The Cotranslational Contacts between Ribosome-bound Nascent Polypeptides and the Subunits of the Hetero-oligomeric Chaperonin TRiC Probed by Photocross-linking

The hetero-oligomeric eukaryotic chaperonin TRiC (TCP-1-ring complex, also called CCT) interacts cotranslationally with a diverse subset of newly synthesized proteins, including actin, tubulin, and luciferase, and facilitates their correct folding. A photocross-linking approach has been used to map the contacts between individual chaperonin subunits and ribosome-bound nascent chains of increasing length. Whereas a cryo-EM study suggests that chemically denatured actin interacts with only two TRiC subunits (δ and either β or ϵ), actin and luciferase chains photocross-link to at least six TRiC subunits (α, β, δ, ϵ, ξ, and θ) at different stages of translation. Furthermore, the photocross-linking of actin, but not luciferase, nascent chains to TRiC subunits ζ and θ was length-dependent. In addition, a single photoreactive probe incorporated at a unique site in actin nascent chains of different lengths reacted covalently with multiple TRiC subunits, thereby indicating that the nascent chain samples the polypeptide binding sites of different subunits. We conclude that elongating actin and luciferase nascent chains contact multiple TRiC subunits upon emerging from the ribosome, and that the TRiC subunits contacted by nascent actin change as it elongates and starts to fold. The hetero-oligomeric eukaryotic chaperonin TRiC (TCP-1-ring complex, also called CCT) interacts cotranslationally with a diverse subset of newly synthesized proteins, including actin, tubulin, and luciferase, and facilitates their correct folding. A photocross-linking approach has been used to map the contacts between individual chaperonin subunits and ribosome-bound nascent chains of increasing length. Whereas a cryo-EM study suggests that chemically denatured actin interacts with only two TRiC subunits (δ and either β or ϵ), actin and luciferase chains photocross-link to at least six TRiC subunits (α, β, δ, ϵ, ξ, and θ) at different stages of translation. Furthermore, the photocross-linking of actin, but not luciferase, nascent chains to TRiC subunits ζ and θ was length-dependent. In addition, a single photoreactive probe incorporated at a unique site in actin nascent chains of different lengths reacted covalently with multiple TRiC subunits, thereby indicating that the nascent chain samples the polypeptide binding sites of different subunits. We conclude that elongating actin and luciferase nascent chains contact multiple TRiC subunits upon emerging from the ribosome, and that the TRiC subunits contacted by nascent actin change as it elongates and starts to fold. Double-ring chaperonin complexes play a fundamental role in cellular protein folding (1Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2435) Google Scholar, 2Frydman J. Annu. Rev. Biochem. 2001; 70: 603-647Crossref PubMed Scopus (944) Google Scholar, 3Hartl F.U. Hayer-Hartl M. Science. 2002; 295: 1852-1858Crossref PubMed Scopus (2799) Google Scholar, 4Spiess M. Meyer A.S. Reissmann S. Frydman J. Trends Cell Biol. 2004; 14: 598-604Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Based on their ability to bind unfolded polypeptides within their ring cavities, chaperonins prevent off-pathway reactions and promote productive protein folding to the native state in a highly cooperative, ATP-dependent manner. Type I chaperonins such as Escherichia coli GroEL are generally homo-oligomeric, require a ring-shaped co-chaperonin complex such as GroES for productive folding, and are found in prokaryotes, mitochondria, and chloroplasts (1Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2435) Google Scholar, 5Hartl F.U. Nature. 1996; 381: 571-580Crossref PubMed Scopus (3130) Google Scholar). GroEL is thought to function primarily in a post-translational manner (1Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2435) Google Scholar, 2Frydman J. Annu. Rev. Biochem. 2001; 70: 603-647Crossref PubMed Scopus (944) Google Scholar, 3Hartl F.U. Hayer-Hartl M. Science. 2002; 295: 1852-1858Crossref PubMed Scopus (2799) Google Scholar), although cotranslational interactions have been reported for some proteins (6Ying B.