Summary
Molecular events accompanying chaperone action are subjects of intensive research. We have a rather elaborate "working-cycle" for the 60 kDa molecular chaperones, which possess a cavity, and are called Anfinsen-cage type chaperones to emphasize the fact that they provide a closed, protected environment to help the folding of their substrates. The involvement of nucleotides and co-chaperones in the folding process has also been largely elucidated. However, our understanding of the molecular mechanism how these chaperones help protein folding is fairly incomplete. The present paper adds two novel elements to the mechanism how Anfinsen-cage type chaperones (members of the 60 kDa chaperone family) aid protein folding by arguing that (1) these chaperones do not generally unfold their targets, but by a multidirectional expansion loosen the tight, inner structure of the collapsed target protein and (2) during the expansion water molecules enter the hydrophobic core of the target, and this percolation is a key step in chaperone action. The paper compares this chaperone-percolator model with existing explanations, such as the iterative annealing, lists experimental evidence supporting the model, and suggests further experiments to test, or refute its validity.
Introduction
Folding of proteins larger than 10-20 kDa often results in a conformationally trapped intermediate after the initial hydrophobic collapse of the extended unfolded structure. Flexibility and conformational mobility of these partially folded proteins are rather restricted, if compared to the unfolded state. On one hand, the initial collapse is very helpful, since it greatly reduces the number of available conformations and therefore alleviates the need for a random search through the astronomical number of initially available conformational states, which would require a much greater time than the age of the Universe (the so-called ‘Levinthal-paradox’). However, hydrophobic collapse in most cases "goes too far", and the comprised intermediate structure (which is only 10 % larger than the tightly packed native structure) makes further rearrangement of the inner hydrophobic core of the protein especially slow, and difficult(1-4). Since the correct and tight packing of the inner core is a key element of protein structure and stability(5,6), restrictions of final folding steps of these inner segments are particularly debilitating for the development of the native structure. Amino acid side chains of protein interiors are rather flexible(7), thus it is not a general "freezing", but the lack of extra space for the inner-core backbone rearrangements, which precludes an easy transition to the native state. Thus the maximal preservation of water structure and its hydrogen bonds is not only the major driving force of the initial hydrophobic collapse, but also acts as a major restrictive force, which does not allow the intensive rearrangement of the inner, hydrophobic segments to reach the native state. The extent of hydrophobic collapse needs adjustment. Therefore most larger proteins need help to complete the organization of their inner core, besides rescuing them from folding traps arising from the improper rearrangement of some specific protein segments and protecting them against aggregation.
All this help is provided by molecular chaperones. Chaperones bind to their target proteins by multiple bonds containing a great variety of interactions between hydrophobic, hydrophilic and charged residues(8-12). These bonds are usually not concentrated to a very small edge of the target protein, but surround a significant portion of one or more folding domain. In case of Anfinsen-cage type chaperones the "encircling" of the target is especially pronounced, where the oligomeric structure of the chaperone sequesters the target to its inner cavity and allows an undisturbed folding process preventing aggregation. Presently the 60 kDa chaperones are the only members of Anfinsen-cage type chaperones(8,12). However, the lacking 3D-description of other multi-chaperone complexes, such as the dynamic complex of Hip/Hsp70/Hop/Hsp90 and other chaperones makes it presently difficult to judge, if similar target "encircling" arrangement, or other common elements of molecular mechanism might occur, or not. The Clp-family of proteases also possess an inner cavity, but the nature and mechanism of the unfolding steps of their action are not clear yet. Due to the incomplete information of the other chaperone classes in the further course of the paper I will describe the proposed model for the Anfinsen-cage type chaperones (and will mean under this term the 60 kDa molecular chaperones), but I will also keep in mind that future structural and functional studies might result in the implication of (at least some elements of) the model to other large chaperone-complexes.
The present paper summarizes a hypothesis, which adds two novel elements to the molecular mechanism of Anfinsen-cage type chaperones: (1) instead of a general unfolding, these chaperones preferentially mobilize, loosen the tight, inner structure of the collapsed target protein and (2) during the chaperone-induced initial expansion of the target, water molecules enter to the hydrophobic interior of the target, and this percolation is a key step in chaperone action. The paper compares this chaperone-percolator model with existing proposals for the molecular mechanism, such as the iterative annealing, lists experimental evidence supporting the model, and suggests further experiments to test, or refute its validity.
