Assisted Protein Folding

The pioneering work of the late Christian Anfinsen and his colleagues (1Anfinsen C.B. Haber E. J. Biol. Chem. 1961; 236: 1361-1363Abstract Full Text PDF PubMed Google Scholar) on the reoxidation of bovine pancreatic ribonuclease (RNase) to a native, biologically active enzyme in vitro after reduction of disulfide bridges and disruption of tertiary structure demonstrated that regeneration of native conformation of a purified protein can occur spontaneously in a test tube without the addition of any other co-factors or helper enzymes. This led to the still valid conclusion that “no special genetic information, beyond that contained in the amino acid sequence, is required for the proper folding of the molecule and for the formation of 'correct’ disulfide bonds” (2Goldberger R.F. Epstein C.J. Anfinsen C.B. J. Biol. Chem. 1963; 238: 628-635Abstract Full Text PDF PubMed Google Scholar). Of course, the story of protein folding goes back much further (reviewed in Ref. 3Dill K.A. Biochemistry. 1990; 29: 7133-7155Crossref PubMed Scopus (3345) Google Scholar). A number of milestones can be noted. In 1911, Chick and Martin found that proteins could be denatured in vitro, and they distinguished that process from aggregation of the protein. In 1929, Wu postulated that protein denaturation was an unfolding process and that native protein structures involved regular, repeated patterns of folding into a three-dimensional network. Anson and Mirsky in 1931 and Anson (1945) showed that hemoglobin folding is reversible and that hemoglobin could be renatured in vitro to a form that had a native-like absorption spectrum, oxygen binding, and tryptic digestion pattern. Studies in the 1950s by Eisenberg and Schwert and by Schellman demonstrated that denaturation and renaturation are thermodynamic processes, involving a change in free energy and large changes in conformation between the denatured and native states. Even the early investigators realized that the protein folding processes that occurred in test tubes, although they could reconstitute native structure, were too slow to work inside cells. For example, even under optimized conditions of protein dilution, pH, and temperature, renaturation of RNase takes about 20 min (2Goldberger R.F. Epstein C.J. Anfinsen C.B. J. Biol. Chem. 1963; 238: 628-635Abstract Full Text PDF PubMed Google Scholar), and RNase is a relatively simple monomeric protein. Renaturation of some multidomain proteins may take several hours in vitro, yet it is clear that all possible conformations could not be sampled on the way to native structure. Levinthal (4Levinthal C. J. Chem. Phys. 1968; 65: 44-45Crossref Google Scholar) summed this up succinctly in the “Levinthal paradox” that can be stated as follows: if a given amino acid can assume approximately 10 different conformations, the total number of possible conformations in a polypeptide chain of 100 residues would be 10100. The time that this could take would be well beyond the life span of an organism if not of the universe, depending on how many conformations could be sampled before a protein reaches native state. Thus, it was realized early on that cells must have special ways to make the process more efficient. Experiments to examine the role of the intracellular environment in protein folding involved the renaturation of proteins such as RNase (2Goldberger R.F. Epstein C.J. Anfinsen C.B. J. Biol. Chem. 1963; 238: 628-635Abstract Full Text PDF PubMed Google Scholar), bovine pancreatic trypsin inhibitor (BPTI) 1The abbreviations used are: BPTIbovine pancreatic trypsin inhibitorERendoplasmic reticulumhCGhuman chorionic gonadotropinPDIprotein disulfide isomeraseHAhemagglutinin. (5Zapun A. Creighton T.E. Rowling P.J.E. Freedman R.B. Proteins Struct. Funct. Genet. 1992; 14: 10-15Crossref PubMed Scopus (52) Google Scholar), or influenza hemagglutinin (6Marquardt T. Hebert D.N. Helenius A. J. Biol. Chem. 1993; 268: 19618-19625Abstract Full Text PDF PubMed Google Scholar) in isolated microsomal fractions. The results indicated that protein folding can be facilitated by proteins contained in the endoplasmic reticulum (ER) of eukaryotic cells. In the case of disulfide bond-containing proteins such as BPTI (5Zapun A. Creighton T.E. Rowling P.J.E. Freedman R.B. Proteins Struct. Funct. Genet. 