Cotranslational Protein Folding (1997)

The problem of how the linear amino acid sequence of a polypeptide folds to assume its unique tertiary structure is one of the most basic and challenging conundrums of contemporary science. Many of the principles and characteristics of protein folding have been learned by studying refolding of denatured polypeptides. However, the problem of protein folding cannot be completely understood without reference to the biological context of protein folding, especially for large, multidomain, and multisubunit proteins. One of the basic differences between biosynthetic protein folding and protein renaturation is cotranslational folding, folding that occurs during synthesis. The elegant idea that the process of protein folding is concomitant with synthesis was articulated, and experimental testing was begun in the early 1960s (1Zipser D. Perrin D. Cold Spring Harbor Symp. Quant. Biol. 1963; 28: 533-537Crossref Google Scholar, 2Kiho Y. Rich A. J. Mol. Biol. 1964; 51: 111-118Google Scholar). Today there is substantial experimental support for the cotranslational folding hypothesis. Both cotranslational and cotranslocational folding, at least when the latter is coupled to translation, share the basic feature of vectorial appearance of the nascent polypeptide from the ribosome or the membrane and the potential initiation of the folding process by the emerging polypeptide. It is true that the same conformations are achieved by polypeptides folded in cells as a consequence of biosynthetic processes and as a result of refolding of the full-length polypeptide from the denatured state. However, identity of the final protein structures does not necessarily mean identity of the pathways leading to their formation (3Baldwin R.L. Annu. Rev. Biochem. 1975; 44: 454-477Crossref Scopus (216) Google Scholar). It is the kinetics of the folding process that establishes the folding pathway(s) and potential partitioning among different final forms and, ultimately, their relative yields. In fact, the biological function that is shared by all proteins is the ability to fold properly, and this function must be executed efficiently by all proteins prior to any other function. This seems to be the essence of the vectorial folding process. Several general patterns and principles of cotranslational folding are summarized in Figs. 1 and2.Figure 2Schematic representation of a section through a protein folding landscape in which the basic funnel concept described by others for refolding polypeptides (67Onuchic J.N. Wolynes P.G. Luthey-Schulten Z. Socci N.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3626-3630Crossref PubMed Scopus (502) Google Scholar, 68Dill K.A. Bromberg S. Yue K. Fiebig K.M. Yee D.P. Thomas P.D. Chan H.S. Protein Sci. 1995; 4: 561-601Crossref PubMed Scopus (1340) Google Scholar) has been adapted to include the processes of cotranslational folding. The energy surface on the left depicts the hypothetical case of protein biosynthesis in the absence of folding (blue arrows). The vertical axis represents conformational energy of the polypeptide, whereas the circumference of the funnel represents the conformational space available to the polypeptide. As the polypeptide emerges from the ribosome, the available conformations will increase (the funnel becomes wider) as the length of the polypeptide increases, and as the polypeptide emerges into the aqueous environment but does not fold, it will move up the surface of the funnel to higher energies. The blue surface represents processes involving covalent bond formation and hydrolysis; the overall process of biosynthesis and folding constitutes movement from left to right. The green surface represents noncovalent interactions associated with protein folding. When the full-length but still unfolded polypeptide is released from the ribosome, it will be free to fold to the native state through the pathways defined by the folding funnel on the right (green arrows). The more realistic model of cotranslational folding is viewed as a tunneling process whereby the nascent polypeptide folds through a series of intermediates as it emerges from the ribosome, thereby retaining a lower energy than would be the case for synthesis without folding. The nascent polypeptide at each stage of biosynthesis will be able to access multiple conformations thus defining a folding funnel similar to that of the full-length polypeptide on the right; we have simplified the figure by showing the most highly populated species at each step of synthesis in the form of a tunnel. When sufficient polypeptide has emerged to begin to assume some structure, we envision the biosynthesis/folding process as leaving the blue funneland "tunneling" to the folding (green) funnel. The intermediates I 1, I 2, and I 3 are as