On the Maximum Size of Proteins to Stay and Fold in the Cavity of GroEL underneath GroES*

GroEL encapsulates non-native protein in a folding cage underneath GroES (cis-cavity). Here we report the maximum size of the non-native protein to stay and fold in thecis-cavity. Using total soluble proteins ofEscherichia coli in denatured state as binding substrates and protease resistance as the measure of polypeptide held in thecis-cavity, it was estimated that thecis-cavity can accommodate up to ∼57-kDa non-native proteins. To know if a protein with nearly the maximum size can complete folding in the cis-cavity, we made a 54-kDa protein in which green fluorescent protein (GFP) and its blue fluorescent variant were fused tandem. This fusion protein was captured in the cis-cavity, and folding occurred there. Fluorescence resonance energy transfer proved that both GFP and blue fluorescent protein moieties of the same fused protein were able to fold into native structures in the cis-cavity. Consistently, simulated packing of crystal structures shows that two native GFPs just fit in the cis-cavity. A fusion protein of three GFPs (82 kDa) was also attempted, but, as expected, it was not captured in thecis-cavity.

[1]  J. Rothman,et al.  Positive cooperativity in the functioning of molecular chaperone GroEL. , 1992, The Journal of biological chemistry.

[2]  M. Chalfie,et al.  Green fluorescent protein as a marker for gene expression. , 1994, Science.

[3]  F. Hartl.  Molecular chaperones in cellular protein folding , 1996, Nature.

[4]  J. Weissman,et al.  Mechanism of GroEL action: Productive release of polypeptide from a sequestered position under groes , 1995, Cell.

[5]  F. Hartl,et al.  Mechanism of chaperonin action: GroES binding and release can drive GroEL‐mediated protein folding in the absence of ATP hydrolysis. , 1996, The EMBO journal.

[6]  Thomas A. Kunkel,et al.  Rapid and efficient site-specific mutagenesis without phenotypic selection. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Bernd Bukau,et al.  The Hsp70 and Hsp60 Chaperone Machines , 1998, Cell.

[8]  G. Lorimer,et al.  Purified chaperonin 60 (groEL) interacts with the nonnative states of a multitude of Escherichia coli proteins , 1992, Protein science : a publication of the Protein Society.

[9]  R. Tsien,et al.  Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer , 1996, Current Biology.

[10]  S. Kain,et al.  Deletions of the Aequorea victoria Green Fluorescent Protein Define the Minimal Domain Required for Fluorescence* , 1997, The Journal of Biological Chemistry.

[11]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[12]  P. Jenö,et al.  Identification of in vivo substrates of the yeast mitochondrial chaperonins reveals overlapping but non‐identical requirement for hsp60 and hsp10 , 1998, The EMBO journal.

[13]  G. Lorimer,et al.  Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfolded state depends on two chaperonin proteins and Mg-ATP , 1989, Nature.

[14]  Neil A. Ranson,et al.  Location of a folding protein and shape changes in GroEL–GroES complexes imaged by cryo-electron microscopy , 1994, Nature.

[15]  Zbyszek Otwinowski,et al.  The crystal structure of the bacterial chaperonln GroEL at 2.8 Å , 1994, Nature.

[16]  Walid A Houry,et al.  In Vivo Observation of Polypeptide Flux through the Bacterial Chaperonin System , 1997, Cell.

[17]  F. Hartl,et al.  Chaperonin-mediated protein folding at the surface of groEL through a 'molten globule'-like intermediate , 1991, Nature.

[18]  F. Hartl,et al.  Protein folding in the central cavity of the GroEL–GroES chaperonin complex , 1996, Nature.

[19]  G. Phillips,et al.  The molecular structure of green fluorescent protein , 1996, Nature Biotechnology.

[20]  H. Taguchi,et al.  Chaperonin-mediated Folding of Green Fluorescent Protein* , 1997, The Journal of Biological Chemistry.

[21]  W. Stemmer,et al.  Improved Green Fluorescent Protein by Molecular Evolution Using DNA Shuffling , 1996, Nature Biotechnology.

[22]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[23]  W. Baumeister,et al.  Chaperonin‐mediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity. , 1992, The EMBO journal.

[24]  A. Horwich,et al.  Distinct actions of cis and trans ATP within the double ring of the chaperonin GroEL , 1997, Nature.

[25]  A. Horwich,et al.  Structure and function in GroEL-mediated protein folding. , 1998, Annual review of biochemistry.

[26]  G. Lorimer,et al.  Dynamics of the chaperonin ATPase cycle: implications for facilitated protein folding. , 1994, Science.

[27]  J. King,et al.  Selective in vivo rescue by GroEL/ES of thermolabile folding intermediates to phage P22 structural proteins. , 1994, The Journal of biological chemistry.

[28]  J. Deisenhofer,et al.  Structural Adaptations in the Specialized Bacteriophage T4 Co-Chaperonin Gp31 Expand the Size of the Anfinsen Cage , 1997, Cell.

[29]  J. Deisenhofer,et al.  The crystal structure of the GroES co-chaperonin at 2.8 Å resolution , 1996, Nature.

[30]  T. Langer,et al.  The reaction cycle of GroEL and GroES in chaperonin-assisted protein folding , 1993, Nature.

[31]  Helen R Saibil,et al.  The Chaperonin ATPase Cycle: Mechanism of Allosteric Switching and Movements of Substrate-Binding Domains in GroEL , 1996, Cell.

[32]  F. Hartl,et al.  Recombination of protein domains facilitated by co-translational folding in eukaryotes , 1997, Nature.

[33]  J. Weissman,et al.  Characterization of the Active Intermediate of a GroEL–GroES-Mediated Protein Folding Reaction , 1996, Cell.

[34]  A. Horwich,et al.  The crystal structure of the asymmetric GroEL–GroES–(ADP)7 chaperonin complex , 1997, Nature.