1992; 14: 10-15Crossref PubMed Scopus (52) Google Scholar) or the human chorionic gonadotropin (hCG)-β subunit (7Huth J.R. Perini F. Lockridge O. Bedows E. Ruddon R.W. J. Biol. Chem. 1993; 268: 16472-16482Abstract Full Text PDF PubMed Google Scholar), the key ER protein involved appears to be protein disulfide isomerase (see below). bovine pancreatic trypsin inhibitor endoplasmic reticulum human chorionic gonadotropin protein disulfide isomerase hemagglutinin. It was soon realized that many polypeptides can reform native structure easily by themselves in vitro (usually small single domain proteins) while others (more complex, multidomain, or oligomeric proteins) fold and assemble efficiently only in the presence of additional proteins that are not constituents of the final native protein itself. These additional proteins have been called “molecular chaperones.” The term molecular chaperone was first used by Laskey et al. (8Laskey R.A. Honda B.M. Mills A.D. Finch J.T. Nature. 1978; 275: 416-420Crossref PubMed Scopus (539) Google Scholar) to describe the role of nucleoplasmin in the assembly of DNA and histones into nucleosomes. The name seemed appropriate because nucleoplasmin promotes histone-histone interactions to form the correct oligomeric form while preventing aggregation. It does so without itself forming part of the nucleosome and without specifying nucleosome structure. Hence nucleoplasmin assumes the role of a chaperone. The term molecular chaperone has been applied by Ellis and Hemmingsen (9Ellis R.J. Hemmingsen S.M. Trends Biochem. Sci. 1989; 14: 339-342Abstract Full Text PDF PubMed Scopus (368) Google Scholar) to the expanding families of proteins of bacterial and eukaryotic compartments involved in protein folding, assembly, and translocation. The term has stuck, and it is now used to define a wide variety of factors that facilitate generation of native protein and nucleic acid structures. There are some similarities as well as differences between intracellular protein folding and protein folding in test tubes. For instance, for the tailspike protein of Salmonella typhimurium phage P22 (10Fuchs A. Seiderer C. Seckler R. Biochemistry. 1991; 30: 6598-6604Crossref PubMed Scopus (79) Google Scholar, 11Mitraki A. Fane B. Haase-Pettingell C. Sturtevant J. King J. Science. 1991; 253: 54-58Crossref PubMed Scopus (172) Google Scholar) and hCG-β subunit (7Huth J.R. Perini F. Lockridge O. Bedows E. Ruddon R.W. J. Biol. Chem. 1993; 268: 16472-16482Abstract Full Text PDF PubMed Google Scholar) intermediates in the folding pathway of the proteins appear to be the same in vivo and in vitro, but the rate and efficiency with which proteins achieve final native state in vivo is higher than that in vitro It must also be kept in mind that, both in vivo and in vitro, correct folding is in competition with misfolding and aggregation. This depends on the protein concentration used for in vitro folding reactions, and in general, very dilute protein concentrations (0.01-0.02 mg/ml) (2Goldberger R.F. Epstein C.J. Anfinsen C.B. J. Biol. Chem. 1963; 238: 628-635Abstract Full Text PDF PubMed Google Scholar, 12Huth J.R. Norton S.E. Lockridge O. Shikone T. Hsueh A.J.W. Ruddon R.W. Endocrinology. 1994; 135: 911-918Crossref PubMed Scopus (30) Google Scholar) are needed to prevent aggregation. This has presented a huge problem to the biotechnology industry in attempts to produce useful amounts of recombinant proteins. The efficiency of folding in vitro can frequently be facilitated by appropriate adjustment of the redox potential (13Lyles M.M. Gilbert H.F. Biochemistry. 1991; 30: 613-619Crossref PubMed Scopus (351) Google Scholar, 14Gilbert H.F. Adv. Enzymol. Relat. Areas Mol. Biol. 1990; 63: 69-172PubMed Google Scholar, 15Huth J.R. Feng W. Ruddon R.W. Biotechnol. Bioeng. 1994; 44: 66-72Crossref PubMed Scopus (14) Google Scholar) or the addition of factors such as protein disulfide isomerase (PDI) for eukaryotic disulfide-bonded proteins (15Huth J.R. Feng W. Ruddon R.W. Biotechnol. Bioeng. 1994; 44: 66-72Crossref PubMed Scopus (14) Google Scholar, 16Creighton T.E. Hillson D.A. Freedman R.B. J. Mol. Biol. 1980; 142: 43-62Crossref PubMed Scopus (145) Google Scholar, 17Weissman J.S. Kim P.S. Nature. 1993; 365: 185-188Crossref PubMed Scopus (187) Google Scholar) or DnaK/DnaJ chaperones for bacterial proteins (reviewed in Refs. 18Wall J.G. Plückthun A. Curr. Opin. Biotechnol. 1995; 6: 507-516Crossref PubMed Scopus (81) Google Scholar and 19Hartl F.U. Nature. 1996; 381: 571-580Crossref PubMed Scopus (3121) Google Scholar). In contrast to what happens in vitro, cells minimize or circumvent the off-pathway events by utilizing molecular chaperones that facilitate the folding process by preventing aggregation and other unfavorable interactions. There is growing interest in what regulates the folding of mammalian proteins in vivo because of the number of human diseases now known to be related to protein folding defects (reviewed in Refs. 20Thomas P.J. Qu B.