Description of the chaperone-percolator model
Early assumtions referred to Anfinsen-cage chaperones, like passive boxes insulating their targets from other proteins and preventing protein aggregation. Though the isolation of the target is a crucial element of their help, recent data indicate a more active role of these chaperones (members of the Hsp60 family) in target folding. Anfinsen-cage chaperones pull away a smaller or larger segment of the target protein. This stretching is helped as the nature of dominant target-binding interactions shifts from mostly hydrophobic to mostly charge-charge interactions and by geometrical changes of the chaperone-target interface (see refs. 9-13, and the movies at the Internet address http://www.cryst.bbk.ac.uk/ ~ ubcg16z/cpn/chaperone.html). These changes may initially loosen the structure of the target, and finally result in its release to the cis chamber of Anfinsen-cage type chaperones. The bona fide folding of the target protein, i.e. the arrangement of its structure to reach the native state may happen after the initial stretching/pulling step. However, the initial expansion of the target is a prerequisite for any subsequent folding process. Quite many times a single pulling/stretching-release-folding attempt is not successful and must be repeated. Therefore chaperones in general do not accelerate folding just make it more efficient. These elements of the molecular mechanism of Anfinsen-cage type chaperone-action have been already proven by numerous experiments and represent a rather consensus view.
The first major element of the present model emphasizes that instead of a general unfolding, Anfinsen-cage type chaperones preferentially loosen the tight, inner structure of the collapsed target protein. Loosening of the hydrophobic core of the target is paralleled by a preferential mobilization of the inner amino acid side chains together with the immobilization of the external segments of the target (Fig. 1). Besides the isolation of their targets from other partially unfolded proteins (and thus prevention of their aggregation) Anfinsen-cage type chaperones also isolate their targets from the bulk of the structured water outside of the chaperone-machine. Thus according to the present model these chaperones reverse the hydrophobic collapse by two mechanisms: (a) by a direct mechanical multidirectional expansion and (b) by the attenuation of the hydrophobic force, i.e. the effects of the structured water to the folding target.
What happens when an Anfinsen-cage chaperone pulls away its target? To avoid vacuum, something must enter to the inside of the target protein. The interior of the Anfinsen-cage type chaperone chamber is packed with water molecules. As a second major element of the present model entry of water molecules to the interior of the folding target protein is postulated (Fig. 1). Water-entry is helped by two mechanisms: (a) by a specific, disordered state of certain water molecules inside the folding cavity and (b) by a massive outflow and consecutive influx of water molecules from the cavity during a single cycle of chaperone action.
Water molecules in the cavity of the chaperone do not resemble completely to the structured water outside their host. Though there are numerous water molecules in the chaperone cavity at each step of the chaperone cycle, water-water hydrogen bonding is limited by geometrical constraints. Water molecules finding themselves in the initially hydrophobic environment of the chaperone may adopt a rather disordered state with special thermodynamic properties(14-17). As an other important difference, the surrounding chaperone may significantly alter the dielectric constant of the "inside water"(18). According to the present model Anfinsen-cage type chaperones lower the energy barriers for the entry of water molecules to the inside of folding proteins. Due to these changes water molecules in the chaperone chamber may find an easier way to enter the inside of the loosened target protein and to fill the "vacuum" generated by its chaperone-mediated expansion. The energy requirement for the transition of water molecules from the outside organized water structure to the inside of the chaperone and to the inside of the target protein reaching 10 to 15 kcal/mol(19) is most probably provided by the conformational changes of the chaperone-machine utilizing the energy of ATP-hydrolysis.
There is a massive traffic of water molecules during chaperone action. The initial volume of the cis cavity (assumed to be 85000 A 3)(11) contains approximately 3000 water molecules. The total volume of a large target protein (such as that of the 52 kDa RuBisCO enzyme) corresponds to 2500 water molecules, thus RuBisCO binding induces a large outflow of water from the chaperone-machine. The amount of chaperone-contained water reaches its minimum during a single cycle (which is in between 500 and 2000 water molecules depending on the the exact geometry of target binding) when ATP binds to the cis ring and induces a large conformational change increasing the cavity volume to 175000 A 3(11). This enlargement is paralleled with a massive influx of water until the total number of water molecules reaches 3500. Further geometrical informations on chaperone-target complexes are necessary to give a more exact estimate of the water molecules present at various steps of the chaperone-cycle. (The volume of the partially unfolded RuBisCO protein has been calculated as only 110 % of the native volume. A more loose initial conformation of the target would result in even larger water fluxes. On the other hand, RuBisCO probably represents the upper size-limit of the proteins able to use the help of the GroE chaperone. Smaller targets induce smaller water fluxes. As a third uncertainity, the volume of water has been assessed assuming the volume of water molecules in the bulk water. Disordered water in the inside of the cavity may occupy a larger volume which also reduces the fluxes during the cycle.) In spite of the uncertainities of the numerical values, it is quite clear that chaperone cycle is paralleled with a massive outflow and influx of water molecules from the folding cavity.
