Hierarchical Formation of Disulfide Bonds in the Immunoglobulin Fc Fragment Is Assisted by Protein-disulfide Isomerase

Antibodies provide an excellent system to study the folding and assembly of all β-sheet proteins and to elucidate the hierarchy of intra/inter chain disulfide bonds formation during the folding process of multimeric and multidomain proteins. Here, the folding process of the Fc fragment of the heavy chain of the antibody MAK33 was investigated. The Fc fragment consists of the CH3 and CH2 domains of the immunoglobulin heavy chain, both containing a single S-S bond. The folding process was investigated both in the absence and presence of the folding catalyst protein-disulfide isomerase (PDI), monitoring the evolution of intermediates by electrospray mass spectrometry. Moreover, the disulfide bonds present at different times in the folding mixture were identified by mass mapping to determine the hierarchy of disulfide bond formation. The analysis of the uncatalyzed folding showed that the species containing one intramolecular disulfide predominated throughout the entire process, whereas the fully oxidized Fc fragment never accumulated in significant amounts. This result suggests the presence of a kinetic trap during the Fc folding, preventing the one-disulfide-containing species (1S2H) to reach the fully oxidized protein (2S). The assignment of disulfide bonds revealed that 1S2H is a homogeneous species characterized by the presence of a single disulfide bond (Cys-130–Cys-188) belonging to the CH3 domain. When the folding experiments were carried out in the presence of PDI, the completely oxidized species accumulated and predominated at later stages of the process. This species contained the two native S-S bonds of the Fc protein. Our results indicate that the two domains of the Fc fragment fold independently, with a precise hierarchy of disulfide formation in which the disulfide bond, especially, of the CH2 domain requires catalysis by PDI. Antibodies provide an excellent system to study the folding and assembly of all β-sheet proteins and to elucidate the hierarchy of intra/inter chain disulfide bonds formation during the folding process of multimeric and multidomain proteins. Here, the folding process of the Fc fragment of the heavy chain of the antibody MAK33 was investigated. The Fc fragment consists of the CH3 and CH2 domains of the immunoglobulin heavy chain, both containing a single S-S bond. The folding process was investigated both in the absence and presence of the folding catalyst protein-disulfide isomerase (PDI), monitoring the evolution of intermediates by electrospray mass spectrometry. Moreover, the disulfide bonds present at different times in the folding mixture were identified by mass mapping to determine the hierarchy of disulfide bond formation. The analysis of the uncatalyzed folding showed that the species containing one intramolecular disulfide predominated throughout the entire process, whereas the fully oxidized Fc fragment never accumulated in significant amounts. This result suggests the presence of a kinetic trap during the Fc folding, preventing the one-disulfide-containing species (1S2H) to reach the fully oxidized protein (2S). The assignment of disulfide bonds revealed that 1S2H is a homogeneous species characterized by the presence of a single disulfide bond (Cys-130–Cys-188) belonging to the CH3 domain. When the folding experiments were carried out in the presence of PDI, the completely oxidized species accumulated and predominated at later stages of the process. This species contained the two native S-S bonds of the Fc protein. Our results indicate that the two domains of the Fc fragment fold independently, with a precise hierarchy of disulfide formation in which the disulfide bond, especially, of the CH2 domain requires catalysis by PDI. Protein folding of disulfide-containing proteins has been studied essentially using small monomeric, one-domain proteins as a model (1Navon A. Ittah V. Scheraga H.A. Haas E. Biochemistry. 2002; 41: 14225-14231Crossref PubMed Scopus (10) Google Scholar, 2Ruoppolo M. Vinci F. Klink T. Raines R.T. Marino G. Biochemistry. 2000; 39: 12033-12042Crossref PubMed Scopus (32) Google Scholar, 3Vinci F. Ruoppolo M. Pucci P. Freedman R.B. Marino G. Protein Sci. 2000; 9: 525-535Crossref PubMed Scopus (26) Google Scholar, 4Iowaka M. Juminaga D. Scheraga H.A. Biochemistry. 