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BIBTEX RIS

Mechanism of Insulin Chain Combination

2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês

10.1074/jbc.m206107200

ISSN

1083-351X

Autores

Qing Hua, Ying Chi Chu, Wenhua Jia, Nelson F. B. Phillips, Run Ying Wang, Panayotis G. Katsoyannis, Michael A. Weiss,

Tópico(s)

Metabolism, Diabetes, and Cancer

Resumo

The A and B chains of insulin combine to form native disulfide bridges without detectable isomers. The fidelity of chain combination thus recapitulates the folding of proinsulin, a precursor protein in which the two chains are tethered by a disordered connecting peptide. We have recently shown that chain combination is blocked by seemingly conservative substitutions in the C-terminal α-helix of the A chain. Such analogs, once formed, nevertheless retain high biological activity. By contrast, we demonstrate here that chain combination is robust to non-conservative substitutions in the N-terminal α-helix. Introduction of multiple glycine substitutions into the N-terminal segment of the A chain (residues A1–A5) yields analogs that are less stable than native insulin and essentially without biological activity. 1H NMR studies of a representative analog lacking invariant side chains IleA2and ValA3 (A chain sequence GGGEQCCTSICSLYQLENYCN; substitutions are italicized and cysteines are underlined) demonstrate local unfolding of the A1–A5 segment in an otherwise native-like structure. That this and related partial folds retain efficient disulfide pairing suggests that the native N-terminal α-helix does not participate in the transition state of the reaction. Implications for the hierarchical folding mechanisms of proinsulin and insulin-like growth factors are discussed. The A and B chains of insulin combine to form native disulfide bridges without detectable isomers. The fidelity of chain combination thus recapitulates the folding of proinsulin, a precursor protein in which the two chains are tethered by a disordered connecting peptide. We have recently shown that chain combination is blocked by seemingly conservative substitutions in the C-terminal α-helix of the A chain. Such analogs, once formed, nevertheless retain high biological activity. By contrast, we demonstrate here that chain combination is robust to non-conservative substitutions in the N-terminal α-helix. Introduction of multiple glycine substitutions into the N-terminal segment of the A chain (residues A1–A5) yields analogs that are less stable than native insulin and essentially without biological activity. 1H NMR studies of a representative analog lacking invariant side chains IleA2and ValA3 (A chain sequence GGGEQCCTSICSLYQLENYCN; substitutions are italicized and cysteines are underlined) demonstrate local unfolding of the A1–A5 segment in an otherwise native-like structure. That this and related partial folds retain efficient disulfide pairing suggests that the native N-terminal α-helix does not participate in the transition state of the reaction. Implications for the hierarchical folding mechanisms of proinsulin and insulin-like growth factors are discussed. circular dichroism distance geometry restrained molecular dynamics monomeric insulin analog containing three substitutions in the B chain (AspB10, LysB28, and ProB29) two-disulfide analog of DKP-insulin containing substitutions SerA7 and SerB7 two-disulfide analog of DKP-insulin containing substitutions SerA6 and SerA11 double-quantum-filtered correlated spectroscopy des-pentapeptide[B26–B30]insulin insulin-like growth factor I nuclear Overhauser effect nuclear Overhauser effect spectroscopy porcine insulin precursor, a single-chain insulin precursor polypeptide reverse phase high performance liquid chromatography specific