The Anfinsen-cage type chaperone machine-mediated entry of water molecules to the interior folding protein is not only a passive consequence of the chaperone-induced pulling/expansion of the target, but may significantly promote the conformational changes required to reach the native state. At low hydration level the unfolding of proteins is severely hindered. Lyophilization of subtilisin or chymotrypsin and their subsequent dissolution in anhydrous solvents freezes the enzyme conformation and generates an "enzyme-memory". This memory can be erased by the addition of water(20). Water induces a larger internal mobility of proteins as judged by neutron scattering, NMR, ESR, Mössbauer spectroscopy, IR, high frequency dielectric measurements, fluorescence and phosphorescence lifetime analysis. These analytical methods cover an enormously large time scale of various protein movements from the psec-scale of neutron scattering to the sec-range of phosphorescence lifetime measurements(21,22). Water molecules were suggested to promote the rapid "flickering" between various conformational states, where the interaction of the peptide backbone with neighboring water molecules results in various transient reverse turn conformations forming a rather smooth path from a certain conformational state to the other(23). Thus Anfinsen-cage type chaperone-induced entry of water molecules to the otherwise hidden interior of the target protein may be essential to make the necessary conformational changes possible. Water-entry to the target is not only a coincident, secondary event, but may be an almost absolute necessity in assisted protein folding. The massive outflow and consecutive influx of water molecules during the chaperone cycle efficiently washes through the target with water. Therefore I suggest to call the above hypothetical mechanism as the chaperone-percolator model, referring to the process of percolation, where a solvent is passed through a permeable substance.
Whether the percolation described by the above model (i.e. the Anfinsen-cage type chaperone-induced increased penetration of the hydrophobic interior of the target proteins by water molecules) is accompanied by a percolation in the sense of an extension of the long range ordered structure of water and development of long range conductivity involving coordinated proton displacement of water molecules in the inside of the target protein(21) remains to be established. However, a – transient – connection of isolated water molecules in the inside of the target protein with molecules of the outer hydrate shell(17) may significantly increase hydrogen bond fluctuation and the subsequent catalysis of conformational rearrangement.
Limitations of the chaperone-percolator model
-- Limitations from the target side
The above chaperone-percolator model assumes that the target protein is relatively large, having a well-developed inner, hydrophobic core, which experiences significant difficulties in its final rearrangements. Fast-folding targets may not need the extensive help described by the present model.
In several large-size targets kinetic barriers of specific final rearrangements may form the rate-limiting steps to reach the native state, and the restriction of the inner-core rearrangement may be only of secondary importance.
-- Limitations from the chaperone side
As noted before, the model requires an Anfinsen-cage type chaperone structure. According to our present knowledge this structural requirement is valid only to the 60 kDa chaperone family. Members of the 70 kDa chaperone family bind small peptide segments(24), where the chaperone-percolator model can not be applied. Multi-chaperone complexes may utilize similar unfolding/percolating steps like those described in the chaperone-percolator model. However, presently we do not have enough information to extend the model beyond the 60 kDa chaperones.
Even in Anfinsen-cage type chaperones some chaperone-target interactions may not be intensive enough, or large-scale enough to cause an increased "breathing" of the inner core of the target.