defined in the legend to Fig. 1. The position on the biosynthesis funnel at which the tunnel begins reflects the length of nascent polypeptide required to stabilize a subset of conformational states; the different tunnels were included to indicate that some polypeptides may require longer N-terminal sequences before any structures become stabilized. The full-length nascent polypeptide,M* folds to native monomer, M n, following release by packing of the C-terminal segment of polypeptide and final isomerization steps. Note that the cotranslational folding pathway maintains a lower barrier than would occur with synthesis in the absence of folding and therefore would be expected to occur faster. It also appears that cotranslational folding would allow the polypeptide to avoid kinetic traps that may be encountered during refolding of full-length polypeptides.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Early Stages of Folding—Early stages of protein folding proceed quickly; secondary structure formation and compaction require much less then 1 s (4Roder H. Colón W. Curr. Opin. Struct. Biol. 1997; 7: 15-28Crossref PubMed Scopus (298) Google Scholar). Formation of compact globular intermediates usually requires no more then a few seconds (5Ptitsyn O.B. Adv. Protein Chem. 1995; 47: 83-229Crossref PubMed Google Scholar). Since polypeptide synthesis requires many seconds (50–300 residues/min for cell-free systems and somewhat faster in vivo; see Ref.6Fedorov A.N. Baldwin T.O. Methods Enzymol. 1998; 290 (in press)PubMed Google Scholar and references therein), compact intermediates must be formed in the process of synthesis. Stereochemical analysis suggests that the nascent polypeptide emerges from the peptidyltransferase center in an α-helical configuration (7Lim V.I. Spirin A.S. J. Mol. Biol. 1986; 188: 565-574Crossref PubMed Scopus (73) Google Scholar). Studies of disulfide bond formation in nascent polypeptides have provided an informative probe of the folding process, since formation of disulfide bonds reflects acquisition of certain tertiary interactions by the polypeptide. Immunoglobin light chains are two-domain polypeptides with two intramolecular disulfide bonds, one in the N-terminal domain and the other in the C-terminal domain. Nascent light chain polypeptides fold in the lumen of the endoplasmic reticulum. The disulfide bond between Cys-35 and Cys-100 of the N-terminal domain starts to form when the nascent chains achieve 15.5 kDa length (8Bergman L.W. Kuehl W.M. J. Biol. Chem. 1979; 254: 8869-8876Abstract Full Text PDF PubMed Google Scholar). Formation of this bond is almost quantitative when the nascent polypeptide has achieved a length of 18 kDa; formation of the disulfide thus requires ∼3 s. It has been shown with a conformation-dependent antibody that Escherichia coli tryptophan synthase β chains begin to fold during translation, even before appearance of the entire N-terminal domain (9Fedorov A.N. Friguet B. Djavadi-Ohaniance L. Alakhov Yu.B. Goldberg M.E. J. Mol. Biol. 1992; 228: 351-358Crossref PubMed Scopus (58) Google Scholar, 10Friguet B. Fedorov A.N. Serganov A. Navon A. Goldberg M.E. Anal. Biochem. 1993; 210: 344-350Crossref PubMed Scopus (10) Google Scholar). No lag was detected between synthesis of the nascent chains and appearance of immunoreactivity (11Tokatlidis K. Friguet B. Deville-Bonne D. Baleux F. Fedorov A.N. Navon A. Djavadi-Ohaniance L. Goldberg M.E. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1995; 348: 89-95Crossref PubMed Scopus (14) Google Scholar). Monoclonal antibody recognizing the structured monomer of bacteriophage P22 tailspike protein reacts with nascent chains (12Friguet B. Djavadi-Ohaniance L. King J. Goldberg M.E. J. Biol. Chem. 1994; 269: 15945-15949Abstract Full Text PDF PubMed Google Scholar). Ribosome-bound firefly luciferase and bovine rhodanese form protease-resistant N-terminal domains (13Frydman J. Nimmesgern E. Ohtsuka K. Hartl F.U. Nature. 1994; 370: 111-117Crossref PubMed Scopus (567) Google Scholar, 14Reid B.G. Flynn G.C. J. Biol. Chem. 1996; 271: 7212-7217Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Folding of ribosome-bound rhodanese and of ricin has been observed through the use of fluorescent probes (15Kudlicki W. Odom O.M. Kramer G. Hardesty B. J. Mol. Biol. 1994; 244: 319-331Crossref PubMed Scopus (52) Google Scholar,16Kudlicki W. Kitaoka Y. Odom O.W. Kramer G. Hardesty B. J. Mol. Biol. 1995; 252: 203-212Crossref PubMed Scopus (24) Google Scholar). Binding of Cofactors and Ligands—Binding of cofactors and ligands often stabilizes protein structure and can affect folding pathways. For the chloroplast reaction center protein D1, binding of several cofactors has been found to occur during synthesis and translocation into the thylakoid membrane (17Kim J. Klein P.G. Mullet J.E. J. Biol. Chem. 1991; 266: 14931-14938Abstract Full Text PDF PubMed Google Scholar). Cotranslational binding of chlorophyll is required to synthesize the full-length protein and prevent degradation of the nascent chains. Glycosylation of influenza hemagglutinin occurs in the lumen of the endoplasmic reticulum (18Chen W. Helenius J. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6229-6233Crossref PubMed Scopus (221) Google Scholar). Upon blockage of oligosaccharide addition, folding of the protein is perturbed, leading to the formation of aggregates. Binding of heme to rabbit α-globin begins when the emerging polypeptide achieves a length of 86 residues (19Komar A.A. Kommer A. Krasheninnikov I.A. Spirin A.S. J. Biol. Chem. 1997; 272: 10646-10651Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Attachment of ligands and cofactors in all the above cases can occur immediately upon or very soon after appearance of the binding sites along the polypeptide chain, thereby stabilizing the tertiary structure of the nascent polypeptide. Later Stages of the Folding Process and Formation of Oligomeric Structures—Rat serum albumin is a secretory protein with 17 disulfide bonds in the native structure which are spread throughout the polypeptide chain. In the nascent polypeptides, about one-half of the cysteinyl residues exist in disulfide bonds, indicating completion of a substantial part of the overall folding process (20Peters T. Davidson L.K. J. Biol. Chem. 1982; 257: 8847-8853Abstract Full Text PDF PubMed Google Scholar). Hemagglutinin-neuraminidase of Newcastle disease virus begins to assume defined structure during the process of synthesis (21McGinnes L.W. Morrison T.G. Virology. 1996; 224: 465-476Crossref PubMed Scopus (8) Google Scholar). Nascent influenza hemagglutinin also forms disulfide bonds cotranslationally, including the critically important 52–277 bond (18Chen W. Helenius J. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6229-6233Crossref PubMed Scopus (221) Google Scholar). Two recent studies have demonstrated formation of enzymatically active forms of rhodanese and firefly luciferase still bound to the ribosomes when these polypeptides are expressed with extended C-terminal segments so that each enzyme was in the bulk solution (22Kudlicki W. Chirgwin J. Kramer G. Hardesty B. Biochemistry. 1995; 34: 14284-14287Crossref PubMed Scopus (54) Google Scholar, 23Makeyev E.V. Kolb V.A. Spirin A.S. FEBS Lett. 1996; 378: 166-170Crossref PubMed Scopus (75) Google Scholar). Polyribosomes from Chironomus salivary gland cells produce giant secretory proteins having compact domain-like structures (24Kiseleva E.V. FEBS Lett. 1989; 257: 251-253Crossref PubMed Scopus (15) Google Scholar). Formation of oligomeric structures involving nascent polypeptides has been reported for several proteins. Formation of the β-galactosidase oligomer from nascent polypeptides was suggested in the pioneering studies of cotranslational folding. Ribosome-bound β-galactosidase chains can complement functionally defective subunits and produce ribosome-bound enzymatically active forms upon coexpression in heterozygous strains of E. coli or by mixing subunits in vitro (1Zipser D. Perrin D. Cold Spring Harbor Symp. Quant. Biol. 1963; 28: 533-537Crossref Google Scholar). Formation of enzymatically active β-galactosidase on ribosomes also was observed following enzyme induction in vivo (2Kiho Y. Rich A. J. Mol. Biol. 1964; 51: 111-118Google Scholar). The modular organization of the monomer and independent folding of each domain provides an explanation for how this large tetrameric complex could be formed with one monomer not yet completely synthesized (25Jacobson R.H. Zhang X.J. DuBose R.F. Matthews B.W. Nature. 1994; 369: 761-766Crossref PubMed Scopus (551) Google Scholar). The authors suggested the possibility of formation of a dimeric complex between nascent polypeptides attached to neighboring ribosomes and then, by a similar mechanism, formation of the tetramer (2Kiho Y. Rich A. J. Mol. Biol. 1964; 51: 111-118Google Scholar). Cotranslational trimerization of the reovirus cell attachment protein via the N-terminal domain has been observed, possibly reflecting trimerization of nascent chains synthesized from adjacent ribosomes in the same polyribosomal complex (26Leone G. Coffey M.C. Gilmore R. Duncan R. Maybaum L. Lee P.W. J. Biol. Chem. 1996; 271: 8466-8471Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The human protein hexabrachion, a hexamer composed of 320-kDa subunits, achieves its folded form upon secretion so efficiently that no intermediate forms involving full-length subunits could be detected in vivo (27Redick S.D. Schwarzbauer J.E. J. Cell Sci. 1995; 108: 1761-1769PubMed Google Scholar). Nascent polypeptides of several eucaryotic cytoskeletal