folding nucleus A-chain analogs containing N-terminal glycine substitutions [GlyA2,GlyA3]DKP-insulin, analog of DKP-insulin in which IleA2 and ValA3 are each replaced by glycine. Amino acids are designated by standard one- and three-letter codes Insulin is a globular protein containing two chains, A (21 residues) and B (30 residues). The monomer in solution (1Hua Q.X., Hu, S.Q. Frank B.H. Jia W. Chu Y.C. Wang S.H. Burke G.T. Katsoyannis P.G. Weiss M.A. J. Mol. Biol. 1996; 264: 390-403Crossref PubMed Scopus (112) Google Scholar, 2Olsen H.B. Ludvigsen S. Kaarsholm N.C. Biochemistry. 1996; 35: 8836-8845Crossref PubMed Scopus (123) Google Scholar) resembles the crystallographic T-state (3Baker E.N. Blundell T.L. Cutfield J.F. Cutfield S.M. Dodson E.J. Dodson G.G. Hodgkin D.M. Hubbard R.E. Isaacs N.W. Reynolds C.D. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1988; 319: 369-456Crossref PubMed Scopus (693) Google Scholar), an α-helix-rich structure stabilized by three disulfide bridges (Fig. 1 A). The hormone is generated in vivo by proteolytic processing of a single-chain precursor, proinsulin (4Steiner D.F. Trans. N. Y. Acad. Sci. 1967; 30: 60-68Crossref PubMed Scopus (94) Google Scholar). Folding proceeds via a preferred disulfide pathway (5Qiao Z.S. Guo Z.Y. Feng Y.M. Biochemistry. 2001; 40: 2662-2668Crossref PubMed Scopus (85) Google Scholar, 6Hua Q.-X. Nakagawa S.H. Jia W., Hu, S.Q. Chu Y.-C. Katsoyannis P.G. Weiss M.A. Biochemistry. 2001; 40: 12299-12311Crossref PubMed Scopus (67) Google Scholar). A peptide model is provided by combination of isolated A and B chains. This reaction, designated insulin chain combination, yields native disulfide pairing (7Katsoyannis P.G. Tometsko A. Proc. Natl. Acad. Sci. U. S. A. 1966; 55: 1554-1561Crossref PubMed Scopus (61) Google Scholar). The absence of disulfide isomers demonstrates that specific folding information resides within the isolated chains (8Tang J.G. Tsou C.L. Biochem. J. 1990; 268: 429-435Crossref PubMed Scopus (46) Google Scholar). Two non-native isomers have been prepared by directed chemical synthesis (9Sieber P.S. Eisler K. Kamber B. Riniker B. Rittel W. Marki F. deGasparo M. Hoppe-Seylers Z. Physiol. Chem. 1978; 359: 113-123PubMed Google Scholar). That such isomers are metastable (converting to insulin in the presence of base; Ref. 9Sieber P.S. Eisler K. Kamber B. Riniker B. Rittel W. Marki F. deGasparo M. Hoppe-Seylers Z. Physiol. Chem. 1978; 359: 113-123PubMed Google Scholar) suggests that the native structure represents the ground state in a space of competing monomeric folds (10Hua Q.X. Gozani S.N. Chance R.E. Hoffmann J.A. Frank B.H. Weiss M.A. Nat. Struct. Biol. 1995; 2: 129-138Crossref PubMed Scopus (120) Google Scholar). Whereas chain combination has enabled the synthesis of many novel insulin analogs (11Slieker L.J. Brooke G.S. DiMarchi R.D. Flora D.B. Green L.K. Hoffmann J.A. Long H.B. Fan L. Shields J.E. Sundell K.L. Surface P.L. Chance R.E. Diabetologia. 1997; 40: S54-S61Crossref PubMed Scopus (138) Google Scholar), including the first commercial recombinant DNA human insulin (12Chance, R. E., Hoffman, J. A., Kroeff, E. P., Johnson, M. G., Schirmer, W. E., Bormer, W. W., Peptides: Synthesis, Structure and Function; Proceedings of the Seventh American Peptide Symposium, Rich, D. H., Gross, E., 1981, 721, 728, Pierce Chemical Co., Rockford, IL.Google Scholar, 13Chance R.E. Frank B.H. Diabetes Care. 1993; 16: 133-142Crossref PubMed Scopus (76) Google Scholar), disulfide pairing can be blocked by specific amino acid substitutions (14Hu S.Q. Burke G.T. Schwartz G.P. Ferderigos N. Ross J.B. Katsoyannis P.G. Biochemistry. 1993; 32: 2631-2635Crossref PubMed Scopus (49) Google Scholar, 15Weiss M.A. Nakagawa S.H. Jia W., Xu, B. Hua Q.X. Chu Y.C. Wang R.Y. Katsoyannis P.G. Biochemistry. 