Abstract
A hallmark of the essential cellular factors known as molecular chaperones is their ability to bind tightly to a broad spectrum of nonnative polypeptides to facilitate protein folding in vivo and in vitro under nonideal or stressful conditions (1, 16). A variety of methods have been used to assess substrate protein binding to the bacterial chaperonin GroEL qualitatively. One of the earliest and most popular approaches involves the inhibition of in vitro refolding of various model proteins, such as ribulose bis-phosphate carboxylase (4,16), citrate synthase (7, 8), rhodanese (9, 10, malate dehydrogenase (11, 13), barnase (14), and glutamine synthetase (15–17). Additional, quantitative procedures have relied on the use of size-exclusion chromatography to separate free radio- labeled proteins from those bound to GroEL (8,9, 18–20), and on the effect of nonnative proteins on the ATPase activity of GroEL (21). However, the most straightforward techniques to measure the affinity and stoichiometry of polypeptide interactions with the chaperonins accurately and precisely have involved a direct or indirect measurement of the change in the concentration of a substrate protein when it partitions to GroEL from solution. Some of these methods have relied on the heat exchanged on polypeptide binding to GroEL (22, 23), on the distribution of bound and free substrates detected by ultracen- trifugation (24, 26), and on changes in surface plasmon resonance using BIAcore (27, 29) 17). Additional, quantitative procedures have relied on the use of size-exclusion chromatography to separate free radio- labeled proteins from those bound to GroEL (8,9, 18–20), and on the effect of nonnative proteins on the ATPase activity of GroEL (21). However, the most straightforward techniques to measure the affinity and stoichiometry of polypeptide interactions with the chaperonins accurately and precisely have involved a direct or indirect measurement of the change in the concentration of a substrate protein when it partitions to GroEL from solution. Some of these methods have relied on the heat exchanged on polypeptide binding to GroEL (22, 23), on the distribution of bound and free substrates detected by ultracen- trifugation (24, 26), and on changes in surface plasmon resonance using BIAcore (27, 29)
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Van Dyk, T. K., Gatenby, A. A., and LaRossa, B. A. (1989) Demonstration by genetic suppression of interaction of GroE products with many proteins. Nature 342, 451–453.
Viitanen, P. V., Gatenby, A. A., and Lorimer, G. H. (1992) Purified chaperonin 60 (groEL) interacts with the nonnative states of a multitude of Escherichia coli proteins. Protein Sci. 1, 363–369.
Horwich, A. L., Low, K. B., Fenton, W. A., Hirschfield, I. N., and Furtak, K. (1993) Folding in vivo of bacterial cytoplasmic proteins: role of GroEL. Cell 74, 909–917.
Goloubinoff, P., Gatenby, A. A., and Lorimer, G. H. (1989) GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxy-lase oligomers in Excherichia coli. Nature 337, 44–47.
Goloubinoff, P., Christeller, J. T., Gatenby, A. A., and Lorimer, G. H. (1989) Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfolded state depends on two chaperonin proteins and MgATP. Nature 342, 884–889.
Viitanen, P. V., Lubben, T. H., Reed, J., Goloubinoff, P., O’Keefe, D. P., and Lorimer, G. H. (1990) Chaperonin-facilitated refolding of ribulosebisphosphate carboxylase and ATP hydrolysis by chaperonin 60 (groEL) Are K+dependent. Biochemistry 29, 5665–5671.
Buchner, J., Schmidt, M., Fuchs, M., Jaenicke, R., Rudolph, R., Schmid, F. X., and Keifhaber, T. (1991) GroE facilitates refolding of citrate synthase by suppressing aggregation. Biochemistry 30, 1586–1591.
Martin, J., Geromanos, S., Tempst, P., and Hartl, F. U. (1993) The reaction cycle of GroEL and GroES in chaperonin-assisted protein folding. Nature 366, 279–282.
Martin, J., Langer, T., Boteva, R., Schramel, A., Horwich, A. L., and Hartl, F. U. (1991) Chaperonin-mediated protein folding at the surface of groEL through a "e;molten globule"e;-like intermediate. Nature 352, 36–42.
Mendoza, J. A., Roger, E., Lorimer, G. H., and Horowitz, P. M. (1991) Chaperonins facilitate the in vitro folding of monomeric mitochondrial rhodanese. J. Biol. Chem. 266, 13,044–13,049.