proteins have been shown to assemble into the corresponding polymeric cytoskeletal structures (28Fulton A.B. L'Ecuyer T. J. Cell Sci. 1993; 105: 867-871PubMed Google Scholar). Formation of the initial complex between immunoglobulin heavy and light chains involves disulfide bond formation between fully synthesized light chains and nascent heavy chains (29Bergman L.W. Kuehl W.M. J. Biol. Chem. 1979; 254: 5690-5694Abstract Full Text PDF PubMed Google Scholar). The Cys residue from the heavy chain that is involved in the disulfide bond is located between two domains, each of which contains a single intradomain disulfide bridge. It appears that formation of this disulfide bridge requires prior folding of two adjacent domains. An intriguing case of cotranslational assembly involves formation of type I procollagen trimer. Association of full-length chains is initiated by interactions between C-terminal propeptides (30Bruckner P. Eikenberry E.F. Prockop D.J. Eur. J. Biochem. 1981; 118: 607-613Crossref PubMed Scopus (68) Google Scholar). The triple helix propagates from the C-terminal propeptide to the N-terminal end. In vivo, a substantial portion of nascent collagen is full-length (31Kirk T.Z. Evans J.S. Veis A. J. Biol. Chem. 1987; 262: 5540-5545Abstract Full Text PDF PubMed Google Scholar) as a consequence of a pause in translation prior to termination. These fully elongated chains can through their propeptides to helix from the to the similar to that for the full-length on the of the collagen to it has been suggested that the initial stages of formation can occur with the nascent collagen chains A. T.Z. J. 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Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, W. Odom O.M. Kramer G. Hardesty B. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). of the ribosome-bound full-length polypeptide on the ribosome to is not by the release requires with at W. Odom O.M. Kramer G. Hardesty B. J. 1995; PubMed Google Scholar). of the potential of in cotranslational folding have of the in between interactions of the with nascent polypeptides from interactions with polypeptides immediately after release into the bulk of the C-terminal segment of amino acid which are the ribosome during can the folding of the released polypeptide as as its interactions with is required to produce rhodanese in an enzymatically active form during synthesis W. Odom O.M. Kramer G. Hardesty B. J. 1995; PubMed Google Scholar). have been for a of the for synthesis of firefly luciferase and of (13Frydman J. Nimmesgern E. Ohtsuka K. Hartl F.U. Nature. 1994; 370: 111-117Crossref PubMed Scopus (567) Google Scholar, J. Hartl F.U. 1996; 272: PubMed Scopus Google Scholar). these a of for folding of these polypeptides, not of the occurs during synthesis or after release of the polypeptide from the The C-terminal sequences of firefly luciferase and are for binding of the polypeptides to (13Frydman J. Nimmesgern E. Ohtsuka K. Hartl F.U. Nature. 1994; 370: 111-117Crossref PubMed Scopus (567) Google Scholar, J. Hartl F.U. 1996; 272: PubMed Scopus Google Scholar). is a of J. 1996; PubMed Scopus Google Scholar) and 1 (13Frydman J. Nimmesgern E. Ohtsuka K. Hartl F.U. Nature. 1994; 370: 111-117Crossref PubMed Scopus (567) Google nascent polypeptides in cell-free systems even of the by these large would be sufficient to a substantial of the nascent polypeptides upon release from the of from E. coli A. M.E. A. Mol. 1994; PubMed Scopus Google Scholar) as as nascent rhodanese chains in vitro B.G. Flynn G.C. J. Biol. 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In this it be that rhodanese and firefly luciferase are proteins that fold in an environment different from that of the and not without the of It would be to the of found for folding of these proteins to all proteins. other have been in binding nascent chains of of proteins. can nascent polypeptides of E. coli secretory folding in the Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). a in the endoplasmic reticulum to some and is required for their folding and It has been found to hemagglutinin nascent chains via attached to the polypeptide (18Chen W. Helenius J. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6229-6233Crossref PubMed Scopus (221) Google Scholar). from the same with nascent procollagen T. K. L. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). of to synthesis of collagen and in its folding. 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The of ribosomes on protein refolding has been to the large to its the and of and eucaryotic S. B. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, W. A. Kramer G. Hardesty B. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). which is involved in the peptidyltransferase center on the large has been in the of the on protein These the ribosomes can an active in biosynthetic protein folding. An of the of cotranslational folding is by the of polypeptide synthesis. For many as the C-terminal segment of amino acid which is by the ribosome prior to the release of the full-length polypeptide into the bulk is for formation of the active folding cannot be before release of the nascent polypeptide from the The kinetics of folding of the polypeptide and appearance of the native form will be a function of the of polypeptide folding of the full-length monomer, and for oligomeric of the of protein can the step of the process and, the kinetics of the overall process. For oligomeric the of synthesized is critically important the reaction is a higher process. Cotranslational folding of the luciferase β is in the formation of the native when is available at a A.N. Baldwin T.O. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar). of subunits to much formation of the native becomes the step A. A.A. R. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). For many proteins for which folding have been observed with nascent cotranslational processes may to the of biosynthetic folding. The of biosynthetic folding cannot be achieved upon renaturation of denatured full-length polypeptides in the of and folding The of in the of folding of ribosome-bound chains has been the possibility that folding occur during the required for analysis of the However, in several folding which occur in vivo, or in vitro prior to have been folding seems to be much faster and more than renaturation for several proteins L.W. Kuehl W.M. J. 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These a for vectorial folding of nascent chains. The of biosynthetic folding relative to renaturation has been for luciferase A.N. Baldwin T.O. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google a protein that contains no disulfide of residues is not in its folding, at least in the refolding of full-length Association of with β the overall of enzyme The β released from the ribosome with the much faster than does which in refolding that the structure of the β when it is released from the ribosome is different from in refolding all begin to at the same upon into native in the there is a of appearance of synthesized polypeptides, which analysis of the A.N. Baldwin T.O. Methods Enzymol. 1998; 290 (in press)PubMed Google Scholar, A.N. Baldwin T.O. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar). It was that the β by biosynthetic folding is a folding intermediate which is a step encountered during refolding of the A.N. Baldwin T.O. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar). The of vectorial folding to the kinetics of polypeptide synthesis has been proteins. It has been that some polypeptides can fold and native However, as the basic that can be by cotranslational folding are kinetics and of the folding process. analysis of the kinetics of refolding of of and domain that the folding of these sequences is and the is lower than that of the Biochemistry. 1996; PubMed Scopus Google Scholar, L. J. Mol. Biol. 1995; PubMed Scopus Google Scholar). For proteins with the and on the of and C-terminal secondary without of the folded of the protein in the into that the final step in folding may binding of the to the surface of the folded Baldwin T.O. J. Mol. Biol. 1996; 257: PubMed Scopus Google Scholar). analysis of more than protein structures has the the length of polypeptide for a residues to with the N-terminal portion of the polypeptide and that the N-terminal on more compact than the C-terminal Protein Sci. 1993; PubMed Scopus (24) Google Scholar). This is with vectorial folding of nascent polypeptides from the and to the In this we have to that cotranslational folding is an of biosynthetic folding of many proteins in This stage of folding may be for the overall kinetics and of folding. Cotranslational folding appears to be especially important for large and multisubunit proteins. all proteins in the into this The of studies of cotranslational protein folding is to the of the pathways The kinetics of the folding process, the partitioning of polypeptides among and the of folded protein are of kinetic partitioning between pathways. The basic differences between cotranslational folding and the refolding of the full-length polypeptide vectorial appearance of the nascent polypeptide and vectorial folding which the potential for interactions and folding by the folded N-terminal segment of a polypeptide which occur with the synthesis of the C-terminal segment of the and attachment of the nascent chain to the large which the potential of the nascent formation of disulfide bonds and which may be more efficiently prior to formation of intermediates in which the Cys and residues are not for the and