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• W2034628566 abstract "Protein evolution is constrained by folding efficiency (“foldability”) and the implicit threat of toxic misfolding. A model is provided by proinsulin, whose misfolding is associated with β-cell dysfunction and diabetes mellitus. An insulin analogue containing a subtle core substitution (LeuA16 → Val) is biologically active, and its crystal structure recapitulates that of the wild-type protein. As a seeming paradox, however, ValA16 blocks both insulin chain combination and the in vitro refolding of proinsulin. Disulfide pairing in mammalian cell culture is likewise inefficient, leading to misfolding, endoplasmic reticular stress, and proteosome-mediated degradation. ValA16 destabilizes the native state and so presumably perturbs a partial fold that directs initial disulfide pairing. Substitutions elsewhere in the core similarly destabilize the native state but, unlike ValA16, preserve folding efficiency. We propose that LeuA16 stabilizes nonlocal interactions between nascent α-helices in the A- and B-domains to facilitate initial pairing of CysA20 and CysB19, thus surmounting their wide separation in sequence. Although ValA16 is likely to destabilize this proto-core, its structural effects are mitigated once folding is achieved. Classical studies of insulin chain combination in vitro have illuminated the impact of off-pathway reactions on the efficiency of native disulfide pairing. The capability of a polypeptide sequence to fold within the endoplasmic reticulum may likewise be influenced by kinetic or thermodynamic partitioning among on- and off-pathway disulfide intermediates. The properties of [ValA16]insulin and [ValA16]proinsulin demonstrate that essential contributions of conserved residues to folding may be inapparent once the native state is achieved. Protein evolution is constrained by folding efficiency (“foldability”) and the implicit threat of toxic misfolding. A model is provided by proinsulin, whose misfolding is associated with β-cell dysfunction and diabetes mellitus. An insulin analogue containing a subtle core substitution (LeuA16 → Val) is biologically active, and its crystal structure recapitulates that of the wild-type protein. As a seeming paradox, however, ValA16 blocks both insulin chain combination and the in vitro refolding of proinsulin. Disulfide pairing in mammalian cell culture is likewise inefficient, leading to misfolding, endoplasmic reticular stress, and proteosome-mediated degradation. ValA16 destabilizes the native state and so presumably perturbs a partial fold that directs initial disulfide pairing. Substitutions elsewhere in the core similarly destabilize the native state but, unlike ValA16, preserve folding efficiency. We propose that LeuA16 stabilizes nonlocal interactions between nascent α-helices in the A- and B-domains to facilitate initial pairing of CysA20 and CysB19, thus surmounting their wide separation in sequence. Although ValA16 is likely to destabilize this proto-core, its structural effects are mitigated once folding is achieved. Classical studies of insulin chain combination in vitro have illuminated the impact of off-pathway reactions on the efficiency of native disulfide pairing. The capability of a polypeptide sequence to fold within the endoplasmic reticulum may likewise be influenced by kinetic or thermodynamic partitioning among on- and off-pathway disulfide intermediates. The properties of [ValA16]insulin and [ValA16]proinsulin demonstrate that essential contributions of conserved residues to folding may be inapparent once the native state is achieved. A fundamental problem in biophysics is posed by the efficiency of protein folding (1.Onuchic J.N. Luthey-Schulten Z. Wolynes P.G. Annu. Rev. Phys. Chem. 1997; 48: 545-600Crossref PubMed Google Scholar, 2.Dobson C.M. Karplus M. Curr. Opin. Struct. Biol. 1999; 9: 92-101Crossref PubMed Scopus (347) Google Scholar). The capability of folding is