Hartman, D. J., Surin, B. P., Dixon, N. E., Hoogenrad, N. J., and Hoj, P. B. (1993) Substoichiometric amounts of the molecular chaperones GroEL and GroES prevent thermal denaturation and aggregation of mammalian mitochondrial malate dehydrogenase in vitro. Proc. Natl. Acad. Sci. USA 90, 2276–2280, and 3775.
Peralta, D., Hartman, D. J., Hoogenraad, N. J., and Hoj, P. B. (1994) Generation of a stable folding intermediate which can be rescued by the chaperonins GroEL and GroES. FEBS Lett. 339, 45–49.
Ranson, N. A., Dunster, N. J., Burston, S. G., and Clarke, A. R. (1995) Chaperonins can catalyze the reversal of early aggregation steps when a protein misfolds. J. Mol. Biol. 250, 581–586.
Gray, T. E., and Fersht, A. R. (1993) Refolding of barnase in the presence of GroE. J. Mol. Biol. 232, 1197–1207.
Fisher, M. T. (1992) Promotion of the in vitro renaturation of dodecameric glutamine synthetase in the presence of GroEL (Chaperonin-60) and ATP. Biochemistry 31, 3955–3963.
Fisher, M. T. (1993) On the assembly of dodecameric glutamine synthetase from stable chaperonin complexes. J. Biol. Chem. 268, 13,777–13,779.
Fisher, M. T. (1994) The effect of groES on the groEL-dependent assembly of dodecameric glutamine synthetase in the presence of ATP and ADP. J. Biol. Chem. 269, 13,629–13,636.
Bochkareva, E. S., Lissin, N. M., Flynn, G. C., Rothman, J. E., and Girshovich, A. S. (1992) Positive cooperativity in the functioning of molecular chaperone GroEL. J. Biol. Chem. 267, 6796–6800.
Viitanen, P. V., Donaldson, G. K., Lorimer, G. H., Lubben, T. H., and Gatenby, A. A. (1991) Complex interactions between the chaperonin 60 molecular chaperone and dihydrofolate reductase. Biochemistry 30, 9716–9723.
Hlodan, R., Tempst, P., and Hartl, F.-U. (1995) Binding of defined regions of a polypeptide to groel and its implications for chaperonin-mediated protein folding nature. Structural Biology 2, 587–595.
Yifrach, O., and Horovitz, A. (1996) Allosteric control by atp of non-folded protein binding to GroEL. J. Mol. Biol. 255, 356–361.
Lin, Z., Schwarz, F. P., and Eisenstein, E. (1995) The hydrophobic nature of groel-substrate binding. J. Biol. Chem. 270, 1011–1014.
Aoki, K., Taguchi, H., Shindo, Y., Yoshida, M., Ogasahara, K., Yutani, K., and Tanaka, N. (1997) Calorimetric observation of a GroEL-protein binding reaction with little contribution of hydrophobic interaction. J. Biol. Chem. 272, 32,158–32,162.
Mendoza, J. A., Demeler, B., and Horowitz, P. M. (1994) Alteration of the quaternary structure of cpn60 modulates chaperonin-assisted folding. J. Biol. Chem. 269, 2447–2451.
Mendoza, J. A., and Horowitz, P. M. (1994) Bound substrate polypeptides can generally stabilize the tetradecameric structure of cpn60 and induce its reassembly from monomers. J. Biol. Chem. 269, 25,963–25,965.
Grunau, C., Dettmer, R., Behlke, J., and Bernhardt, R. (1995) Bovine adreno-doxin-a mitochondrial iron-sulphur protein-binds to chaperonin GroEL. Biochem. Biophys. Res. Commun. 210, 1001–1008.
Zahn, R., Axmann, S. E., Rucknagel, K.-P., Jaeger, E., Laminet, A. A., and Pluckthun, A. (1994) Thermodynamic partitioning model for hydrophobic binding of polypeptides by GroEL. I. GroEL recognizes the signal sequences of β-lactamase precursor. J. Mol. Biol. 242, 150–164.
Murai, N., Taguchi, H., and Yoshida, M. (1995) Kinetic analysis of interactions between GroEL and reduced a-lactalbumin. J. Biol. Chem. 270, 19,957–19,963.
Lin, Z., and Eisenstein, E. (1996) Nucleotide binding-promoted conformational changes release a nonnative polypeptide from the Escherichia coli chaperonin GroEL. Proc. Natl. Acad. Sci. USA 93, 1977–1981.
