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J. Biol. Chem., Vol. 281, Issue 38, 28131-28142, September 22, 2006
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1
From the
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana 46285, and ¶Amgen, Inc., Thousand Oaks, California 91320
Received for publication, March 20, 2006 , and in revised form, July 19, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-helices are nonetheless observed at 4 °C. Furthermore, 13C-edited nuclear Overhauser effects establish transient formation of a native-like partial core; no non-native nuclear Overhauser effects are observed. Together, these observations suggest that early events in the folding of insulin-related polypeptides are nucleated by a native-like molten subdomain containing CysA20 and CysB19. We propose that nascent interactions within this subdomain orient the A20 and B19 thiolates for disulfide bond formation and stabilize the one-disulfide intermediate once formed. Substitutions in the corresponding region of insulin are associated with inefficient chain combination and impaired biosynthetic expression. The intrinsic conformational propensities of a flexible disulfide-linked peptide thus define a folding nucleus, foreshadowing the structure of the native state. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-helical domains and disulfide bridges (1–9). Canonical cystines (using an insulin-based nomenclature) are A6–A11, A7–B7, and A20–B19 (Fig. 1A); corresponding cystines in IGF-I span residues 47–52, 6–48, and 18–61 (Fig. 1B). Whereas insulin and relaxin contain two chains (designated A and B) as a consequence of post-translational processing (3–5, 13), IGFs are single-chain polypeptides containing A- and B-domains, an intervening connecting (C) domain, and C-terminal D-domain (1, 2). In this article we describe the design, synthesis, and conformational repertoire of a peptide model of a key IGF-I folding intermediate (14, 15). Designated IGF-p, the disulfide-linked peptide contains about half of the IGF-I sequence (Fig. 2, A and B). Although not well ordered, IGF-p exhibits an unexpected richness of nascent structure. Its conformational propensities, foreshadowing the structure of the native state, suggest a model for an IGF-I folding nucleus. This model is likely to generalize to other members of the insulin-related superfamily, including proinsulin.
Our studies are motivated by general principles of protein folding and their application to the insulin-related superfamily. The native state of a globular protein may be viewed as the coalescence of discrete subdomains (16). Folding trajectories exhibit transient formation and stabilization of native-like elements of secondary structure (17).3 Coalescence of resulting microdomains is consistent with classical diffusion-collision and framework mechanisms (18). Although the existence of funnel-like free-energy landscapes suggest the importance of parallel events in folding (19), preferred trajectories may exist (20), effectively defining predominant classes of structural intermediates. Associated transition states and intermediates have been probed by respective analyses of residue-specific 
values (21) and hydrogen-deuterium exchange (22). Structural insights have been obtained from equilibrium models of protein-folding intermediates (23, 24), including peptide fragments (25, 26).
Studies of the disulfide-coupled refolding of small globular proteins have exploited chemical trapping of preferred kinetic intermediates (27). Refolding of IGF-I and a single-chain proinsulin analog (mini-proinsulin, also designated porcine insulin precursor (PIP) (28)) is notable for populated one- and two-disulfide intermediates (14, 29, 30). A key role is played in each case by formation of the canonical A20–B19 disulfide bridge (cystine 18–61 in IGF-I). In the native states of insulin and IGF-I (Fig. 1) this bridge connects the C-terminal -helix of the A-domain to the central -helix of the B-domain, packing within a cluster of conserved aliphatic and aromatic side chains in the hydrophobic core (Fig. 1; Refs. 31–33). In the IGF-I pathway near neutral pH this is the only one-disulfide species to accumulate (34).4 Cystine A20–B19 in the insulin-related superfamily is thus proposed to stabilize a specific and polarized folding nucleus (specific folding nucleus (35, 36)).
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-helix content. As successive disulfide bridges are introduced in these equilibrium models, 1H NMR spectra exhibit a progressive increase in chemical-shift dispersion, suggesting stepwise stabilization of structure (15). Although resonance assignments were not obtained in these studies, comparison to the assigned spectrum of native IGF-I (37, 38) suggest formation of a native-like subdomain near cystine A20–B19 (IGF-I residues 18–61; Ref. 15). Additional evidence for the critical role of this cystine has been obtained through structural analysis of insulin analogs lacking either cystine A6–A11 or A7–B7 (36, 39, 40). Because information required for the folding of proinsulin is contained within the A and B chains (41, 42), these analogs may be regarded as peptide models of corresponding two-disulfide proinsulin intermediates. Although of low thermodynamic stability and partially unfolded, their predominant conformations contain a native-like cluster of non-polar side chains surrounding the A20–B19 disulfide bridge. Structures of analogous one-disulfide insulin analogs have not been described. In this article we describe the synthesis and characterization of a one-disulfide-linked peptide model of the populated 18–61, IGF-I intermediate (14, 29). The pattern of polar and non-polar residues in the polypeptide (IGF-p; Table 1) is conserved among insulin, IGF-II, relaxin, and relaxin-related proteins (cysteines are boxed, and the critical residues Cys18 and Cys61 are marked by asterisks; top line of Fig. 2, A and B). An analogous one-disulfide PIP analog containing cystine A20–B19 has been described by Feng and co-workers (43). Although the reduced polypeptide could efficiently form the A20–B19 disulfide bridge in vitro, its folding and secretion in Saccharomyces cerevisiae were severely impaired. The PIP analog was found to exhibit marked attenuation of CD-defined helix content5 and non-cooperative reduction in a glutathione redox buffer (43). These findings provide evidence of major perturbations but do not resolve possible regions of native-like or non-native structure.