an evolved property of biological sequences, i.e. not broadly representative of polypeptides as a class of heteropolymers (3.Shakhnovich E. Farztdinov G. Gutin A.M. Karplus M. Phys. Rev. Lett. 1991; 67: 1665-1668Crossref PubMed Scopus (253) Google Scholar). A funnel-shaped free-energy landscape enables an efficient conformational search leading by multiple trajectories to the native state (4.Onuchic J.N. Socci N.D. Luthey-Schulten Z. Wolynes P.G. Fold. Des. 1996; 1: 441-450Abstract Full Text Full Text PDF PubMed Google Scholar, 5.Dill K.A. Chan H.S. Nat. Struct. Biol. 1997; 4: 10-19Crossref PubMed Scopus (1912) Google Scholar, 6.Lazaridis T. Karplus M. Science. 1997; 278: 1928-1931Crossref PubMed Scopus (472) Google Scholar). What distinguishes foldable from nonfoldable sequences (7.Mirny L.A. Abkevich V.I. Shakhnovich E.I. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 4976-4981Crossref PubMed Scopus (159) Google Scholar), and how do productive trajectories avoid bottlenecks (8.Dinner A.R. Abkevich V. Shakhnovich E. Karplus M. Proteins. 1999; 35: 34-40Crossref PubMed Scopus (45) Google Scholar, 9.Tiana G. Broglia R.A. Shakhnovich E.I. Proteins. 2000; 39: 244-251Crossref PubMed Scopus (55) Google Scholar, 10.Oliveberg M. Wolynes P.G. Q. Rev. Biophys. 2005; 38: 245-288Crossref PubMed Scopus (222) Google Scholar)? Despite the centrality of these questions, experimental approaches are limited. Here, we combine biophysical and cellular studies to identify a residue critical to the efficiency of oxidative folding. A model is provided by insulin, a globular protein central to the regulation of vertebrate metabolism (11.Dodson G. Steiner D. Curr. Opin. Struct. Biol. 1998; 8: 189-194Crossref PubMed Scopus (395) Google Scholar). The insulin gene encodes a single-chain precursor, preproinsulin (Fig. 1A, top) (12.Steiner D.F. Chan S.J. Horm. Metab. Res. 1988; 20: 443-444Crossref PubMed Google Scholar). A signal peptide (Fig. 1A, gray bar) is cleaved on translocation into the endoplasmic reticulum (ER) 5The abbreviations used are: ERendoplasmic reticulumDKP-insulininsulin analogue containing three substitutions in B-chain (AspB10, LysB28 and ProB29)DMdiabetes mellitusHPLChigh performance liquid chromatographyUPRunfolded protein responseTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycinePDBProtein Data Bank. Amino acids are designated by three-letter code. to yield proinsulin. The precursor contains successive sequence motifs, defining a B-domain (Fig. 1A, blue bar), connecting domain (black bar), and A-domain (red bar) (13.Steiner D.F. Trans. N. Y. Acad. Sci. 1967; 30: 60-68Crossref PubMed Google Scholar). The translocated polypeptide is reduced and unfolded. Biophysical studies suggest that folding in the ER yields a well organized A-B (insulin-like) core and disordered C-domain (Fig. 1B, dashed black segment) (14.Frank B.H. Veros A.J. Biochem. Biophys. Res. Commun. 1968; 32: 155-160Crossref PubMed Google Scholar, 15.Pekar A.H. Frank B.H. Biochemistry. 1972; 11: 4013-4016Crossref PubMed Google Scholar, 16.Frank B.H. Pekar A.H. Veros A.J. Diabetes. 1972; 21: 486-491Crossref PubMed Google Scholar, 17.Snell C.R. Smyth D.G. J. Biol. Chem. 1975; 250: 6291-6295Abstract Full Text PDF PubMed Google Scholar, 18.Brems D.N. Brown P.L. Heckenlaible L.A. Frank B.H. Biochemistry. 1990; 29: 9289-9293Crossref PubMed Google Scholar, 19.Weiss M.A. Frank B.H. Khait I. Pekar A. Heiney R. Shoelson S.E. Neuringer L.J. Biochemistry. 1990; 29: 8389-8401Crossref PubMed Google Scholar). Folding of proinsulin is coupled to disulfide pairing (Fig. 1, A and B, gold A6–A11, A7–B7, and A20–B19). Two cystines provide interior struts (A20–B19 and A6–A11), whereas the third forms an external staple (A7–B7). These structural elements are required for stability and activity (20.Narhi 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 Google Scholar, 21.Hua 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 (106) Google Scholar, 22.Dai Y. Tang J.G. Biochim. Biophys. Acta. 1996; 1296: 63-68Crossref PubMed Scopus (35) Google Scholar, 23.Hober S. Uhlén M. Nilsson B. Biochemistry. 