1998; 37: 4490-4501Crossref PubMed Scopus (48) Google Scholar, 5Kim P.S. Baldwin R.L. Annu. Rev. Biochem. 1992; 51: 247-265Google Scholar, 6Creighton T.E. Creighton T.E. Protein Folding. W. H. Freeman and Co., New York1992: 301-351Google Scholar, 7Weissman J.S. Kim P.S. Science. 1991; 253: 1386-1390Crossref PubMed Scopus (488) Google Scholar). The analysis of the folding process of multimeric or multidomains proteins containing intra- and interchain disulfide bonds is hampered by several factors including the presence of an increasing number of folding intermediates and the superimposition of different folding events. Antibodies are multimeric proteins consisting of different domains characterized by two antiparallel β-sheets linked by an intradomain disulfide bond (8Huber R. Deisenhofer J. Colman P.M. Matsushima M. Palm W. Nature. 1976; 264: 415-420Crossref PubMed Scopus (312) Google Scholar, 9Deisenhofer J. Biochemistry. 1981; 20: 2361-2370Crossref PubMed Scopus (1357) Google Scholar, 10Augustine J.G. de la Calle A. Knarr G. Buchner J. Frederick C.A. J. Biol. Chem. 2001; 276: 3287-3294Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). A characteristic feature of antibodies is that the intradomain disulfide bond, which connects residues far apart in sequence, is completely buried in the core of the protein. These proteins provide an excellent system to study the folding and assembly of all β-sheet proteins and to elucidate the hierarchy of intra/interchain disulfide bond formation during the folding process of multimeric and multidomain proteins (11Goto Y. Hamaguchi Y. J. Biochem. (Tokyo). 1979; 86: 1433-1441Crossref PubMed Scopus (136) Google Scholar, 12Goto Y. Hamaguchi Y. J. Mol. Biol. 1982; 156: 911-926Crossref PubMed Scopus (88) Google Scholar, 13Lilie H. Mc Laughlin S. Freedman R. Buchner J. J. Biol. Chem. 1994; 269: 14290-14296Abstract Full Text PDF PubMed Google Scholar, 14Frisch C. Kolmar H. Schmidt A. Klemann G. Reinhardt A. Pohl E. Uson L. Schneider T.R. Fritz H.J. Fold. Des. 1996; 1: 431-440Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 15Thies M.J.W. Mayer J. Augustine J.G. Frederick C.A. Lilie H. Buchner J. J. Mol. Biol. 1999; 293: 67-79Crossref PubMed Scopus (77) Google Scholar). The β-sheet folding is usually much slower than the α-helix formation because amino acid residues, which are far apart in the polypeptide chain, must interact correctly in the three-dimensional space to form stabilizing interactions (16Clark P.L. Liu Z.P. Gierasch L.M. Biol. PubMed Scopus Google Scholar). folding of were carried out antibody and single antibody domains (11Goto Y. Hamaguchi Y. J. Biochem. (Tokyo). 1979; 86: 1433-1441Crossref PubMed Scopus (136) Google Scholar, 12Goto Y. Hamaguchi Y. J. Mol. Biol. 1982; 156: 911-926Crossref PubMed Scopus (88) Google Scholar, 13Lilie H. Mc Laughlin S. Freedman R. Buchner J. J. Biol. Chem. 1994; 269: 14290-14296Abstract Full Text PDF PubMed Google Scholar, 15Thies M.J.W. Mayer J. Augustine J.G. Frederick C.A. Lilie H. Buchner J. J. Mol. Biol. 1999; 293: 67-79Crossref PubMed Scopus (77) Google Scholar, J. R. 1991; 9: PubMed Scopus Google Scholar). the model system is the by the domain containing a single intramolecular disulfide bond. The of disulfide the and of CH3 has been M.J.W. F. Mayer M. S. Ruoppolo M. Marino G. Buchner J. J. Mol. Biol. 2002; PubMed Scopus Google Scholar). has been that formation is to the to form the disulfide bond and to the of is and M.J.W. F. Mayer M. S. Ruoppolo M. Marino G. Buchner J. J. Mol. Biol. 2002; PubMed Scopus Google Scholar). native disulfide bond formation and is that a number of different and a in protein disulfide bond formation J.S. Science. 