-W. Taguchi H. Kondo M. Ueda T. J. Biol. Chem. 2005; 280: 12035-12040Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Type II chaperonins are found in Archaea and eukaryotes, are hetero-oligomeric, and do not require a GroES-like cofactor (4Spiess M. Meyer A.S. Reissmann S. Frydman J. Trends Cell Biol. 2004; 14: 598-604Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 7Gutsche I. Essen L.O. Baumeister W. J. Mol. Biol. 1999; 293: 295-312Crossref PubMed Scopus (182) Google Scholar). The eukaryotic chaperonin named TRiC 1The abbreviations used are: TRiC, TCP-1 ring complex; ϵANB, Nϵ-(5-azido-2-nitrobenzoyl); CCT, chaperonin containing tail-less complex polypeptide 1; RNC, ribosome-nascent chain complex. (for TCP-1 ring complex) or CCT (for chaperonin containing TCP1) consists of eight different, yet homologous, subunits per ring that are designated either α-θ or 1-8. Based on an analysis of chaperonin subcomplexes (8Liou A.K. Willison K.R. EMBO J. 1997; 16: 4311-4316Crossref PubMed Scopus (159) Google Scholar), a clockwise order for the arrangement of subunits in the ring has been proposed to be, moving clockwise, α/1, ϵ/5, ζ/6, β/2, γ/3, θ/8, δ/4, and η/7. All TRiC subunits are essential for viability in yeast (4Spiess M. Meyer A.S. Reissmann S. Frydman J. Trends Cell Biol. 2004; 14: 598-604Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar), and this raises a fundamental question: why did TRiC evolve different subunits if prokaryotes can mediate folding with homo-oligomeric chaperonins? One possibility is that certain TRiC subunits or combinations of subunits interact with substrates that have specific structural features or motifs, and that eight unique subunits provide a mechanism for accommodating a wider variety of substrates (9Llorca O. McCormack E.A. Hynes G. Grantham J. Cordell J. Carrascosa J.L. Willison K.R. Fernandez J.J. Valpuesta J.M. Nature. 1999; 402: 693-696Crossref PubMed Scopus (233) Google Scholar, 10Llorca O. Martin-Benito J. Grantham J. Ritco-Vonsovici M. Willison K.R. Carrascosa J.L. Valpuesta J.M. EMBO J. 2001; 20: 4065-4075Crossref PubMed Scopus (117) Google Scholar). But in fact, little is known about how TRiC interacts with its substrates. Several studies have examined how purified TRiC interacts in vitro with chemically denatured substrates, including actin and actin-derived peptides (10Llorca O. Martin-Benito J. Grantham J. Ritco-Vonsovici M. Willison K.R. Carrascosa J.L. Valpuesta J.M. EMBO J. 2001; 20: 4065-4075Crossref PubMed Scopus (117) Google Scholar, 11Meyer A.S. Gillespie J.R. Walther D. Millet I.S. Doniach S. Frydman J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). cryo-EM studies of complexes by purified and chemically denatured actin with purified TRiC that actin is in to only two subunits, and either β or (9Llorca O. McCormack E.A. Hynes G. Grantham J. Cordell J. Carrascosa J.L. Willison K.R. Fernandez J.J. Valpuesta J.M. Nature. 1999; 402: 693-696Crossref PubMed Scopus (233) Google Scholar). proposed that of actin only to the to either β or have two cryo-EM analysis on that within the can only chaperonin to and in the TRiC binding sites in the to that are either not or are the single and are not chemically unfolded polypeptides not to in J. H. F.U. Biol. 1999; PubMed Scopus Google Scholar). has been that proteins, such as luciferase, to cotranslationally the of the polypeptide has been synthesized and its is the J. H. F.U. Biol. 1999; PubMed Scopus Google Scholar). Furthermore, such as and TRiC J. F.U. Nature. PubMed Scopus Google Scholar, H. Frydman J. J. Cell Biol. PubMed Scopus Google bind to nascent chains as from the ribosome, and it has been proposed that protein and folding to the nascent chains within a folding Frydman EMBO J. 1999; PubMed Scopus Google Scholar). raises the possibility that interactions and nascent chain that folding are different from between chemically proteins and purified how TRiC to its substrates in the it is to how proteins interact with the chaperonin as from the A approach to this is to ribosome-bound polypeptides and thereby by photocross-linking the proteins to the nascent chain at the of We have used this approach to the of nascent chains the Cell. 2004; Full Text Full Text PDF PubMed Scopus Google and cotranslational to S. PubMed Scopus Google Scholar), J. Cell Biol. PubMed Scopus Google Scholar), and J. Cell Biol. PubMed Scopus Google Scholar, H. D. J. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google the of the In addition, photocross-linking that ribosome-bound actin and luciferase nascent chains interact cotranslationally with TRiC H. Frydman J. J. Cell Biol. PubMed Scopus Google Scholar). to newly synthesized chaperonin substrates interact with or only a subset of TRiC subunits, the photocross-linking approach to subunits of the hetero-oligomeric chaperonin complex are in contact with polypeptide actin and luciferase chains of lengths to TRiC and its contacts examined the TRiC complex and with actin and luciferase nascent chains found to photocross-link to at least of the TRiC subunits. Furthermore, actin to some TRiC subunits upon nascent chain thereby that the interactions with the chaperonin as the chain In addition, a single site in of actin to multiple subunits, the actin nascent chains to contact and different TRiC subunits the folding a probe to TRiC emerging from the TRiC in to the site the stages of a that is with a folding and for and luciferase H. Frydman J. J. Cell Biol. PubMed Scopus Google the and the of was by of lengths as J. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). was as S. PubMed Scopus Google Scholar, J. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar), as was the an J. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, J.J. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Cell to and as Frydman J. Mol. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google that in and not essential in TRiC by on and with as and vitro in as H. Frydman J. J. Cell Biol. PubMed Scopus Google in the of of and either of or of on for a J. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). samples with and by to of A to the TRiC complex Willison K.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was to a of and of the at for purified the β, δ, ϵ, and θ TRiC subunits a unique for Grantham J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), from to of protein in A containing for at least at with of and in of and a and H. Frydman J. J. Cell Biol. PubMed Scopus Google that ribosome-bound actin nascent chains photocross-link to the in that study native the TRiC subunits and it was not known TRiC subunits to the nascent A of nascent chain by TRiC nascent chain interactions with individual TRiC subunits and also the nascent chain of with nascent chains of a in vitro by that in the the of such but not of the of a The nascent chain to the as a in a ribosome-nascent chain complex the of the nascent polypeptide is by the of the can nascent chain by of different the nascent chain to a TRiC the two polypeptides contact and to an can a photocross-linking approach to nascent chain to individual TRiC subunits at a specific of We incorporated a probe at or specific sites in the nascent chain by the in the of and either or that either a or an a probe in an complex covalently for an of with protein to the nascent Whereas the of a only that the nascent chain was to that TRiC at the of probe the of photocross-linking between two proteins between two Furthermore, by the of the probe in the nascent as as the of the nascent in the of the nascent chain can approach a of the and interactions of the nascent chain with individual TRiC subunits at different stages the folding in a at a of the nascent chain and binding to individual TRiC subunits in the complexes that the of TRiC native did not the TRiC complex its subunits H. Frydman J. J. Cell Biol. PubMed Scopus Google Scholar), the of that the TRiC complex. this approach two the and by with subunits the and TRiC with and the by by native with the β not containing TRiC used to that the TRiC subunits, but binding to their reported Willison K.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), a TRiC and to the subunits of the unique peptides G. H. Willison K.R. PubMed Scopus (54) Google also the TRiC subunits the that the to the of and in the of the and that with the or not at to the and subunits, as the subunits do not of and the in the of the in is not In addition, in to the in of the is But for this study and its of the subunits that photocross-link to the nascent six of the eight TRiC subunits that TRiC, as by the different of the subunits and the of only a single in used for reported the of that the of its TRiC from a not of to TRiC TRiC subunits are and to an actin nascent chain folding, with a actin nascent chain synthesized in a containing TRiC, and a was and analysis by in photocross-linking between the actin and the and β subunits of the photocross-linking of the actin to was this nascent chain of of the actin to ζ or θ was also between the actin and was upon nascent actin is in to multiple TRiC subunits at this of and not only to as from the cryo-EM