1997; 36: 4616-4622Crossref PubMed Scopus (29) Google Scholar, 24.Weiss M.A. Hua Q.X. Jia W. Chu Y.C. Wang R.Y. Katsoyannis P.G. Biochemistry. 2000; 39: 15429-15440Crossref PubMed Scopus (56) Google Scholar, 25.Guo Z.Y. Feng Y.M. Biol. Chem. 2001; 382: 443-448Crossref PubMed Scopus (48) Google Scholar, 26.Feng Y. Liu D. Wang J. J. Mol. Biol. 2003; 330: 821-837Crossref PubMed Google Scholar, 27.Jia X.Y. Guo Z.Y. Wang Y. Xu Y. Duan S.S. Feng Y.M. Protein Sci. 2003; 12: 2412-2419Crossref PubMed Scopus (24) Google Scholar, 28.Yan H. Guo Z.Y. Gong X.W. Xi D. Feng Y.M. Protein Sci. 2003; 12: 768-775Crossref PubMed Scopus (32) Google Scholar, 29.Hua Q.X. Mayer J.P. Jia W. Zhang J. Weiss M.A. J. Biol. Chem. 2006; 281: 28131-28142Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Insulin disulfide isomers exhibit molten structures of marginal stability and low activity (30.Sieber P. Eisler K. Kamber B. Riniker B. Rittel W. Märki F. de Gasparo M. Hoppe-Seylers Z. Physiol. Chem. 1978; 359: 113-123PubMed Google Scholar, 31.Hua 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 (111) Google Scholar, 32.Hua Q.X. Jia W. Frank B.H. Phillips N.F. Weiss M.A. Biochemistry. 2002; 41: 14700-14715Crossref PubMed Scopus (66) Google Scholar). Proinsulin isomers have been observed at low concentration in β-cells (33.Liu M. Li Y. Cavener D. Arvan P. J. Biol. Chem. 2005; 280: 13209-13212Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), and their accumulation may be linked to β-cell dysfunction (34.Ron D. J. Clin. Invest. 2002; 109: 443-445Crossref PubMed Google Scholar, 35.Liu M. Hodish I. Rhodes C.J. Arvan P. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 15841-15846Crossref PubMed Scopus (110) Google Scholar). Misfolding of proinsulin variants is associated with non-native aggregation, leading to β-cell dysfunction and in turn diabetes mellitus (36.Støy J. Edghill E.L. Flanagan S.E. Ye H. Paz V.P. Pluzhnikov A. Below J.E. Hayes M.G. Cox N.J. Lipkind G.M. Lipton R.B. Greeley S.A. Patch A.M. Ellard S. Steiner D.F. Hattersley A.T. Philipson L.H. Bell G.I. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 15040-15044Crossref PubMed Scopus (373) Google Scholar, 37.Colombo C. Porzio O. Liu M. Massa O. Vasta M. Salardi S. Beccaria L. Monciotti C. Toni S. Pedersen O. Hansen T. Federici L. Pesavento R. Cadario F. Federici G. Ghirri P. Arvan P. Iafusco D. Barbetti F. Early Onset Diabetes Study Group of the Italian Society of Pediatric Endocrinology and DiabetesJ. Clin. Invest. 2008; 118: 2148-2156PubMed Google Scholar, 38.Edghill E.L. Flanagan S.E. Patch A.M. Boustred C. Parrish A. Shields B. Shepherd M.H. Hussain K. Kapoor R.R. Malecki M. MacDonald M.J. Støy J. Steiner D.F. Philipson L.H. Bell G.I. Hattersley A.T. Ellard S. Diabetes. 2008; 57: 1034-1042Crossref PubMed Scopus (256) Google Scholar, 39.Molven A. Ringdal M. Nordbø A.M. Raeder H. Støy J. Lipkind G.M. Steiner D.F. Philipson L.H. Bergmann I. Aarskog D. Undlien D.E. Joner G. Søvik O. Bell G.I. Njølstad P.R. Diabetes. 2008; 57: 1131-1135Crossref PubMed Scopus (136) Google Scholar). endoplasmic reticulum insulin analogue containing three substitutions in B-chain (AspB10, LysB28 and ProB29) diabetes mellitus high performance liquid chromatography unfolded protein response N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine Protein Data Bank. Amino acids are designated by three-letter code. Following the folding of proinsulin in the ER, the biosynthetic pathway of insulin imposes additional structural constraints. After transit through the Golgi apparatus and entry into immature secretory granules (40.Huang X.F. Arvan P. J. Biol. Chem. 1994; 269: 20838-20844Abstract Full Text PDF PubMed Google Scholar), a specific set of prohormone convertases proteolytically excises the C-peptide at conserved dibasic sites (BC and CA junctions; Fig. 1, A and B, green), liberating