2000; PubMed Scopus Google Scholar, A. F. C.A. Biol. 2001; PubMed Scopus Google Scholar, R.B. P. 2002; PubMed Scopus Google Scholar). The native disulfide bond formation in proteins which are disulfide bond formation is linked to in protein These are by proteins belonging to the protein-disulfide isomerase PDI, protein-disulfide electrospray mass oxidized mass PDI, protein-disulfide electrospray mass oxidized mass which formation and of disulfide bonds the native is to R.B. Mc Laughlin Scholar). been that in the disulfide bond formation in a the of the residues during the folding process to of the of H. Mc Laughlin S. Freedman R. Buchner J. J. Biol. Chem. 1994; 269: 14290-14296Abstract Full Text PDF PubMed Google Scholar). The immunoglobulin heavy protein with in the folding of the and fragment M. R. Buchner J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). the immunoglobulin and in a in which residues are to PDI. the folding process of the Fc fragment of the heavy chain of the antibody MAK33 the of was investigated in the absence and in the presence of PDI. The Fc fragment consists of the CH3 and CH2 domains of the immunoglobulin heavy chain, both containing a single S-S bond. The evolution and the of intermediates in the folding of the Fc fragment were by electrospray mass spectrometry. The disulfide bonds present in the folding mixture at different times were by mass mapping to determine the of disulfide bonds formation in the presence or absence of PDI. The results indicate a precise hierarchy in the formation of disulfide bonds during the folding process and the by in the folding of the Fc and oxidized were and were was Science. and were were of the was as M. R. Buchner J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). and of Fc Fc fragment of MAK33 was a by the F. 1979; PubMed Scopus Google containing the Full Text PDF PubMed Scopus Google was with the The was in at and with at an of were by later and in at of in a the was the by at The Fc fragment was in the as were to R. G. Lilie H. R. Creighton T.E. Protein A The of was of the The protein was in at the was to and the was at and at The protein was as Biochem. 1976; PubMed Scopus Google Scholar). antibody J. R. 1991; 9: PubMed Scopus Google Scholar, J. 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F. Mayer M. S. Ruoppolo M. Marino G. Buchner J. J. Mol. Biol. 2002; PubMed Scopus Google Scholar, Protein Sci. 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The protein was and to in with showed that the Fc fragment is a with an mass of The Fc fragment was characterized by far in the protein a an immunoglobulin domain. The of to with at and that the protein is The at result the of amino to the as the CH3 domain M.J.W. F. Mayer M. S. Ruoppolo M. Marino G. Buchner J. J. Mol. Biol. 2002; PubMed Scopus Google Scholar). the and protein a with The of the and Fc fragment that the is in the native This is a feature of antibody domains that results the of a to the disulfide bond. The of the of to and that the buried the that Fc is and The Fc fragment was by folding The to the Fc fragment was by The analysis revealed the presence of a single a mass of in with the the of amino acid The were in the folding of the Fc of the Fc fragment the fully and protein to the native was carried out at a protein of in the presence of a of and a that or species J. Biochem. 1992; PubMed Scopus Google Scholar, R. H. in Protein and de Co., Scholar). of the folding were at different and the intermediates present in were by of the and by to the species The to trap the the mass of the intermediates by a the by mass of intermediates containing different of disulfide analysis the of the of folding intermediates that the different M.J.W. F. Mayer M. S. Ruoppolo M. Marino G. Buchner J. J. Mol. Biol. 2002; PubMed Scopus Google Scholar, M. M. M. Pucci P. A. Marino G. E. Biochemistry. 1998; 37: PubMed Scopus Google Scholar). the electrospray mass of the folding mixture of in the of intermediates is characterized by a different number of intramolecular disulfide bonds as with the and The number of to the number of present in the folding intermediates and as throughout the The revealed the presence of a to the fully A protein species a of and residues was in the The of in electrospray is to the number and of the protein V. J. Chem. Scopus Google Scholar, 1991; Scholar, V. 1991; PubMed Scopus Google Scholar). The species showed a of with to The of by the species was to that the a and The electrospray were at different times of folding and showed the evolution of the intermediates containing different of disulfide The of the different folding intermediates was of in The species to a of and The species containing one intramolecular disulfide and two (1S2H) in the stages of the and predominated the process. the fully oxidized