2002; 41: 809-819Crossref PubMed Scopus (32) Google Scholar). Marked variations in yield of biosynthetic expression of variant single-chain precursor polypeptides have also been observed in engineered strains of Saccharomyces cerevisiae (16Kristensen C. Kjeldsen T. Wiberg F.C. Schaffer L. Hach M. Havelund S. Bass J. Steiner D.F. Andersen A.S. J. Biol. Chem. 1997; 272: 12978-12983Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Because the mechanism of folding is not well characterized, it is not known in either case why some analogs are readily prepared while others are not. We have recently shown that LeuA16 makes an essential contribution to the efficiency of insulin chain combination (15Weiss M.A. Nakagawa S.H. Jia W., Xu, B. Hua Q.X. Chu Y.C. Wang R.Y. Katsoyannis P.G. Biochemistry. 2002; 41: 809-819Crossref PubMed Scopus (32) Google Scholar). Invariant among vertebrate insulins and insulin-like growth factors (Refs. 3Baker E.N. Blundell T.L. Cutfield J.F. Cutfield S.M. Dodson E.J. Dodson G.G. Hodgkin D.M. Hubbard R.E. Isaacs N.W. Reynolds C.D. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1988; 319: 369-456Crossref PubMed Scopus (693) Google Scholar and 17Steiner D.F. Chan S.J. Horm. Metab. Res. 1988; 20: 443-444Crossref PubMed Scopus (33) Google Scholar; see arrow in Fig. 1 B), LeuA16 projects between A and B chains to anchor the C-terminal α-helix (Fig. 1 C). Its structural environment is constrained on opposite sides by the internal disulfide bridges of the protein (A6–A11 and A20–B19) and is otherwise bounded by the conserved side chains of IleA2, TyrA19, LeuB11, and LeuB15 (3Baker E.N. Blundell T.L. Cutfield J.F. Cutfield S.M. Dodson E.J. Dodson G.G. Hodgkin D.M. Hubbard R.E. Isaacs N.W. Reynolds C.D. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1988; 319: 369-456Crossref PubMed Scopus (693) Google Scholar). Non-polar substitutions (IleA16, ValA16, and PheA16) were shown to impair disulfide pairing and perturb the thermodynamic stability of the hormone. Once formed, however, A16 analogs retain substantial receptor binding activity, suggesting that LeuA16 does not itself contact the receptor 1It is not known whether impaired chain combination of A16 analogs reflects their thermodynamic instability or a kinetic block to disulfide pairing. Efforts to obtain an AlaA16 analog through biosynthetic expression of a single-chain precursor have been unsuccessful (16Kristensen C. Kjeldsen T. Wiberg F.C. Schaffer L. Hach M. Havelund S. Bass J. Steiner D.F. Andersen A.S. J. Biol. Chem. 1997; 272: 12978-12983Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). The yield of a ValA16 mini-proinsulin analog expressed in S. cerevisiae is likewise negligible (see Footnote 7). (15Weiss M.A. Nakagawa S.H. Jia W., Xu, B. Hua Q.X. Chu Y.C. Wang R.Y. Katsoyannis P.G. Biochemistry. 2002; 41: 809-819Crossref PubMed Scopus (32) Google Scholar). A complementary approach toward dissecting determinants of disulfide pairing is provided by "protein undesign": segmental destabilization of discrete structural elements (18Weiss M.A. Hua Q.-X. Jia W. Chu Y.-C. Wang R.-Y. Katsoyannis P.G. Biochemistry. 2000; 39: 15429-15440Crossref PubMed Scopus (61) Google Scholar). In this article we investigate whether the N-terminal α-helix of the A chain is necessary for the success of chain combination. Inspection of crystal structures suggests that this helix is an integral component of the insulin fold (3Baker E.N. Blundell T.L. Cutfield J.F. Cutfield S.M. Dodson E.J. Dodson G.G. Hodgkin D.M. Hubbard R.E. Isaacs N.W. Reynolds C.D. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1988; 319: 369-456Crossref PubMed Scopus (693) Google Scholar). Introduction of multiple glycine substitutions into the A1–A5 segment (highlighted in red in Fig.1 B) yields analogs that are inactive and less stable than native insulin. Such substitutions shave side-chain contacts and attenuate helical propensities (19Luo Y. Baldwin R.L. Biochemistry. 2001; 40: 5283-5289Crossref PubMed Scopus (35) Google Scholar). Glycine substitutions are studied in the context of a variant B chain designed to prevent formation of insulin dimers and hexamers. The engineered monomer (designated DKP-insulin) exhibits enhanced stability and activity (20Weiss M.A. Frank B.H. Khait I. Pekar A. Heiney R. Shoelson S.E. Neuringer L.J. Biochemistry. 1990; 29: 8389-8401Crossref PubMed Scopus (93) Google Scholar, 21Shoelson S.E., Lu, Z.X. Parlautan L. Lynch C.S. Weiss M.A. Biochemistry. 1992; 31: 1757-1767Crossref PubMed Scopus (75) Google Scholar, 22Brems D.N. Brown P.L. Bryant C. Chance R.E. Green L.K. Long H.B. Miller A.A. Millican R. Shields J.E. Frank B.H. Protein Eng. 1992; 5: 519-525Crossref PubMed Scopus (43) Google Scholar). To correlate synthetic yields with structure, circular dichroism (CD)2 and two-dimensional 1H NMR studies of a representative analog lacking invariant side chains IleA2 and ValA3(A chain sequence GGGEQCCTSICSLYQLENYCN; substitutions underlined and cysteines in bold) are presented. We demonstrate that chain combination is robust to multiple glycine substitutions in the N-terminal segment of the A chain and thus that this segment does not participate in the transition state of the pairing reaction. The solution structure of [GlyA2,GlyA3]DKP-insulin exhibits local unfolding of the A1–A5 segment in an otherwise native-like structure. Although the supersecondary structure of the B chain is not significantly perturbed by the variant A chain, conformational broadening of amide resonances is reduced relative to DKP-insulin. These observations suggest that packing of the native A1–A8 α-helix influences the time scale of fluctuations in the B chain. Non-cooperative detachment of the A1–A5 segment is in accord with the modular partial folds of populated disulfide intermediates in the oxidative folding pathway of proinsulin (1Hua Q.X., Hu, S.Q. Frank B.H. Jia W. Chu Y.C. Wang S.H. Burke G.T. Katsoyannis P.G. Weiss M.A. J. Mol. Biol. 1996; 264: 390-403Crossref PubMed Scopus (112) Google Scholar, 6Hua Q.-X. Nakagawa S.H. Jia W., Hu, S.Q. Chu Y.-C. Katsoyannis P.G. Weiss M.A. Biochemistry. 2001; 40: 12299-12311Crossref PubMed Scopus (67) Google Scholar, 18Weiss M.A. Hua Q.-X. Jia W. Chu Y.-C. Wang R.-Y. Katsoyannis P.G. Biochemistry. 2000; 39: 15429-15440Crossref PubMed Scopus (61) Google Scholar) and insulin-like growth factors (23Narhi L.O. Hua Q.X. Arakawa T. Fox G.M. Tsai L. Rosenfeld R. Holst P. Miller J.A. Weiss M.A. Biochemistry. 1993; 32: 5214-5221Crossref PubMed Scopus (71) Google Scholar, 24Hua Q.X. Narhi L. Jia W. Arakawa T. Rosenfeld R. Hawkins N. Miller J.A. Weiss M.A. J. Mol. Biol. 1996; 259: 297-313Crossref PubMed Scopus (66) Google Scholar). Although of critical importance in receptor recognition (1Hua Q.X., Hu, S.Q. Frank B.H. Jia W. Chu Y.C. Wang S.H. Burke G.T. Katsoyannis P.G. Weiss M.A. J. Mol. Biol. 1996; 264: 390-403Crossref PubMed Scopus (112) Google Scholar, 5Qiao Z.S. Guo Z.Y. Feng Y.M. Biochemistry. 2001; 40: 2662-2668Crossref PubMed Scopus (85) Google Scholar, 25Nakagawa S.H. Tager H.S. Biochemistry. 