the bioactive hormone (41.Steiner D.F. Clark J.L. Nolan C. Rubenstein A.H. Margoliash E. Aten B. Oyer P.E. Recent Prog. Horm. Res. 1969; 25: 207-282PubMed Google Scholar, 42.Galloway J.A. Hooper S.A. Spradlin C.T. Howey D.C. Frank B.H. Bowsher R.R. Anderson J.H. Diabetes Care. 1992; 15: 666-692Crossref PubMed Google Scholar, 43.Steiner D.F. Curr. Opin. Chem. Biol. 1998; 2: 31-39Crossref PubMed Google Scholar). Insulin thus contains two chains, A (21 residues) and B (30 residues), and is stored as Zn2+-stabilized hexamers within specialized secretory granules (Fig. 1C) (44.Huang X.F. Arvan P. J. Biol. Chem. 1995; 270: 20417-20423Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The hexamers dissociate upon secretion into the portal circulation, enabling the circulating hormone to function as a Zn2+-free monomer (Fig. 1C, right). This study investigates a mutant proinsulin defective in folding and yet active and well organized once the native state is achieved. Substitution of LeuA16 by Val (45.Weiss 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 (29) Google Scholar) reduces the efficiency of disulfide pairing in vitro and leads in mammalian cells to accelerated degradation associated with ER stress (46.Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Google Scholar, 47.Yoshida H. FEBS J. 2007; 274: 630-658Crossref PubMed Scopus (782) Google Scholar). Despite these perturbations, the crystal structure of the variant (as a mutant insulin) is essentially identical to wild type (48.Baker 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 Google Scholar, 49.Wan Z. Xu B. Huang K. Chu Y.C. Li B. Nakagawa S.H. Qu Y. Hu S.Q. Katsoyannis P.G. Weiss M.A. Biochemistry. 2004; 43: 16119-16133Crossref PubMed Scopus (29) Google Scholar). We propose that ValA16 allows formation of off-pathway oxidative intermediates and/or destabilizes on-pathway intermediates (50.Weiss M.A. J. Biol. Chem. 2009; 284: 19159-19163Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Such perturbations are similar to those proposed to underlie a newly recognized syndrome of toxic protein misfolding, permanent neonatal-onset diabetes mellitus (DM) due to mutations in proinsulin (36.Støy J. Edghill E.L. Flanagan S.E. Ye H. Paz V.P. Pluzhnikov A. Below J.E. Hayes M.G. Cox N.J. Lipkind G.M. Lipton R.B. Greeley S.A. Patch A.M. Ellard S. Steiner D.F. Hattersley A.T. Philipson L.H. Bell G.I. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 15040-15044Crossref PubMed Scopus (373) Google Scholar, 37.Colombo C. Porzio O. Liu M. Massa O. Vasta M. Salardi S. Beccaria L. Monciotti C. Toni S. Pedersen O. Hansen T. Federici L. Pesavento R. Cadario F. Federici G. Ghirri P. Arvan P. Iafusco D. Barbetti F. Early Onset Diabetes Study Group of the Italian Society of Pediatric Endocrinology and DiabetesJ. Clin. Invest. 2008; 118: 2148-2156PubMed Google Scholar, 38.Edghill E.L. Flanagan S.E. Patch A.M. Boustred C. Parrish A. Shields B. Shepherd M.H. Hussain K. Kapoor R.R. Malecki M. MacDonald M.J. Støy J. Steiner D.F. Philipson L.H. Bell G.I. Hattersley A.T. Ellard S. Diabetes. 2008; 57: 1034-1042Crossref PubMed Scopus (256) Google Scholar, 39.Molven A. Ringdal M. Nordbø A.M. Raeder H. Støy J. Lipkind G.M. Steiner D.F. Philipson L.H. Bergmann I. Aarskog D. Undlien D.E. Joner G. Søvik O. Bell G.I. Njølstad P.R. Diabetes. 2008; 57: 1131-1135Crossref PubMed Scopus (136) Google Scholar). Although to date clinical mutations (excluding insertion or removal of cysteines) have clustered in the B-domain, our results demonstrate that the A-domain is also required for efficient folding. The aberrant properties of [ValA16]proinsulin thus provide a model for the molecular genetics of a disease of protein misfolding. Insulin was provided by Eli Lilly and Co.; S-sulfonate B-chain derivatives were obtained by oxidative sulfitolysis (51.Hu S.Q. Burke G.T. Schwartz G.P. Ferderigos N. Ross J.B. Katsoyannis P.G. Biochemistry. 