species at never than during the entire folding process. the containing one S-S bond and one disulfide with the of the fully oxidized species was at and throughout the entire process. the species and the formation of the intramolecular S-S bond The analysis of the of a in the of that the folding process a of the Fc in the of an the the folding of the Fc fragment was carried out in the presence of the the electrospray mass of the intermediates mixture of in the presence of PDI. The of the fully oxidized species the whereas the one disulfide-containing (1S2H) was as a The species showed a of to This is different that both the and species and the number of suggests the of a and the The 1S2H species showed a of to that the the protein. This result suggests that the 1S2H has an and the of the process. A of was throughout the entire process. The protein and the 1S2H species predominated to the fully oxidized protein to The species in the stages of the process and a This formation of the species in the presence of the was the the uncatalyzed and the process. of in the of the Fc of the uncatalyzed folding mixture of the Fc fragment were at and of and as the S-S bonds present in the folding the were with and the were by an the of the the at The at was to the linked to the fragment by the S-S and This disulfide bond is the native in the CH3 domain. Moreover, was in a disulfide with as by the at to the linked to a the showed the presence of a at that was to the containing the the at to the with by the S-S bonds identified in the at different of the analysis showed the at to the disulfide and that formation of the in the folding of Fc of folding, mass analysis identified the presence of the S-S bond, to the native of the CH3 as This disulfide was the intramolecular S-S bond to stages of and was present of These results that the 1S2H species the in the folding process as by kinetic analysis Moreover, the assignment of S-S bonds in the folding intermediates that 1S2H is a species containing the CH3 intradomain of disulfide bonds during the uncatalyzed folding of Fc fragment in a the of the mixture the at stages of the process revealed the presence of a at to the and linked by the S-S This disulfide to the native intradomain in the CH2 and presence the of the fully oxidized protein in the kinetic analysis The assignment of were by of the with that the mass to mass by of The at to the with linked to the was to to the linked to The at to to the and linked by the S-S were in the at and at and These were to the (1Navon A. Ittah V. Scheraga H.A. Haas E. Biochemistry. 2002; 41: 14225-14231Crossref PubMed Scopus (10) Google Scholar, 2Ruoppolo M. Vinci F. Klink T. Raines R.T. Marino G. Biochemistry. 2000; 39: 12033-12042Crossref PubMed Scopus (32) Google Scholar, 3Vinci F. Ruoppolo M. Pucci P. Freedman R.B. Marino G. Protein Sci. 2000; 9: 525-535Crossref PubMed Scopus (26) Google Scholar, 4Iowaka M. Juminaga D. Scheraga H.A. Biochemistry. 1998; 37: 4490-4501Crossref PubMed Scopus (48) Google Scholar, 5Kim P.S. Baldwin R.L. Annu. Rev. Biochem. 1992; 51: 247-265Google Scholar, 6Creighton T.E. Creighton T.E. Protein Folding. W. H. Freeman and Co., New York1992: 301-351Google Scholar, 7Weissman J.S. Kim P.S. Science. 1991; 253: 1386-1390Crossref PubMed Scopus (488) Google Scholar, R. Deisenhofer J. Colman P.M. Matsushima M. Palm W. Nature. 1976; 264: 415-420Crossref PubMed Scopus (312) Google Scholar, 9Deisenhofer J. Biochemistry. 1981; 20: 2361-2370Crossref PubMed Scopus (1357) Google Scholar, 10Augustine J.G. de la Calle A. Knarr G. Buchner J. Frederick C.A. J. Biol. Chem. 2001; 276: 3287-3294Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, Y. Hamaguchi Y. J. Biochem. (Tokyo). 1979; 86: 1433-1441Crossref PubMed Scopus (136) Google Scholar, 12Goto Y. Hamaguchi Y. J. Mol. Biol. 1982; 156: 911-926Crossref PubMed Scopus (88) Google Scholar, 13Lilie H. Mc Laughlin S. Freedman R. Buchner J. J. Biol. Chem. 1994; 269: 14290-14296Abstract Full Text PDF PubMed Google Scholar, 14Frisch C. Kolmar H. Schmidt A. Klemann G. Reinhardt A. Pohl E. Uson L. Schneider T.R. Fritz H.J. Fold. Des. 1996; 1: 431-440Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 15Thies M.J.W. Mayer J. Augustine J.G. Frederick C.A. Lilie H. Buchner J. J. Mol. Biol. 1999; 293: 67-79Crossref PubMed Scopus (77) Google Scholar, P.L. Liu Z.P. Gierasch L.M. Biol. PubMed Scopus Google Scholar, J. R. 1991; 9: PubMed Scopus Google Scholar, M.J.W. F. Mayer M. S. Ruoppolo M. Marino G. Buchner J. J. Mol. Biol. 2002; PubMed Scopus Google Scholar, J.S. Science. 