 min.sup.-1 within 5-10 min 
of addition. Increasing the concentration of groES did not further reduce 
the rate of ATP-hydrolysis. A saturating concentration of casein caused a 
2-fold activation of the groEL ATPase in the absence of groES (FIG. 4a). 
Binding of unfolded rhodanese also resulted in a continuous stimulation to 
a rate of approximately 17 ATP hydrolyzed per min. This is consistent with 
the finding that, in the absence of groEs, the substrate protein and groEL 
undergo cycles of complete, Mg-ATP-dependent release and re-binding 
without folding. Under these conditions, the competitor casein will 
displace 50% of bound rhodanese from groEL within 15 sec of initiation of 
ATP hydrolysis (FIG. 4b). It is estimated that approximately 27 molecules 
of ATP are hydrolyzed per molecule of rhodanese released in the absence of 
groES. 
Addition of unfolded rhodanese to groEL and groES initially resulted in an 
approximately 40-fold increase of the groES-suppressed rate of 
ATP-hydrolysis (FIG. 4b). The ATPase activity then gradually decreased as 
reactivation of rhodanese approached completion. It was calculated that 
100.+-.20 moles of ATP were hydrolyzed per mole of rhodanese folded (FIG. 
4b). Due to the faster kinetics of reactivation, DHFR showed a 2 to 3-fold 
lower hydrolysis of ATP during groE-dependent folding. In contrast, casein 
caused a continuous stimulation of the groEL ATPase even in the presence 
of groES (FIG. 4A). The "native" protein casein appears to be unable to 
vary structural elements interacting with groEL. 
In summary, these results lead to the conclusion that groES couples the 
groEL ATPase with the folding reaction at the surface of the chaperonin, 
and that GroES coordinates Mg-ATP-dependent conformational changes of 
GroEL and thereby allows correct folding to occur by a process of 
step-wise release. GroES prevents the "whole-sale" dissociation of the 
substrate protein from groEL which is unproductive for folding. 
The conformation of groEL-bound polypeptides resembles that of the 
so-called "molten globule" or "compact intermediate", a transient, early 
folding state that rapidly interconverts with the fully unfolded form. It 
is characterized by containing all or part of the secondary structure of 
the protein in conjunction with a relatively compact but unordered, 
flexible tertiary structure internalizing a "molten", hydrophobic core. 
The groEL-stabilized intermediates, DHFR-11 and RHO-11, fulfill these 
criteria: i) compared to the fully unfolded state, their tryptophans are 
in a less polar environment and are only partially exposed; ii) their 
hydrophobic interior is much less densely packed than that of the native 
conformation as demonstrated by its solvent accessibility and the typical 
incorporation of the hydrophobic fluorescent probe ANS; and (iii) their 
structural flexibility is reflected in the high sensitivity towards 
protease. 
Substrate proteins acquire a more rigid structure while maintaining the 
interaction with the groEL scaffold. The decrease in fluorescence 
intensity during conversion of DHFR-11 to DHFR-12 is likely due to the 
formation of the natural, hydrophobic environment of Trp 24. This is based 
on findings with a mutant form of human HDHFR in which Trp 24 is changed 
to Phe; the mutant protein does not show the increase in Trp fluorescence 
intensity typically observed upon unfolding. DHFR12 and RHO-12 represent 
more compact conformations on the folding pathway. Apparently, their 
hydrophobic interior is already more densely packed than in the molten 
globule-state, explaining the reduced accessibility for the fluorescent 
probe ANS. The native state of the protein is reached during or shortly 
after the final release from groEL. 
The large groEL 14mer appears to interact with only 1 to 2 substrate 
molecules. Folding occurs as an active process at the interacting surface 
between groEL and the substrate protein, relying initially on binding of 
two (or more) segments of the protein substrate and releasing them 
sequentially upon ATP hydrolysis. This latter step requires the action of 
groES, which couples the hydrolysis of ATP with folding at groEL, 
coordinating the function of (adjacent) parts of the substrate 
binding-region, belonging to different subunits of groEL, for example, by 
modulating the time-span during which they are refractory for rebinding to 
segments of the folding protein. This prevents the release of the complete 
protein in a single step, and gives a (sub)domain sufficient time to 
organize to a more compact structure which is no longer able to re-bind. 
Rhodanese folding is fully dependent on such a mechanism while DHFR, at 
least in vitro, is not. Nevertheless, the observations made with DHFR are 
consistent with the above considerations: In the absence of groES, folding 
is retarded by increasing the concentration of groEL as a result of cycles 
of unproductive binding and release. This is not the case in the presence 
of groES, however, apparently because the protein is then forced onto a 
folding pathway that involves a staged release during a single round of 
interaction. 
The amount of ATP hydrolyzed by groEL per protein molecule folded is in the 
range of 5 to 10% of the total required for synthesis, suggesting that the 
structural organization of a polypeptide at the surface of groEL requires 
multiple rounds of ATP hydrolysis by the groEL 14mer. The energy of the 
ATP hydrolyzed is not conserved in the final folded structure but is 
probably required to promote conformational changes of groEL resulting in 
(sequential) release of the bound substrate. The chaperonins are essential 
for folding in vivo, i.e., they "catalyze" protein folding in a biological 
sense. 
Modifications and variations of the compositions and methods of use thereof 
to fold or refold proteins will be obvious to those skilled in the art 
from the foregoing detailed description. Such modifications and variations 
are intended to come within the scope of the appended claims.