1993; 32: 2631-2635Crossref PubMed Google Scholar). A- and B-chain analogues were otherwise prepared by solid-phase synthesis (51.Hu S.Q. Burke G.T. Schwartz G.P. Ferderigos N. Ross J.B. Katsoyannis P.G. Biochemistry. 1993; 32: 2631-2635Crossref PubMed Google Scholar). Insulin analogues were prepared by chain combination as described previously (45.Weiss 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 (29) Google Scholar). Predicted molecular masses were confirmed by mass spectrometry. Wild-type and variant proinsulins were expressed in Escherichia coli (52.Mackin R.B. Choquette M.H. Protein Expr. Purif. 2003; 27: 210-219Crossref PubMed Scopus (14) Google Scholar); wild-type and variant mini-proinsulins (53-residue construct) were expressed in folded secreted form in Pichia pastoris (53.Wang Y. Liang Z.H. Zhang Y.S. Yao S.Y. Xu Y.G. Tang Y.H. Zhu S.Q. Cui D.F. Feng Y.M. Biotechnol. Bioeng. 2001; 73: 74-79Crossref PubMed Scopus (0) Google Scholar). HEK293T cells were cultured at 37 °C in high glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 0.1% penicillin/streptomycin with 5% CO2. CHO-CLA14 cells were maintained in low glucose Dulbecco's modified Eagle's medium plus 400 μg/ml G418 and 200 μg/ml hygromycin at 37 °C with 5% CO2 (54.Liu M. Ramos-Castañeda J. Arvan P. J. Biol. Chem. 2003; 278: 14798-14805Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). For metabolic labeling, cells were plated into 6-well plates 1 day before transfection. Plasmid DNA (2 μg) was transfected into each well using Lipofectamine (Invitrogen). For studies of the unfolded protein response (UPR), cells were seeded into 24-well plates 1 day before transfection. Plasmid DNAs containing proinsulin (wild type or variant) were co-transfected with plasmids (provided by R. Kaufman) encoding luciferase (driven by BiP promoter) and β-galactosidase (driven by CMV promoter) at 5:2:1 ratio using Lipofectamine; assays were performed in triplicate. At 40 h post-transfection, cells were preincubated in methionine/cysteine-deficient medium for 30 min, metabolically labeled in the same medium containing 35S-labeled Met and Cys for 1 h, washed once with complete medium, and chased for the indicated times. To examine degradation, labeled cells were chased for 4 h with or without 20 μm MG132 or lactacystin. After chase media were collected, cells were lysed in 100 mm NaCl, 1% Triton X-100, 0.2% sodium deoxycholate, 0.1% SDS, 10 mm EDTA, and 25 mm Tris-HCl (pH 7.4). Lysates and chase media were immunoprecipitated with guinea pig anti-insulin antiserum (LINCO Diagnostics, Inc.) and analyzed by Tris-Tricine/urea-SDS-PAGE under reducing and nonreducing conditions (33.Liu M. Li Y. Cavener D. Arvan P. J. Biol. Chem. 2005; 280: 13209-13212Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 55.Hua Q.X. Liu M. Hu S.Q. Jia W. Arvan P. Weiss M.A. J. Biol. Chem. 2006; 281: 24889-24899Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The UPR assay (see supplemental Fig. S1 and supplemental Table S1) was performed as described previously (56.Tirasophon W. Welihinda A.A. Kaufman R.J. Genes Dev. 1998; 12: 1812-1824Crossref PubMed Google Scholar). At 40 h post-transfection cells were washed three times with phosphate-buffered saline, lysed in luciferase assay buffer, and transferred to a 96-well plate; luminometry was performed in a Turner Biosystems plate luminometer using the Dual-Light assay system (Applied Biosystems). Statistical analysis employed a randomized block design analysis of variance model after checking for appropriateness of a normality assumption on errors using a Q-Q plot with Wilk-Shapiro test. Testing (two-tailed) was done at a significance level of α = 0.05. When significant findings were discovered, a nonparametric version of analysis of variance was run to ensure consistency. CD spectra, obtained using an Aviv spectropolarimeter equipped with a titration unit for denaturation studies, were measured at a protein concentration of 5 μm at 4 °C (57.Hua Q.X. Chu Y.C. Jia W. Phillips N.F. Wang R.Y. Katsoyannis P.G. Weiss M.A. J. Biol. Chem. 2002; 277: 43443-43453Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). 