2000; PubMed Scopus Google Scholar, A. F. C.A. Biol. 2001; PubMed Scopus Google Scholar, R.B. P. 2002; PubMed Scopus Google Scholar, R.B. Mc Laughlin Scholar, M. R. Buchner J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, F. 1979; PubMed Scopus Google Scholar, Full Text PDF PubMed Scopus Google Scholar, R. G. Lilie H. R. Creighton T.E. Protein A Scholar, Biochem. 1976; PubMed Scopus Google Scholar, J. Biochem. 1992; PubMed Scopus Google Scholar, Protein Sci. PubMed Scopus Google Scholar, R. H. in Protein and de Co., Scholar, M. M. M. Pucci P. A. Marino G. E. Biochemistry. 1998; 37: PubMed Scopus Google Scholar, V. J. Chem. Scopus Google and (1Navon A. Ittah V. Scheraga H.A. Haas E. Biochemistry. 2002; 41: 14225-14231Crossref PubMed Scopus (10) Google Scholar, 2Ruoppolo M. Vinci F. Klink T. Raines R.T. Marino G. Biochemistry. 2000; 39: 12033-12042Crossref PubMed Scopus (32) Google Scholar, 3Vinci F. Ruoppolo M. Pucci P. Freedman R.B. Marino G. Protein Sci. 2000; 9: 525-535Crossref PubMed Scopus (26) Google Scholar, 4Iowaka M. Juminaga D. Scheraga H.A. Biochemistry. 1998; 37: 4490-4501Crossref PubMed Scopus (48) Google Scholar, 5Kim P.S. Baldwin R.L. Annu. Rev. Biochem. 1992; 51: 247-265Google Scholar, 6Creighton T.E. Creighton T.E. Protein Folding. W. H. Freeman and Co., New York1992: 301-351Google Scholar, 7Weissman J.S. Kim P.S. Science. 1991; 253: 1386-1390Crossref PubMed Scopus (488) Google Scholar, R. Deisenhofer J. Colman P.M. Matsushima M. Palm W. Nature. 1976; 264: 415-420Crossref PubMed Scopus (312) Google Scholar, 9Deisenhofer J. Biochemistry. 1981; 20: 2361-2370Crossref PubMed Scopus (1357) Google Scholar, 10Augustine J.G. de la Calle A. Knarr G. Buchner J. Frederick C.A. J. Biol. Chem. 2001; 276: 3287-3294Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, Y. Hamaguchi Y. J. Biochem. (Tokyo). 1979; 86: 1433-1441Crossref PubMed Scopus (136) Google Scholar, 12Goto Y. Hamaguchi Y. J. Mol. Biol. 1982; 156: 911-926Crossref PubMed Scopus (88) Google Scholar, 13Lilie H. Mc Laughlin S. Freedman R. Buchner J. J. Biol. Chem. 1994; 269: 14290-14296Abstract Full Text PDF PubMed Google Scholar, 14Frisch C. Kolmar H. Schmidt A. Klemann G. Reinhardt A. Pohl E. Uson L. Schneider T.R. Fritz H.J. Fold. Des. 1996; 1: 431-440Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 15Thies M.J.W. Mayer J. Augustine J.G. Frederick C.A. Lilie H. Buchner J. J. Mol. Biol. 1999; 293: 67-79Crossref PubMed Scopus (77) Google Scholar, P.L. Liu Z.P. Gierasch L.M. Biol. PubMed Scopus Google Scholar, J. R. 1991; 9: PubMed Scopus Google Scholar, M.J.W. F. Mayer M. S. Ruoppolo M. Marino G. Buchner J. J. Mol. Biol. 2002; PubMed Scopus Google Scholar, J.S. Science. 2000; PubMed Scopus Google Scholar, A. F. C.A. Biol. 2001; PubMed Scopus Google Scholar, R.B. P. 2002; PubMed Scopus Google Scholar, R.B. Mc Laughlin Scholar, M. R. Buchner J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, F. 1979; PubMed Scopus Google Scholar, Full Text PDF PubMed Scopus Google Scholar, R. G. Lilie H. R. Creighton T.E. Protein A Scholar, Biochem. 1976; PubMed Scopus Google Scholar, J. Biochem. 1992; PubMed Scopus Google Scholar, Protein Sci. PubMed Scopus Google Scholar, R. H. in Protein and de Co., Scholar, M. M. M. Pucci P. A. Marino G. E. Biochemistry. 1998; 37: PubMed Scopus Google linked to and in the CH2 domain form a disulfide with at the stages of the process. of in the was to the disulfide bonds in the folding intermediates of the The mass of the of the the at is in mass were at and The was to the and linked by the S-S whereas the was as the containing the intramolecular S-S bond, The mass of the the presence of S-S bond throughout the at to the and linked by the disulfide The mass mapping analysis in that both native of Fc fragment at the stages of the in the presence of PDI. S-S bonds were never throughout the entire process. Moreover, the assignment of disulfide bonds of the folding intermediates that and were in with These two as the of the two Fc of disulfide bonds during the folding of Fc fragment in a Antibodies an