of TRiC β from different actin containing actin with a single at or and containing the β by and are by the to β of actin nascent chains of the lengths with only at or are by in and with We that is to with for protein in vitro and that an of about is incorporated for J. Cell Biol. PubMed Scopus Google Scholar). the of actin it is that such nascent chain or if a single nascent chain contacts two or TRiC subunits at the it is that upon a nascent chain covalently with TRiC In fact, on the of some the photocross-linking of a single actin nascent chain to two TRiC subunits although the has not yet from the in the actin nascent chain is to and interacts with the β, δ, ϵ, and θ subunits of TRiC, and a single nascent chain is to at a of the of actin to a TRiC on the of the nascent containing nascent actin chains between and in in the of TRiC, and the of nascent chain photocross-linking to TRiC subunits by nascent actin chains of or to δ, although the of photocross-linking for only the are Whereas this from a nascent chain in actin with a in the of nascent chain to the as the nascent chain the is that the nascent chain in to the stages of folding to TRiC subunits in the or of chain the of the of in a containing two TRiC subunits not the nascent chain was a of in the actin nascent chain to the of multiple photoreactive a single nascent only the actin nascent chains a that can by two or subunits. to as the nascent actin as was of containing only the The nascent chain of actin photocross-linking to β, and was to that of δ, and the are in I. But photocross-linking to ζ and θ was only the actin nascent chain photocross-linking to ζ and θ was with containing the actin at an of actin folding TRiC, the nascent chain is to β, δ, and ϵ, but not to ζ or it that nascent actin interacts with the TRiC subunits at the folding to TRiC by is as TRiC actin folding (4Spiess M. Meyer A.S. Reissmann S. Frydman J. Trends Cell Biol. 2004; 14: 598-604Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). is to the or of the of the actin nascent chain to individual actin subunits, as by the or of is for containing actin nascent chains of different lengths was and was with to the was in the of the of actin to individual TRiC subunits was examined by and in the or of either or the photocross-linking of a actin nascent chain to the of TRiC, but did not the of nascent actin to the of different nascent actin chains to β, ϵ, and θ not by the or of Whereas nascent chain to individual TRiC subunits was not to this from the to the of to the of nascent chain interactions with individual TRiC subunits, photocross-linking to TRiC was examined lengths of nascent luciferase, folding of the approach as with actin nascent the β and subunits of TRiC found to photocross-link to luciferase nascent chains as as and as as either in the or of of lengths of luciferase nascent chains also to δ, and θ either in the or of to TRiC subunits in the or of chain the of the of in a of nascent luciferase was to of the six TRiC subunits the of nascent photocross-linking was not is in the of as a function of nascent chain for β and Furthermore, the of nascent luciferase photocross-linking to β and as the nascent chain the of β and for the luciferase the of and photocross-linking to β to A with that as the nascent chain it interacts to different with the individual TRiC subunits, the subunits have for different of the nascent In addition, the for that nascent chain to and contact with individual TRiC subunits as the nascent chain and folding the of luciferase, its it is that this folding the of the photocross-linking in A to One TRiC that the actin nascent chains to multiple TRiC subunits suggests a of in the of the binding sites within the subunits. in the a nascent chain photocross-link to a TRiC from of within the nascent chain incorporated in of in the a single site in the actin nascent chain interact with multiple subunits, a photoreactive probe at in the nascent this a single was the actin in of the at is within the proposed binding site of (9Llorca O. McCormack E.A. Hynes G. Grantham J. Cordell J. Carrascosa J.L. Willison K.R. Fernandez J.J. Valpuesta J.M. Nature. 1999; 402: 693-696Crossref PubMed Scopus (233) Google Scholar). synthesized in vitro an that an and an at that with an as as J.J. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). chains at the by the of the do not a probe and do not photocross-linking only that the to the of the to a nascent chain of the length. the probe at this single to TRiC subunits, a of actin with the at was and of the with actin nascent chains to β, ϵ, and with the with the β at this of protein folding TRiC, this in the actin nascent chains is in contact with at least different TRiC subunits. this in binding the of nascent interactions the of actin folding, the of photocross-linking on nascent chain and probe The in that in a of different TRiC subunits (α, β and to of different sites the nascent actin chain it was or the nascent chain have from the ribosome, different actin are to different TRiC subunits in a of complexes as the nascent chain is from to of specific in nascent actin chains of different lengths to TRiC chain at to the TRiC β not to the TRiC to the TRiC not in a of to the of specific sites in the nascent chain to a a single the actin in of of the at or actin containing a single probe at to β examined the of the probe at on the with this samples of actin that only in the of the probe in the nascent at or in the of TRiC, actin was found to photocross-link to the β of TRiC, although with different that at this of actin folding, and are to β, and are to β are the actin The nascent chain of photocross-linking β from of actin was Whereas nascent chains only to β, probe in actin nascent chains between and in was to β of the actin was to contact β in the TRiC complex as the nascent chain in by a of the nascent chain not to at a within the TRiC the folding The in the photocross-linking to β of probe in the nascent actin is of the cotranslational of in the of actin that the probe TRiC in to the to the with nascent chains as from the nascent chain and the in that a probe at in a nascent chain to the β of an in the nascent chain only from the site is to can TRiC to the as it and the nascent containing a actin nascent chain with a probe at and found by to covalently with TRiC β A probe in the nascent chain only from the can covalently with the mechanism by a chaperonin the folding of a nascent chain as it is synthesized by the has not yet been it is to the of the nascent chain the chaperonin and a of interactions with some or of the chaperonin subunits to a protein or protein But in fact, on the and of nascent chains with chaperonin subunits, that interactions are by We to the to that and fundamental of of the nascent chain by a a nascent chain to chaperonin subunits, or it contact only a nascent chain to a chaperonin change as and folding a nascent chain interact with two chaperonin subunits different nascent chains interact with the the nascent chain interact with individual chaperonin by or nascent chains of actin and luciferase as synthesized by a a of complexes in nascent chain the and a at the TRiC with nascent chains their H. Frydman J. J. Cell Biol. PubMed Scopus Google Scholar), to the photocross-linking of the nascent chain to chaperonin and thereby provide a of nascent chain to individual subunits at the of folding. the nascent chain to only or a subunits, to only a of nascent The in this study and in that actin nascent chains of or are to at least of the TRiC subunits at the of and I and a single nascent chain to subunits, at least a of the nascent chain in the is to of the subunits at nascent luciferase chains of or are to of the TRiC subunits at the of Furthermore, this in the of nascent chains between TRiC subunits was as the actin and luciferase nascent chains to and and the photocross-linking approach did not a of and nascent chain with specific TRiC subunits, it is that the nascent chain not a within the it is to only a of the subunits. In fact, a actin nascent chain only a single probe of different and its to that of the nascent chain to bind and photocross-link to only or two subunits, the nascent chains to TRiC subunits is in the nascent chain binding of the individual subunits. to the that nascent chain within the chaperonin is in a is to of the TRiC subunits, and from to within the of the of the nascent it is also from that nascent interactions are not and is from in the of nascent photocross-linking as the of the nascent chain and as the of the probe as as from the different of actin photocross-linking to TRiC subunits it that are different of individual TRiC subunits for specific nascent chain and that the interactions to photocross-linking and the of suggests that such are and only to a the of of nascent interactions is at with the proposed for that a polypeptide interacts with specific subunits within the The from cryo-EM that to that chemically unfolded actin polypeptides the examined in this a and highly within the and was in to only two TRiC subunits, and either β or (9Llorca O. McCormack E.A. Hynes G. Grantham J. Cordell J. Carrascosa J.L. Willison K.R. Fernandez J.J. Valpuesta J.M. Nature. 