1H NMR spectra were obtained at 700 MHz in D2O solution at pH 7.0 and 25 °C (21.Hua 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 (106) Google Scholar). CD-detected guanidine denaturation data (222 nm) were fitted by nonlinear least squares to a two-state model (supplemental Table S2) as described previously (58.Sosnick T.R. Fang X. Shelton V.M. Methods Enzymol. 2000; 317: 393-409Crossref PubMed Google Scholar). In brief, CD data, θ(x), were fitted by a nonlinear least-squares program according to Equation 1, θ(x)=θA+θBe(-ΔGH2O0-mx)/RT1+e(-ΔGH2O0-mx)/RT(Eq. 1) where x is the concentration of guanidine hydrochloride, and where 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) = θAH2O + mAx and θBH2O + mBx. Fitting the original CD data and base lines simultaneously circumvents artifacts associated with linear plots of DG as a function of denaturant according to ΔG0(x) = ΔGH2O0 + m0x (for reviews see Refs. 58.Sosnick T.R. Fang X. Shelton V.M. Methods Enzymol. 2000; 317: 393-409Crossref PubMed Google Scholar, 59.Pace C.N. Shaw K.L. Proteins. 2000; 4: 1-7Crossref PubMed Google Scholar). Crystals were grown by hanging-drop vapor diffusion with a 1:2.5 ratio of Zn2+ to protein monomer and 3.7:1 ratio of phenol to protein monomer (49.Wan Z. Xu B. Huang K. Chu Y.C. Li B. Nakagawa S.H. Qu Y. Hu S.Q. Katsoyannis P.G. Weiss M.A. Biochemistry. 2004; 43: 16119-16133Crossref PubMed Scopus (29) Google Scholar). Drops consisted of 1 μl of protein solution (10 mg/ml in 0.02 m HCl) mixed with 1 μl of reservoir solution (0.02 m Tris-HCl, 0.05 m sodium citrate, 5% acetone, 0.03% phenol, and 0.01% zinc acetate (pH 8.0)). Crystals (space group R3) were obtained at room temperature after 2 weeks. Data were collected from single crystals flash-frozen to 100 K. Reflections from 22.58 to 1.8 Å were measured using synchrotron radiation (Chess, Cornell University). Data were processed with programs DENZO (version 1.9.6) and SCALEPACK (version 1.9.6). The structure was determined by molecular replacement using CNS; initial model was the wild-type TRf dimer (PDB identifier 1RWE) following removal of water, zinc, and chloride ions. A translation-function search was performed following rotation-function analysis of data between 15.0 and 4.0 Å. Rigid-body refinement using CNS, employing overall anisotropic temperature factors and bulk-solvent correction, yielded values of 0.31 and 0.30 for R and Rfree, respectively, for data between 19.2 and 3.0 Å resolution. Between refinement cycles, 2Fo − Fc and Fo − Fc maps were calculated using data to 3.0 Å resolution; zinc, chloride ions, and phenol molecules were built using O (60.Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (12931) Google Scholar). Water molecules were calculated and checked using DDQ (61.van den Akker F. Hol W.G. Acta Crystallogr. D Biol. Crystallogr. 1999; 55: 206-218Crossref PubMed Google Scholar). The geometry was monitored with PROCHECK (62.Laskowski R.A. Macarthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar); zinc ions and water molecules were built as refinement proceeded. Further refinement employed CNS (63.Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16713) Google Scholar), which implements maximum-likelihood torsion-angle dynamics and conjugate-gradient refinement. Statistical data are provided in supplemental Table S3. Potential packing defects were calculated with Surfnet (64.Laskowski R.A. J. Mol. Graph. 1995; 13 (307–308): 323-330Crossref PubMed Scopus (766) Google Scholar). The hydrophobic core of insulin is conserved among vertebrates (12.Steiner D.F. Chan S.J. Horm. Metab. Res. 1988; 20: 443-444Crossref PubMed Google Scholar); the neighborhood of cystine A20–B19 is structurally invariant (48.Baker 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. 