Relationship of the chaperone-percolator model with other chaperone mechanisms, such as the iterative-annealing mechanism
There is a rather detailed description of the "working-cycle" of the 60 kDa molecular chaperones. However, the description of the molecular details how Anfinsen-cage type chaperones help the refolding of their target proteins lags behind. The "threading" described by Hubbard and Sander(25) may be operational in chaperones, which bind their targets in an extended conformation. However, up till now, there is no direct experimental evidence which verifies this model. The other possible alternative molecular mechanism of Hubbard and Sander(25), "plucking" is much closer to the present model, but also involves the "straightening out" of the peptide backbone. The iterative annealing mechanism of Todd et al.(26) describes the 60 kDa molecular chaperones as "they repeatedly bind kinetically trapped conformers, randomly disrupt their structure, and release them in less folded states, allowing substrate proteins multiple opportunities to find pathways leading to the most thermodynamically stable state." As a further development, Chan and Dill(27) describe the chaperone-machine as a protein "that helps pull apart an incorrectly folded protein so it can try again to fold." The expression "pulling apart" in Chan and Dill’s paper refers mostly to the disruption of hydrophobic contacts(27). The various pulling processes described by these mechanisms is similar to the above chaperone-percolator model. However, the chaperone-percolator model makes the physical meaning of the pulling process mediated by Anfinsen-cage type molecular chaperones clearer by emphasizing that instead of a general unfolding, Anfinsen-cage type chaperones preferentially loosen the tight, inner structure of the collapsed target protein. The chaperone-percolator model also emphasizes the importance of intensive interactions between water molecules and the peptide backbone of the target protein. The present model gives new elements to explain the dramatic changes of the energy landscape of protein folding upon chaperone action postulated by Gulukota and Wolynes(28) and describes a mechanism where the chaperone does not act as a direct catalyst of unfolding as predicted by Schmid and co-workers (29).
Experimental evidence supporting chaperone-percolator model
Evidence for a multidirectional pulling process, which mobilizes the interior of the target
Alkaline phosphatase provides one of the best examples for the slow rearrangement of the inner core during refolding, where the formation of the crystalline-like inner structure may last for weeks as judged by phosphorescence analysis of an inner tryptophane residue(30). This slow refolding emphasizes the necessity of a special mechanism able to loosen the slowly rearranging inner structure of the target folding intermediate.
3D structures of complexes of Anfinsen-cage type chaperones with their substrates show a rather extended conformation of the target(12,31) supporting the notion that chaperones interact with their targets using multiple bonds surrounding the target molecule. The structure of GroEL is significantly stabilized after binding of rhodanese which gives a further proof for the multiple, large scale chaperone-target interactions(32). However, these data do not allow us to judge whether an actual "inflation" of the globular target occurs.
Indirect evidence from Ullrich Hartl’s lab(33) suggests that GroEL "pulls apart" the two domains of rhodanese exposing the hydrophobic interdomain contacts of the protein.
A direct experimental evidence supporting the chaperone-percolator model came from the studies of the group of Art Horwich(34). Analyzing the fluorescence anisotropy of the RuBisCO tryptophanes after the addition of GroE they found a fast drop of the anisotropy value followed by a gradual increase. The fast drop in the anisotropy corresponding to the mobilization of the inner core of the RuBisCO protein, and the gradual increase reflecting the slow rearrangement and tightening of the inner structure(12) is in excellent agreement with the prediction of the internal target mobilization by the chaperone-percolator model.
During the formulation of the present manuscript a paper of Persson et al.(35) has been published, which analyzed the interaction between human carbonic anhydrase and GroEL using spin-labeled variants of the target. The chaperone-percolator model would predict the mobilization of the inner segments of carbonic anhydrase with a concomitant immobilization of its peripheral portions involved in binding to GroEL. Exactly this, what could be observed by the great variety of spin-labeled probes used by Persson et al.(35) giving an excellent proof for the predictive power of the model.
Evidence for water percolation
Reduction of the hydrophobic force (i.e. the sequestration of hydrophobic segments by the water structure) by detergents and other chemical chaperones, may contribute to the efficiency of these small molecules in refolding of several target proteins. Besides their increase of the "solvophobic effect" on the peptide backbone(36) the disorganization of water structure induced by chemical chaperones may result in an increased "breathing" of the inner core of the target and a smoother entry of water molecules to the interior of the target alleviating the need for a complex chaperone-structure.
The acceleration of the loss of tritium atoms from tritiated RuBisCO protein(37) upon the addition of an active GroE complex may be interpreted as a partial unfolding of the molecule but may also be the consequence of accelerated water penetration to the inner segments of the protein.