1999; 402: 693-696Crossref PubMed Scopus (233) Google Scholar). In that newly actin polypeptides contact multiple subunits in the TRiC complex. why do the photocross-linking and cryo-EM different We that the from the different and from the of the samples Whereas photocross-linking reported in the and and nascent chains per (9Llorca O. McCormack E.A. Hynes G. Grantham J. Cordell J. Carrascosa J.L. Willison K.R. Fernandez J.J. Valpuesta J.M. Nature. 1999; 402: 693-696Crossref PubMed Scopus (233) Google in their (9Llorca O. McCormack E.A. Hynes G. Grantham J. Cordell J. Carrascosa J.L. Willison K.R. Fernandez J.J. Valpuesta J.M. Nature. 1999; 402: 693-696Crossref PubMed Scopus (233) Google Scholar). cryo-EM the of and in their unfolded actin polypeptides TRiC are not and such are not in the thereby the by this of in to the in the of the in cryo-EM and photocross-linking the two studies in the of complexes examined and in the of of complexes in the also that cryo-EM that actin is in a highly analysis of complexes that actin chains are in a A.S. Gillespie J.R. Walther D. Millet I.S. Doniach S. Frydman J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). between the two studies the The of nascent chain in the photocross-linking studies is covalently to a in the and nascent chain is to the the in the photocross-linking nascent chain have its of the to the TRiC and the ribosome, the of the nascent chain is to different and folding In the substrates examined in the cryo-EM study are to the chaperonin and their to different and is not by the and in the the of the it is not to the and that and in only a of the chemically denatured actin is by TRiC, that of the actin chains interact in a 11Meyer A.S. Gillespie J.R. Walther D. Millet I.S. Doniach S. Frydman J. Cell. Full Text Full Text PDF PubMed Scopus Google and G. Nature. PubMed Scopus Google Scholar), of the newly actin is and by TRiC Frydman EMBO J. 1999; PubMed Scopus Google and J. F.U. Science. 1996; PubMed Scopus Google Scholar). the folding and folding of the folding from purified Whereas the photocross-linking that at are a of different nascent interactions and in a of it is also to that the photocross-linking some structural an actin not covalently with TRiC and a actin covalently with six of the TRiC subunits a actin covalently with only the β, δ, and subunits it that nascent actin the TRiC the nascent chain is not to the θ and ζ subunits. a photoreactive probe is at in an nascent actin, this probe is to covalently with the β but not with the and subunits Based on that on nascent chain it that is a for the nascent chain as it the although are to the of this the interactions with individual TRiC subunits for actin and luciferase nascent such a upon the as nascent chain a of folding and within the that probe to a the of containing two TRiC subunits and that a single nascent chain can contact two different subunits at the that the nascent chains contact subunits, but this has yet to ATP-dependent in the photocross-linking of nascent chains to different subunits, but and not in the of as yet the of the of nascent chain to the different TRiC subunits. as have H. Frydman J. J. Cell Biol. PubMed Scopus Google Scholar), TRiC is to with the nascent chain it from its have found that a actin with a probe at is to photocross-link to the TRiC β, and subunits a actin with a probe at can photocross-link the β a probe only nascent chain from the site is to photocross-link to We have between two different incorporated the nascent chain that proteins are within the nascent chain Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). that the probe is about from the site and the of the nascent chain is on the order of it that the TRiC β is within of the site in the cotranslational complex. In the analysis of nascent chain to individual TRiC subunits suggests that nascent polypeptides contact multiple chaperonin subunits upon emerging from the TRiC subunits to the of the with different in to the of highly specific interactions that provide for interactions between the polypeptide and the Whereas different subunits some in the of nascent interactions at of folding We are to and as as and of the for