Hemmingsen, S. M., Woolford, C., van der Vies, S. M., Tilly, K., Dennis, D. T., Georgopoulos, C. P., Hendrix, R. W., and Ellis, R. J. (1988) Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature 333, 330–334.
Hayer-Hartl, M. K., Ewbank, J. J., Creighton, T. E., and Hartl, F. U. (1994) Conformational specificity of the chaperonin GroEL for the compact folding intermediates of a-lactalbumin. EMBO J. 13, 3192–3202.
Hoshino, M., Kawata, Y., and Goto, Y. (1996) Interaction of GroEL with conformational states of horse cytochrome c. J. Mol. Biol. 262, 575–587.
Corrales, F. J., and Fersht, A. R. (1995) The folding of GroEL-bound barnase as a model for chaperonin-mediated protein folding. Proc. Natl. Acad. Sci. USA 92, 5326–5330.
Katsumata, K., Okazaki, A., and Kuwajima, K. (1996) Effect of GroEL on the re-folding kinetics of a-lactalbumin. J. Mol. Biol. 258, 827–838.
Sparrer, H., Lilie, H., and Buchner, J. (1996) Dynamics of the GroEL-protein complex: effects of nucleotides and folding mutants. J. Mol. Biol. 258, 74–87.
Goldberg, M. S., Zhang, J., Sondek, S., Matthews, C. R., Fox, R. O., and Horwich, A. L. (1997) Native-like structure of a protein-folding intermediate bound to the chaperonin GroEL. Proc. Natl. Acad. Sci. USA 94, 1080–1085.
Ross, J. B. A., Szabo, A. G., and Hogue, C. W. V. (1997) Enhancement of protein spectra with tryptophan analogs: fluorescence spectroscopy of protein-protein interactions. Meth. Enzymol. 278, 151–202.
Rye, H. S., Burston, S. G., Fenton, W. A., Beecham, J. M., Xu, Z., Sigler, P. B., and Horwich, A. L. (1997) Distinct actions of cis and trans ATP within the double ring of the chaperonin GroEL. Nature 388, 792–798.
Shortle, D. (1995) Staphylococcal Nuclease: A ahowcase of m-value effects. Adv. Protein Chem. 46, 217–247.
Green, S. M., Meeker, A. K., and Shortle, D. (1992) Contributions of the polar, uncharged amino acids to the stability of staphylococcal nuclease: evidence for muta-tional effects on the free energy of the denatured state. Biochemistry 31, 5717–5728.
Ybarra, J., and Horowitz, P. (1995) Inactive GroEL monomers can be isolated and reassembled to functional tetradecamers that contain few bound polypeptides. J. Biol. Chem. 270, 22,962–22,967.
Clark, A. C., Hugo, E., and Frieden, C. (1996) Determination of regions in the dihydrofolate reductase structure that interact with the molecular chaperone GroEL. Biochemistry 35, 5893–5901.
Todd, M. J., and Lorimer, G. H. (1998) Criteria for assessing the purity and quality of GroEL. Meth. Enzymol. 290, 135–141.
Shortle, D., and Meeker, A. K. (1989) Residual structure in large fragments of ataphylococcal nuclease: effects of amino acid substitutions. Biochemistry 28, 936–944.
Gill, S. C., and von Hippel, P. H. (1989) Calculation of protein extinction coefficients from amino acid sequence data analytical. Biochemistry 182, 319–326.
Johnson, M. L., and Fraser, S. G. (1985) Nonlinear least-squares analysis. Meth. Enzymol. 117, 301–342.
Tanford, C. (1961) Physical Chemistry of Macromolecules, John Wiley and Sons, New York.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2000 Humana Press Inc.
About this protocol
Cite this protocol
Hartmann, W.K., Eisenstein, E. (2000). Interaction of Nonnative Polypeptide Substrates with the Escherichia coli Chaperonin GroEL. In: Schneider, C. (eds) Chaperonin Protocols. Methods in Molecular Biology, vol 140. Humana, Totowa, NJ. https://doi.org/10.1385/1-59259-061-6:97
Download citation
DOI: https://doi.org/10.1385/1-59259-061-6:97
Publisher Name: Humana, Totowa, NJ
Print ISBN: 978-0-89603-739-7
Online ISBN: 978-1-59259-061-2
eBook Packages: Springer Protocols