1992; 31: 3204-3214Crossref PubMed Scopus (84) Google Scholar, 26Olsen H.B. Ludvigsen S. Kaarsholm N.C. J. Mol. Biol. 1998; 284: 477-488Crossref PubMed Scopus (38) Google Scholar, 27Xu B. Hua Q.X. Nakagawa S.H. Jia W. Chu Y.C. Katsoyannis P.G. Weiss M.A. J. Mol. Biol. 2002; 316: 435-441Crossref PubMed Scopus (42) Google Scholar), the N-terminal α-helix of the A chain plays a peripheral role in the specification of disulfide pairing. Hierarchical folding of structural elements (28Baldwin R.L. Rose G.D. Trends Biochem. Sci. 1999; 24: 26-33Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar, 29Baldwin R.L. Rose G.D. Trends Biochem. Sci. 1999; 24: 77-83Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar) may enable discrete surfaces of insulin to reorganize independently on receptor binding (27Xu B. Hua Q.X. Nakagawa S.H. Jia W. Chu Y.C. Katsoyannis P.G. Weiss M.A. J. Mol. Biol. 2002; 316: 435-441Crossref PubMed Scopus (42) Google Scholar, 30Derewenda U. Derewenda Z. Dodson E.J. Dodson G.G. Bing X. Markussen J. J. Mol. Biol. 1991; 220: 425-433Crossref PubMed Scopus (194) Google Scholar, 31Hua Q.X. Shoelson S.E. Kochoyan M. Weiss M.A. Nature. 1991; 354: 238-241Crossref PubMed Scopus (235) Google Scholar). 4-Methylbenzhydrylamine resin (0.6 mmol of amine/g; Star Biochemicals, Inc.) was used as solid support for synthesis of A chain analogs; (N-butoxycarbonyl,O-benzyl)-threonine-PAM resin (0.56 mmol/g; Bachem, Inc.) was used as solid support for synthesis of the DKP B chain analog. tert-Butoxycarbonyl-amino acids and derivatives were obtained from Bachem and Peninsula Laboratories;N,N′-dicyclohexylcarbodiimide andN-hydroxybenzotriazole (recrystallized from 95% ethanol) were from Fluka. Amino acid analyses of synthetic chains and insulin analogs were performed after acid hydrolysis; protein determinations were carried out by the Lowry method using native insulin as standard. Chromatography resins were pre-swollen microgranular carboxymethylcellulose (Whatman CM52), DE53 cellulose (Whatman), and Cellex E (Ecteola cellulose; Sigma); solvents were high performance liquid chromatography (HPLC)-grade. Six insulin analogs were prepared by solid-phase synthesis (Ref. 32Merrifield R.B. Vizioli L.D. Boman H.G. Biochemistry. 1982; 21: 5020-5031Crossref PubMed Scopus (290) Google Scholar; Table I). Crude S-sulfonated A chains were purified by chromatography on a Cellex E column (1.5 × 47 cm) as described (14Hu S.Q. Burke G.T. Schwartz G.P. Ferderigos N. Ross J.B. Katsoyannis P.G. Biochemistry. 1993; 32: 2631-2635Crossref PubMed Scopus (49) Google Scholar, 33Chu Y.C., Hu, S.Q. Zong L. Burke G.T. Gammeltoft S. Chan S. Steiner D.F. Katsoyannis P.G. Biochemistry. 1994; 33: 11278-11285Crossref PubMed Scopus (17) Google Scholar), dialyzed against distilled water, and lyophilized to yield purified A chain S-sulfonate analogs. Crude S-sulfonated DKP B chain was likewise purified on a cellulose DE53 column (1.5 × 47 cm), dialyzed and lyophilized. Chain combination was implemented by the method of Chance and colleagues (12Chance, R. E., Hoffman, J. A., Kroeff, E. P., Johnson, M. G., Schirmer, W. E., Bormer, W. W., Peptides: Synthesis, Structure and Function; Proceedings of the Seventh American Peptide Symposium, Rich, D. H., Gross, E., 1981, 721, 728, Pierce Chemical Co., Rockford, IL.Google Scholar) for 24 h in 0.1 m glycine (pH 10.6) at 4 °C using S-sulfonate-modified A and B chains as described (14Hu S.Q. Burke G.T. Schwartz G.P. Ferderigos N. Ross J.B. Katsoyannis P.G. Biochemistry. 1993; 32: 2631-2635Crossref PubMed Scopus (49) Google Scholar). Concentrations of A and B chains were 1.2 and 0.4 mm, respectively; a molar excess of A chain favors A-B pairing (relative to B chain aggregation) and compensates for formation of cyclic A chains. S-Sulfonate modification enhances peptide solubility and renders peptides refractory to disulfide chemistry. Specific A-B pairing is initiated by addition of dithiothreitol in quantity stoichiometric to the concentration of S-sulfonate groups. Relative yields are given in Table II. In a mock chain-combination reaction lacking the A chain, 10% of the B chain mass remained as HPLC-recoverable monomers after 24 h, whereas 