-H. Pedersen P.L. Trends Biochem. Sci. 1995; 20: 456-459Abstract Full Text PDF PubMed Scopus (484) Google Scholar and 21Ruddon R.W. Sherman S.A. Bedows E. Protein Sci. 1996; 5: 1443-1452Crossref PubMed Scopus (70) Google Scholar). This includes cystic fibrosis, α1-antitrypsin deficiency, Alzheimer's disease, Creutzfeld-Jacob disease, neurodegenerative diseases such as Huntington's chorea, and cancer. The intracellular folding pathway of only a few proteins has been studied in detail. These include the S. typhimurium phage P22 tailspike protein (11Mitraki A. Fane B. Haase-Pettingell C. Sturtevant J. King J. Science. 1991; 253: 54-58Crossref PubMed Scopus (172) Google Scholar), hCG-β subunit (22Ruddon R.W. Krzesicki R.F. Norton S.E. Saccuzzo-Beebe J. Peters B.P. Perini F. J. Biol. Chem. 1987; 262: 12533-12540Abstract Full Text PDF PubMed Google Scholar, 23Saccuzzo-Beebe J. Mountjoy K. Krzesicki R.F. Perini F. Ruddon R.W. J. Biol. Chem. 1990; 265: 312-317Abstract Full Text PDF PubMed Google Scholar, 24Huth J.R. Mountjoy K. Perini F. Ruddon R.W. J. Biol. Chem. 1992; 267: 8870-8879Abstract Full Text PDF PubMed Google Scholar, 25Bedows E. Huth J.R. Ruddon R.W. J. Biol. Chem. 1992; 267: 8880-8886Abstract Full Text PDF PubMed Google Scholar), luciferase (26Waddle J.J. Johnston T.C. Baldwin T.O. Biochemistry. 1987; 26: 4917-4921Crossref PubMed Scopus (46) Google Scholar), influenza hemagglutinin (27Braakman I. Hoover-Litty H. Wagner K.R. Helenius A. J. Cell Biol. 1991; 114: 401-411Crossref PubMed Scopus (252) Google Scholar, 28Chen W. Helenius J. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6229-6233Crossref PubMed Scopus (221) Google Scholar), and the HIV type 1 envelope glycoprotein (29Otteken A. Earl P.L. Moss B. J. Virol. 1996; 70: 3407-3415Crossref PubMed Google Scholar). Where the pathways have been determined both in vitro and in vivo, for example for the phage P22 tailspike protein (10Fuchs A. Seiderer C. Seckler R. Biochemistry. 1991; 30: 6598-6604Crossref PubMed Scopus (79) Google Scholar, 11Mitraki A. Fane B. Haase-Pettingell C. Sturtevant J. King J. Science. 1991; 253: 54-58Crossref PubMed Scopus (172) Google Scholar) and the hCG-β subunit (7Huth J.R. Perini F. Lockridge O. Bedows E. Ruddon R.W. J. Biol. Chem. 1993; 268: 16472-16482Abstract Full Text PDF PubMed Google Scholar, 22Ruddon R.W. Krzesicki R.F. Norton S.E. Saccuzzo-Beebe J. Peters B.P. Perini F. J. Biol. Chem. 1987; 262: 12533-12540Abstract Full Text PDF PubMed Google Scholar, 23Saccuzzo-Beebe J. Mountjoy K. Krzesicki R.F. Perini F. Ruddon R.W. J. Biol. Chem. 1990; 265: 312-317Abstract Full Text PDF PubMed Google Scholar, 24Huth J.R. Mountjoy K. Perini F. Ruddon R.W. J. Biol. Chem. 1992; 267: 8870-8879Abstract Full Text PDF PubMed Google Scholar, 25Bedows E. Huth J.R. Ruddon R.W. J. Biol. Chem. 1992; 267: 8880-8886Abstract Full Text PDF PubMed Google Scholar), the in vitro and in vivo folding pathways proceed through the same respective folding intermediates. A diagram of the folding pathway of the hCG-β subunit is shown in Fig. 1. Many of the eukaryotic proteins whose folding and assembly have been studied in vivo are membrane or secreted proteins. They follow a similar route to the cell surface. (i) Synthesis is carried out in the rough ER. (ii) Nascent proteins are translocated into the cisternal space of the ER where the signal peptide is cleaved; initial co-translational folding involving secondary structure and some native tertiary structure occurs; addition of high mannose N-linked oligosaccharides and initial processing of N-linked oligosaccharide chains (for glycoproteins) takes place; formation of disulfide bonds occurs, and for multimeric proteins, oligomerization or subunit assembly is attained along with achievement of native structure. (iii) The proteins destined for the cell surface or secretion are translocated to the Golgi apparatus, further processed, and then either translocated to the cell surface or packaged into secretory vesicles for secretion. It has been clear for a long time that the in vitro folding of proteins targeted for secretion is facilitated by folding in the presence of microsomal extracts (2Goldberger R.F. Epstein C.J. Anfinsen C.B. J. Biol. Chem. 1963; 238: 628-635Abstract Full Text PDF PubMed Google Scholar). It is now known that microsomes contain many chaperones that foster protein folding (30Gething M.-J. J. Nature. 1992; PubMed Scopus Google Scholar) as well as the to a redox potential for the formation of disulfide bonds (13Lyles M.M. Gilbert H.F. Biochemistry. 1991; 30: 613-619Crossref PubMed Scopus (351) Google Scholar, C. H.F. Science. 1992; PubMed Scopus Google Scholar). For many secreted proteins, disulfide bonds are for of tertiary structure and for assembly into multimeric structures R. R. J. 1992; 6: PubMed Scopus Google Scholar). is a key disulfide For example, the rate of of BPTI folding by microsomal extracts is to the by the of extracts (5Zapun A. Creighton T.E. Rowling P.J.E. Freedman R.B. Proteins Struct. Funct. Genet. 