excellent model system to the folding of multidomains proteins. These proteins both intra- and disulfide the of the hierarchy of intra/interchain disulfide bond formation during the folding process. the analysis of the folding of the entire antibody is hampered by several including The analysis of immunoglobulin folding was by the folding process of different of the antibody The of the folding of the immunoglobulin domain CH3 containing a single S-S bond been the of a at the folding and of different of the folding of CH3 the formation of the single disulfide the as the of the M.J.W. F. Mayer M. S. Ruoppolo M. Marino G. Buchner J. J. Mol. Biol. 2002; PubMed Scopus Google Scholar). Here, the was to the of the folding of the Fc consisting of the CH2 and CH3 domain of the heavy chain, containing a single disulfide bond. The folding process was in both in the absence and presence of PDI, that in the The of the and the of the folding intermediates were by electrospray mass spectrometry. The analysis of the folding of the Fc fragment showed that the species containing one intramolecular disulfide (1S2H) predominated throughout the entire process, whereas the fully oxidized Fc fragment never accumulated in significant amounts. This result suggests the presence of a kinetic trap during that the formation of the S-S bond the 1S2H The assignment of disulfide bonds in the intermediates present at different times during the process revealed that the disulfide and the at the stages of the and at stages the 1S2H was the species present in the present in the CH2 domain much slower with the Moreover, the 1S2H species was to a homogeneous containing the disulfide belonging to the CH3 domain. were never the of the the kinetic trap in the Fc folding to two different factors or a of the The is the different of the residues in the two was identified as the in the Fc in with results in the study of the folding of the CH3 domain M.J.W. F. Mayer M. S. Ruoppolo M. Marino G. Buchner J. J. Mol. Biol. 2002; PubMed Scopus Google Scholar). The by the formation of an of the which is the in the formation of the S-S bond of the Fc because the of disulfide formation is the and the of the a in the CH2 domain in the two residues at the and in the This to a of the CH2 which the formation of the S-S bond at the of the process. The of in the that 1S2H species has an and of a number of that the CH2 domain is When the disulfide bond the species a with a much number of the of the a model the folding of the Fc fragment is as in The in the process is the of with the This disulfide is by to form the intradomain disulfide to the formation of 1S2H the CH3 disulfide the disulfide and and is to the to the native fully oxidized Fc The assignment of S-S bonds in different experiments that a precise hierarchy of is present domain. and are the the CH3 and the CH2 are in disulfide with at different whereas the of and is to the disulfide to form the intramolecular S-S bond. The results that the two domains present in the Fc fragment A hierarchy of in the process, with the disulfide of the CH3 domain than the CH2 S-S bond. the stages of folding the CH3 domain a that is to the two in the to form the disulfide bond and to the as has been CH3 M.J.W. F. Mayer M. S. Ruoppolo M. Marino G. Buchner J. J. Mol. Biol. 2002; PubMed Scopus Google Scholar). the CH2 domain to a that the formation of the S-S bond. The in the Fc folding process the of the two of the CH2 domain. folding in the absence of folding at a This is in with results in the study of the folding of fragment M. R. Buchner J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). These the that antibodies the of folding to folding Here, in the folding of the process at the formation of the CH2 The native species containing both native disulfide bonds of the Fc to and predominated at stages of the process. Moreover, assignment of disulfide bonds at different times during the revealed that both and at the folding the S-S bonds identified in the process, as with different protein F. Ruoppolo M. Pucci P. Freedman R.B. Marino G. Protein Sci. 2000; 9: 525-535Crossref PubMed Scopus (26) Google Scholar). This study that the folding process of two immunoglobulin domains is the is the that the domains of antibodies to different folding and the immunoglobulin fold