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The conformational propensities of IGF-p foreshadow the native structure of IGF-I. Although these features represent only transient interactions within an otherwise unfolded ensemble, they may be central to the mechanism of oxidative folding. We propose that within the insulin superfamily an (A20–B19)-related microdomain orients these thiolates for initial disulfide bond formation, stabilizes the one-disulfide intermediate once formed, and provides a platform for non-random formation of subsequent disulfide bridges. Implications for the foldability of insulin and related proteins are discussed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-mercaptoethanol, water, and phenol in a ratio of 80:5:5:5:5, respectively. After stirring for 4 h at room temperature, the suspension was filtered, the filtrate was concentrated using a rotary evaporator, and the crude material was precipitated by addition of diethyl ether. Isotopic Labeling of Peptides—Labeled peptides were assembled manually by t-Boc/benzyl methodology to enable realtime monitoring of coupling efficiencies by the ninhydrin reaction (47). Starting with either 4-4-methylbenzylhydrylamine resin (48), the sequences were assembled by a manual dicyclohexylcarbodiimide/N-hydroxybenzotriazole-mediated first coupling followed by a routine second coupling. At labeled residues the first coupling involved activation of the labeled material with dicyclohexylcarbodiimide/N-hydroxybenzotriazole followed by an extended coupling period of several hours. To conserve the labeled material, an additional coupling at that position was carried out using the unlabeled form of the same amino acid. Following removal of the N-terminal t-Boc group, peptide resins were cleaved under high HF conditions for 60 min at 0 °C using 5% p-thiocresol and 5% m-cresol as scavengers. Sites of specific isotopic labeling are summarized in Table 1.
Peptide Purification—Reverse-phase high-performance liquid chromatography (RP-HPLC) was used to purify both Fmoc- and t-Boc-derived materials. Crude peptides were applied to a Vydac C-18 column and eluted with a linear acetonitrile gradient in 0.1% trifluoro-acetic acid. Fractions exhibiting the best analytical profile were pooled and lyophilized. Yields of purified peptides were in the 10–20% range, based on initial resin substitution levels.
Disulfide Pairing—To obtain IGF-p, chain combination reactions were performed by mixing equimolar amounts of the individual chains at a concentration of 1.0–1.5 mg/ml in distilled water/acetonitrile (2:1) at pH 9.5 for 48–72 h. HPLC analysis of reaction mixtures revealed three peaks (each 
30% of the total), corresponding to the two homodimers as well as the desired heterodimer. To optimize the yield of labeled material following preparative RP-HPLC isolation of the heterodimer, labeled homodimers were reduced in the presence of dithiothreitol, re-purified, and used in another chain combination. Recycling labeled material resulted in an eventual yield of 10–20 mg of each heterodimer. Each heterodimer was subjected to amino acid analysis and mass spectroscopy; results coincided closely with expected values. As a control for disulfide-dependent structural features, spectroscopic studies were in part repeated following reduction of the inter-chain disulfide bridge by dithiothreitol; perdeuterated dithiothreitol was purchased from Cambridge Isotopes, Inc. (Woburn, MA).
Gel Permeation Chromatography—To assess its oligomeric status, IGF-p was fractionated with Superdex® Peptide column HR 10/30 (30 cm x 10 mm; Amersham Biosciences) using a Waters 515 HPLC system. The column was equilibrated in 5% acetic acid (pH 2.5) in 50 mM KCl and run at a flow rate of 0.5 ml/min. IGF-p and control samples (25 µl) were introduced through a Waters 717-plus autoinjector. A range of molecular weight standards was employed to calibrate the column: mellitin (2.9 kDa), a 34-residue fragment of PTH-rp (a monomeric and disordered peptide of similar length to IGF-p), the dimer of HNF-p1 (the dimerization domain of hepatocyte nuclear factor-1, 7.7 kDa), human proinsulin (86 residues), and cytochrome c (12.5 kDa). IGF-p was loaded at a polypeptide concentration of 200 µM; elution was monitored at 280 nm. Samples and running buffer were cooled to 4 °C prior to loading; at elution the temperature was about 15 °C.