1992; 14: 10-15Crossref PubMed Scopus (52) Google Scholar). the rate of folding of BPTI in vitro by several but has on disulfide formation of BPTI disulfide bonds that form efficiently in the of J.S. Kim P.S. Nature. 1993; 365: 185-188Crossref PubMed Scopus (187) Google Scholar). The pioneering work of Creighton and his colleagues (reviewed in Ref. T.C. J. J. 1996; PubMed Scopus Google Scholar) the formation of disulfide bonds as a to the folding pathway of This acid protein has disulfide and and folding pathway can be in vitro by the of the disulfide with acid to free Creighton et al. T.C. J. J. 1996; PubMed Scopus Google Scholar) a folding pathway involving intermediates that in and type of disulfide They also the of a of intermediates disulfide bonds not in the native the formation of which is the of disulfide There is also that disulfide protein folding in cells. For example, in the intracellular folding pathway of the hCG-β subunit determined by J.R. Mountjoy K. Perini F. 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Mountjoy K. Perini F. Bedows E. Ruddon R.W. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). many membrane and secretory proteins are it is to the role of in protein folding, assembly, and secretion. oligosaccharides of the high mannose are to the of proteins in the ER. of N-linked is to facilitate protein folding and N-linked chains are by of residues in or by of cells with that addition of N-linked or many and the ER (reviewed in Ref. A. Mol. Biol. 1994; 5: PubMed Scopus Google Scholar). to fold and be translocated efficiently without N-linked The only that to is that more have more folding if N-linked are The role of N-linked oligosaccharide chains in intracellular folding of the hCG-β subunit has been determined by the of folding in cells with or hCG-β or both of the W. M.M. Mountjoy K. Bedows E. Ruddon R.W. I. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). of hCG-β both N-linked was and with the slow formation of the disulfide bonds and to form in the hCG-β folding hCG-β was secreted from and subunit folding intermediates in cells for more than were into a hCG-β of which is required for formation of the biologically active folding and formation of disulfide bonds and of hCG-β N-linked that the presence of subunit subunit folding and assembly, because the subunit can a chaperone for subunit In the molecular chaperones and were found in a with hCG-β and may be involved in the folding of this form W. M.M. Mountjoy K. Bedows E. Ruddon R.W. I. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). These that N-linked oligosaccharides hCG-β subunit folding by disulfide by the and of the native structure that disulfide The role of molecular chaperones in protein folding, assembly, and intracellular has been the of a number of F.U. 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Nature. 1992; PubMed Scopus Google of of structures in in of structures and of to secretory of folding and of structures in of of of folding of folding membrane or of for of cell or 1 of of slow protein folding disulfide and of disulfide of and secretory before in with to native protein of chain to to for form of and form of protein to protein folding intermediates and promotes of denatured assembly in a of the ER of chaperones include as an protein the name J. Cell Biol. PubMed Scopus Google S. Sherman J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar), J. S. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar), Nature. 1993; PubMed Scopus Google Scholar), and R.A. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). chaperones to be and The protein disulfide isomerase R. J. Biol. 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Huth J.R. I. Ruddon R.W. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). are a number of where protein misfolding to protein in the ER and molecular chaperones appear to be involved in proteins for in the ER. is of For example, chains that are folding and in the ER of cells are to as disulfide-bonded and then chains that are more and secreted only with S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar). The that chaperones are needed to protein folding in cells does not the of Anfinsen and others that proteins can fold spontaneously in only on contained in amino acid the the in vitro in vivo folding pathways for proteins that have been studied in this for example the S. typhimurium phage P22 tailspike protein (10Fuchs A. Seiderer C. Seckler R. Biochemistry. 1991; 30: 6598-6604Crossref PubMed Scopus (79) Google Scholar, 11Mitraki A. Fane B. Haase-Pettingell C. Sturtevant J. King J. 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