60% was sedimented by microcentrifugation; in a mock reaction lacking B chain, the oxidized A chains remained soluble and recoverable by reverse-phase HPLC (RP-HPLC).Table IReceptor binding activitiesAnalogActivity%Human insulin100DKP-insulinaDKP-insulin contains three substitutions in the B chain to prevent self-assembly; the classical dimer interface is destabilized by substitutions ProB28 → Lys and LysB29→ Pro, whereas the hexamer interface is destabilized by HisB10 → Asp (21, 56). The stability of DKP-insulin is greater than that of the native insulin monomer (Ref. 22; see Table II).161 ± 19 (4)GlyA2-DKP-insulin (2G)0.13 ± 0.06 (3)GlyA3-DKP-insulin0.15 ± 0.03 (3)5G-insulin0.03 ± 0.01 (3)3G-DKP-insulin0.04 ± 0.01 (4)4G-DKP-insulinND5G-DKP-insulinNDActivity is defined by affinity for the human placental insulin receptor relative to human insulin (100%); number of assays is given in parenthesis with standard deviations provided. Under these conditions the K d for native insulin is 0.48 ± 0.06 nM. ND, not determined.a DKP-insulin contains three substitutions in the B chain to prevent self-assembly; the classical dimer interface is destabilized by substitutions ProB28 → Lys and LysB29→ Pro, whereas the hexamer interface is destabilized by HisB10 → Asp (21Shoelson S.E., Lu, Z.X. Parlautan L. Lynch C.S. Weiss M.A. Biochemistry. 1992; 31: 1757-1767Crossref PubMed Scopus (75) Google Scholar, 56Weiss M.A. Hua Q.X. Lynch C.S. Frank B.H. Shoelson S.E. Biochemistry. 1991; 30: 7373-7389Crossref PubMed Scopus (97) Google Scholar). The stability of DKP-insulin is greater than that of the native insulin monomer (Ref. 22Brems D.N. Brown P.L. Bryant C. Chance R.E. Green L.K. Long H.B. Miller A.A. Millican R. Shields J.E. Frank B.H. Protein Eng. 1992; 5: 519-525Crossref PubMed Scopus (43) Google Scholar; see Table II). Open table in a new tab Table IIProperties of insulin analogsProteinYieldaYield is defined following purification of analogs by CM-cellulose chromatography. Values pertain to chain combination at basic pH and are provided in both sections A and B for the convenience of the reader. In control synthesis of DKP-insulin a mixture of 60 mg of A chain-S-sulfonate and 30 mg of DKP B chain-S-sulfonate yields 12 mg of monocomponent DKP-insulin.ΔG ubΔGu (kcal/mol) indicates apparent change in free energy on denaturation in guanidine-HCl as extrapolated to zero denaturant concentration by a two-state model (36).ΔΔG ucΔΔGu (kcal/mol) indicates difference in ΔGu values relative to parent structure (DKP-insulin or human insulin). Uncertainties in two-state fitting parameters do not include possible systematic error due to non-two-state behavior.C middCMid is defined as that concentration of guanidine-HCl at which 50% of the protein is unfolded.m eThe m value is the slope obtained in plotting unfolding free energy ΔGu versus molar concentration of denaturant; in some cases this slope may be proportional to the protein surface area exposed on unfolding.A. Neutral pH%mkcal/mol/mHuman insulin1004.4 ± 0.15.3 ± 0.10.84 ± 0.01DKP-insulin1104.9 ± 0.15.8 ± 0.10.84 ± 0.01GlyA2-DKP-insulin106GlyA3-DKP-insulin (2G)882.9 ± 0.2−2.0 ± 0.25.0 ± 0.10.57 ± 0.073G-DKP-insulin782.8 ± 0.1−2.1 ± 0.24.4 ± 0.10.64 ± 0.014G-DKP-insulin962.4 ± 0.1−2.5 ± 0.24.0 ± 0.10.61 ± 0.015G-DKP-insulin1012.7 ± 0.1−2.2 ± 0.24.2 ± 0.10.63 ± 0.015G-insulin1032.0 ± 0.1−2.4 ± 0.24.2 ± 0.10.60 ± 0.01DKP-des-[A6-A11]SerfLack of sigmoidicity in transitions makes uncertain fitting of pre-transition base line; only upper bounds to stability have been estimated (6).90< 2.2>−3.0 ± 0.4< 3.20.65 ± 0.07DKP-des-[A7-B7]Ser33< 1.0>−4.4 ± 0.2< 1.50.46 ± 0.03B. Basic pHHuman insulin1003.2 ± 0.14.3 ± 0.10.75 ± 0.02DKP-insulin1103.9 ± 0.15.3 ± 0.10.73 ± 0.023G-DKP-insulin783.4 ± 0.1−0.5 ± 0.14.6 ± 0.10.74 ± 0.024G-DKP-insulin963.5 ± 0.1−0.4 ± 0.14.6 ± 0.10.76 ± 0.025G-DKP-insulin1013.2 ± 0.1−0.7 ± 0.13.5 ± 0.10.70 ± 0.025G-insulin1032.5 ± 0.1−0.7 ± 0.23.6 ± 0.10.58 ± 0.02a Yield is defined following purification of analogs by CM-cellulose chromatography. Values pertain to chain combination at basic pH and are provided in both sections A and B for the convenience of the reader. In control synthesis of DKP-insulin a mixture of 60 mg of A chain-S-sulfonate and 30 mg of DKP B chain-S-sulfonate yields 12 mg of monocomponent DKP-insulin.b ΔGu (kcal/mol) indicates apparent change in free energy on denaturation in guanidine-HCl as extrapolated to zero denaturant concentration by a two-state model (36Sosnick T.R. Fang X. Shelton V.M. Methods Enzymol. 2000; 317: 393-409Crossref PubMed Google Scholar).c ΔΔGu (kcal/mol) indicates difference in ΔGu values relative to parent structure (DKP-insulin or human insulin). Uncertainties in two-state fitting parameters do not include possible systematic error due to non-two-state behavior.d CMid is defined as that concentration of guanidine-HCl at which 50% of the protein is unfolded.e The m value is the slope obtained in plotting unfolding free energy ΔGu versus molar concentration of denaturant; in some cases this slope may be proportional to the protein surface area exposed on unfolding.f Lack of sigmoidicity in transitions makes uncertain fitting of pre-transition base line; only upper bounds to stability have been estimated (6Hua Q.-X. Nakagawa S.H. Jia W., Hu, S.Q. Chu Y.-C. Katsoyannis P.G. Weiss M.A. Biochemistry. 2001; 40: 12299-12311Crossref PubMed Scopus (67) Google Scholar). Open table in a new tab Activity is defined by affinity for the human placental insulin receptor relative to human insulin (100%); number of assays is given in parenthesis with standard deviations provided. Under these conditions the K d for native insulin is 0.48 ± 0.06 nM. ND, not determined. Insulin analogs were isolated from the combination mixture as described (14Hu S.Q. Burke G.T. Schwartz G.P. Ferderigos N. Ross J.B. Katsoyannis P.G. Biochemistry. 1993; 32: 2631-2635Crossref PubMed Scopus (49) Google Scholar, 33Chu Y.C., Hu, S.Q. Zong L. Burke G.T. Gammeltoft S. Chan S. Steiner D.F. Katsoyannis P.G. Biochemistry. 1994; 33: 11278-11285Crossref PubMed Scopus (17) Google Scholar) and purified on a 0.9 × 23-cm carboxymethylcellulose chromatography and RP-HPLC on a Vydac 218 TP column (0.46 × 25 cm); the latter used a flow rate of 0.5 ml/min with 20–80% linear gradient of 80% aqueous acetonitrile containing 0.1% trifluoroacetic acid over 80 min. Re-chromatography of this material on RP-HPLC under the same conditions in each case gave a single sharp peak. Amino acid analyses and mass spectrometry in each case gave expected values. The purity of the insulin analogs was in each case greater than 98% as evaluated by analytical reverse-phase HPLC. Electrospray mass spectra revealed no anomalous molecular masses as contaminants. Native disulfide pairing of GlyA3-insulin was determined by x-ray crystallography in an R6 zinc hexamer. 3G. D. Smith, S. Nakagawa, H. S. Tager, and M. A. Weiss, manuscript in preparation. Native disulfide pairing of G3-DKP-insulin was verified by two-dimensional NMR spectroscopy as follows. A native-like nuclear Overhauser effect (NOE) on the protein surface is observed between the β proton of CysA7 and the α proton of CysB7; a native-like NOE in the core is observed between the β proton of CysA6 and the side chain of LeuB6; the positions of CysA11, CysA19, and CysB20 are well defined in the core by a native-like network of inter-residue NOEs involving LeuA16, LeuB11, and PheB24. Imposition of non-native pairing schemes in molecular models would violate one or more of these NOEs. Correct pairing of the remaining analogs was assumed. 