Data obtained from NMR and ESI-MS measurements show a 103- to 106-fold increased deuterium-hydrogen exchange of peptide amide groups of the chaperone-bound target protein. The degree of exchange varies from one chaperone-target pair to the other and was regarded as a measure from partial to almost complete unfolding of the target(38-42). The varying degree of unfolding experienced in these studies may also reflect the various levels of percolation of the extended inner structure by water molecules. The dramatic increase in water-peptide bond interactions even in the inside of the target and the resemblance of the partially protected target peptide bonds to the native globular structure of the target(42) provide a further strong support for the chaperone-percolator model.
As it is clear from the above pieces of evidence, the second part of the model (water-entry to the interior of the target) is more hypothetical at present than the first assumption (multidirectional expansion and mobilization of the interior of the target). In the following section I will suggest some experiments to test the validity model.
Suggested experiments to test the model
With the limitations mentioned above, let me summarize again the major consequences of the chaperone-percolator model:
target proteins undergo a multidirectional expansion during their interactions with Anfinsen-cage type molecular chaperones;
during the interaction the hydrophobic core of the target gets more flexible, while its outer, charged residues gets more immobilized than in its unbound state;
chaperone cycle is paralleled with a massive water outflow and consecutive influx of water molecules which contribute to the percolation of the target protein;
water molecules reside in a special, "high-energy" state in the inside of the chaperone chamber and penetrate to the inside of the target protein during its expansion significantly easing its conformational transitions.
The first of the above assumptions is difficult to test. Geometry of chaperone-target interactions may vary from one chaperone-target pair to the other. The expansion of target proteins might be too small to allow easy verification. Thus experiments to test the chaperone-percolator model should mostly concentrate to the parallel analysis of target and water mobility in the absence and presence of chaperones. Some suggestions:
lifetime of target tryptophane phosphorescence as well as its fluorescence anisotropy should decrease after Anfinsen-cage type chaperone binding (the first anisotropy observation has been already made by the group of Art Horwich(34));
ESR-probes on the surface and in the interior of the target protein should report different changes during chaperone-target interactions (the first observations has been already made by Persson et al. (35));
the amount and distribution of nuclear magnetic relaxation rates of water protons or oxygens (similar to those described in ref. 43) should change upon target binding to Anfinsen-cage type chaperones due to the appearance of a significant amount of "percolating water" with residence times in the microsecond range;
Raman spectra of O–H and N–H vibrational bands should also report increasing amount of "peculiar" hydrogen-bonding when Anfinsen-cage type chaperones bind their target;
target surfaces should be less sensitive, while the hydrophobic cores of the target should be more sensitive to the decomposition by hydroxyl radicals in the presence than in the absence of Anfinsen-cage type chaperones (common sense would dictate that Anfinsen-cage type chaperones protect all parts of the sequestered targets equally).
If neither NMR nor Raman experiments report any unusual water molecules during the "working cycle" of Anfinsen-cage type chaperones with appropriate (i.e. relatively large) targets, the second major assumption of the chaperone-percolator model (i.e. the penetration of water molecules to the inside of the target protein) will not hold.
Conversely, if the above experiments (and perhaps others) will validate the chaperone-percolator model in case of the only Anfinsen-cage type chaperones presently known, the 60 kDa molecular chaperones, the same experiments might be used to test the folding mechanism of target proteins with other chaperone machineries, e.g. with the Hsp90-organized foldosome (Hip/Hsp70/Hop/Hsp90 complex and its other co-chaperones). If similar results will be obtained, this might suggest a transient Anfinsen-cage geometry of this larger chaperone complexes. At present this possibility is rather unlikely, but can not be fully excluded.
Conclusions
As a conclusion the present paper describes a novel molecular mechanism of chaperone action, the mobilization of the inner, hydrophobic core of the target protein by Anfinsen-cage type chaperones via multiple interactions with the outer side chains of the target and its consequent percolation by water molecules which allow easier transitions between the individual conformations of the folding protein (Fig. 1). This "chaperone-percolator" model is supported by several pieces of theoretical and experimental evidence and can be easily tested by further experiments.
Acknowledgments
The author is an International Research Scholar of the Howard Hughes Medical Institute and acknowledges the useful comments of Drs. Ken Dill (Department of Pharmaceutical Biochemistry, University of California, San Francisco CA, USA) Peter G. Wolynes (School of Chemical Sciences, University of Illinois, Urbana IL, USA), Csaba Sõti (Department of Medical Chemistry, Semmelweis University, Budapest, Hungary) and all the anonymous referees of the manuscript.
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