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LeuA16, inaccessible in the native state (Fig. 2A, red), adjoins TyrA19 and CysA20 (Fig. 2B, blue and gold, respectively) along the inner surface of the A12–A19 α-helix and projects between CysA11 and LeuB15 (gold and gray). Hydrophobic collapse of these side chains in the nascent polypeptide is proposed to orient CysA20 and CysB19 for initial pairing (20.Narhi 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 Google Scholar, 29.Hua Q.X. Mayer J.P. Jia W. Zhang J. Weiss M.A. J. Biol. Chem. 2006; 281: 28131-28142Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 57.Hua Q.X. Chu Y.C. Jia W. Phillips N.F. Wang R.Y. Katsoyannis P.G. Weiss M.A. J. Biol. Chem. 2002; 277: 43443-43453Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Insulin chain combination provides a peptide model of proinsulin refolding (70.Katsoyannis P.G. Science. 1966; 154: 1509-1514Crossref PubMed Google Scholar). The reaction leads to native disulfide pairing, demonstrating that information required for proinsulin folding is contained within the A and B sequences (71.Tang J.G. Tsou C.L. Biochem. J. 1990; 268: 429-435Crossref PubMed Google Scholar). Yield is limited by off-pathway products under kinetic control (disulfide-linked cyclic A-chains, cyclic B-chains, B-chain dimers, and B-chain polymers). The robustness of chain combination to amino acid substitutions has enabled synthesis of hundreds of analogues in past decades. As an initial test of the importance of LeuA16 in disulfide pairing, we prepared a variant A-chain containing ValA16. Reactions employed 80 mg of wild-type A-chain and 40 mg of B-chain (each as S-sulfonate derivatives). Whereas this protocol ordinarily yields 8–9 mg of wild-type insulin (following purification by reverse-phase high performance liquid chromatography (HPLC) (51.Hu S.Q. Burke G.T. Schwartz G.P. Ferderigos N. Ross J.B. Katsoyannis P.G. Biochemistry. 1993; 32: 2631-2635Crossref PubMed Google Scholar, 57.Hua Q.X. Chu Y.C. Jia W. Phillips N.F. Wang R.Y. Katsoyannis P.G. Weiss M.A. J. Biol. Chem. 2002; 277: 43443-43453Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 72.Chance 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.in: Rich D.H. Gross E. Pierce Chemical Co., Rockford, IL1981: 721-728Google Scholar)), no product was detectable in three attempted reactions. Because the threshold of HPLC detection is <40 μg, the efficiency of chain combination was reduced by >200-fold. Refolding of proinsulin is more efficient than chain combination (73.Steiner D.F. Diabetes. 1978; 27: 145-148Crossref PubMed Google Scholar). Accordingly, we sought to prepare ValA16-human proinsulin (position 81 of the polypeptide) by recombinant expression in E. coli. Bacterial expression gave rise to inclusion bodies containing reduced and unfolded proinsulin; following purification, oxidative refolding ordinarily yields native disulfide pairing (52.Mackin R.B. Choquette M.H. Protein Expr. Purif. 2003; 27: 210-219Crossref PubMed Scopus (14) Google Scholar, 74.Frank B.H. Chance R.E. MMW Munch. Med. Wochenschr. 1983; 125: S14-S20Google Scholar). Expression of wild-type and variant polypeptides within inclusion bodies achieved similar levels; the reduced polypeptides were purified in similar yield. Although refolding of wild-type proinsulin was robust (52.Mackin R.B. Choquette M.H. Protein Expr. Purif. 2003; 27: 210-219Crossref PubMed Scopus (14) Google Scholar), however, the yield of [ValA16]proinsulin was negligible. The refolding mixture gave rise to a broad and inhomogeneous HPLC elution profile, suggesting the presence of multiple products. Cellular folding efficiencies of variant proinsulins were evaluated in CLA14 cells (a subclone of the Chinese hamster ovary cell line) (54.Liu" @default.
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• W2034628566 date "2009-12-01" @default.
• W2034628566 modified "2023-09-27" @default.
• W2034628566 title "Crystal Structure of a “Nonfoldable” Insulin" @default.
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