4Insulin chain combination in each case was observed to generate a unique product and not a mixture of isomers, which typically exhibit very different HPLC mobilities (10Hua Q.X. Gozani S.N. Chance R.E. Hoffmann J.A. Frank B.H. Weiss M.A. Nat. Struct. Biol. 1995; 2: 129-138Crossref PubMed Scopus (120) Google Scholar). To our knowledge, in no instance have mutations in insulin caused mispairing in synthesis. Gross substitution of the entire insulin B chain by the B domain of insulin-like growth factor I (IGF-I) yields native and non-native isomers in equilibrium (57Guo Z.Y. Shen L. Feng Y.M. Biochemistry. 2002; 41: 1556-1567Crossref PubMed Scopus (34) Google Scholar) in accord with the anomalous refolding properties of IGF-I (49Hober S. Forsberg G. Palm G. Hartmanis M. Nilsson B. Biochemistry. 1992; 31: 1749-1756Crossref PubMed Scopus (104) Google Scholar, 50Miller J.A. Narhi L.O. Hua Q.X. Rosenfeld R. Arakawa T. Rohde M. Prestrelski S. Lauren S. Stoney K.S. Tsai L. Weiss M.A. Biochemistry. 1993; 32: 5203-5213Crossref PubMed Scopus (104) Google Scholar). Radiolabeled [125I-TyrA14]human insulin was purchased fromAmersham Biosciences. Receptor binding assays were performed as described (34Cara J.F. Mirmira R.G. Nakagawa S.H. Tager H.S. J. Biol. Chem. 1990; 265: 17820-17825Abstract Full Text PDF PubMed Google Scholar) with minor modifications. Human placental cell membranes were prepared (35Marshall R.N. Underwood L.E. Voina S.J. Foushee D.B. Van Wyk J.J. J. Clin. Endocrinol. Metab. 1974; 39: 283-292Crossref PubMed Scopus (239) Google Scholar), stored at −80 °C in small aliquots, and thawed prior to use. Membrane fragments (0.025 mg of protein/tube) were incubated with 125I-labeled insulin (∼30,000 cpm) in presence of selected concentrations of unlabeled peptide for 18 h at 4 °C in a final volume of 0.25 ml of 0.05 m Tris-HCl and 0.25% (w/v) bovine serum albumin at pH 8. Subsequent to incubation, each mixture was diluted with 1 ml of ice-cold buffer and centrifuged (10,000 × g) for 5 min at 4 °C. The supernatant was then removed by aspiration, and the membrane pellet counted for radioactivity. Data were corrected for nonspecific binding (amount of radioactivity remaining membrane-associated in the presence of 1 μm human insulin). Each determination was performed with three or four replicates (see Table I); values are reported as mean and standard deviation. Far-ultraviolet (UV) CD spectra of each analog were obtained using an Aviv spectropolarimeter equipped with thermister temperature control and automated titration unit for guanidine denaturation studies. CD samples for wavelength spectra contained 25–50 μm insulin analog in (a) 50 mm KCl and 10 mm potassium phosphate (pH 7.4) or (b) 100 mm KCl and 10 mm glycine (pH 10.5); samples were diluted to 5 μm for equilibrium denaturation studies. Data were obtained at 4 °C unless otherwise indicated. Guanidine denaturation data were fitted by non-linear least squares (36Sosnick T.R. Fang X. Shelton V.M. Methods Enzymol. 2000; 317: 393-409Crossref PubMed Google Scholar). In brief, CD data were fitted to Equation 1. θ(x)=θA+θBe(−ΔGH2Oo−mx)/RT1+e−(ΔGH2Oo−mx)/RTEquation 1 x is the concentration of guanidine hydrochloride, and θA and θB are base-line values in the native and unfolded states. These base lines were approximated by pre- and post-transition lines θA(x) = θ AH2O +mAx and θB(x) = θ BH2O +mBx . Fitting CD data and base lines simultaneously circumvents artifacts associated with linear plots of ΔG as a function of denaturant according to ΔGo(x) = ΔG H2Oo + mBx (for review, see Ref. 36Sosnick T.R. Fang X. Shelton V.M. Methods Enzymol. 2000; 317: 393-40

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