One-disulfide Analog of Intact IGF-I—Design, recombinant expression, and purification of a one-disulfide IGF-I analog containing cystine-(18–61) has previously been described (15). The analog, which contain pairwise substitution of cystine-(47–52) by Ala and cystine-(6–48) by Ser, is designated IGF-(18–61).
Circular Dichroism—Far ultraviolet CD spectra were acquired using an Aviv spectropolarimeter equipped with thermister control. Samples were made 30–50 µM in dilute HCl (pH 2); spectra were acquired at 4 and 25 °C in quartz cuvettes with 1-mm path length. Spectra were also obtained after addition of 20% ethanol and 20% trifluoroethanol (TFE), chosen as helicogenic organic cosolvents to mimic possible effects of 20% deuteroacetic acid in NMR studies. Spectra were also obtained in phosphate-buffered saline (pH 7.4). Helix contents were estimated by Selcon-3 (49).
NMR Studies of IGF-p—Spectra were obtained at 600 and 700 MHz in 20% deuteroacetic acid at 4–25 °C. These conditions, previously employed in NMR studies of IGF-I analogs (15) and insulin (33, 35, 50), enhance solubility relative to neutral pH. Resonance assignment was based on homonuclear two-dimensional NOESY (mixing times 80 and 250 ms), total correlation spectroscopy (TOCSY; mixing time 55 ms), and double-quantum filtered correlated spectroscopy spectra. In key cases resonance overlap was resolved based on selective 2H and 13C labeling; 1H-13C HSQC and 13C-edited NOESY spectra of labeled samples (Table 1) were obtained at 700 MHz. Control spectra were obtained in the absence of acetic acid at pH 2 (0.01 N HCl) and on progressive dilution in 20% acetic acid to a polypeptide concentration of 50 µM. Amide proton exchange was monitored in a D2O solution containing 20% deuteroacetic acid (51).
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NMR Studies of IGF-(18–61)—1H NMR spectra of the corresponding one-disulfide analog of IGF-I were obtained in 10 and 20% deuteroacetic acid as described (15). Presumptive selected resonance assignments, proposed by Narhi et al. (15) by analogy to the assigned spectrum of native IGF-I, were verified by explicit sequential assignment. Assignments were obtained in the regions of IGF-(18–61) spanned by IGF-p but incomplete elsewhere due to degeneracy. Spectra were obtained at 600, 700, and 750 MHz, the latter were acquired at Varian Instruments Inc. (Palo Alto, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-helix of the B-domain spans residues 8–18 in IGF-I, corresponding to canonical helix B9–B19 in insulin). Peptide PB (19 amino acids; IGF-I residues 7–25) thus spans canonical residues B8–B26. In the structure of native IGF-I (37, 38, 52, 53) this region forms a U-shaped helix-turn-strand super-secondary structure. Peptide PAD (18 amino acids) spans IGF-I residues 53–70 (positions A12–A21 and D1–D8). In native IGF-I this region comprises the C-terminal A-chain helix (residues 53–60) and less well organized D-domain extension (52, 53). Gel-permeation chromatography under conditions similar to those employed for NMR studies indicates that IGF-p is monomeric at a polypeptide concentration of 200 µM. 1H NMR spectra are unaffected by polypeptide concentrations in the 50 µM to 1.5 mM range.
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-helix in crystal structures of IGF-I (25 of 70 residues, or 36%; Ref. 52 and 53). This discrepancy, which presumably reflects the flexibility of IGF-I in solution (37, 38), highlights the limitations of CD deconvolution algorithms trained on the basis of well ordered globular proteins (49). CD spectra of the one-disulfide analog IGF-(18–61) has features intermediate between the spectra of IGF-p and native IGF-1 (dashed lines in Fig. 3, A and B). Helical CD features of both IGF-p and IGF-I are markedly enhanced on addition of TFE (Fig. 3, C and D, respectively). In 20% TFE (v/v) the spectrum of IGF-p exhibits a distinct minimum at 208 nm and shoulder at 222 nm (dashed line in Fig. 3C). These features, similar to those of IGF-(16–81) in the absence of TFE (dashed lines in Fig. 3, A and B), correspond to an estimated helix content of about 13%. Helical CD features are further accentuated in 40, 60, and 80% TFE; these spectra, nearly identical (Fig. 3C), correspond to helix contents of 25– 27%. The CD spectrum of IGF-I also exhibits accentuated helical features in the presence of TFE (Fig. 3D). Unlike in studies of IGF-p, successive addition of TFE in the 40–80% range is associated with a progressive increase in the magnitude of helix-related features at 195, 208, and 222 nm. In 80% TFE the CD spectrum of IGF-I is consistent with its crystal structure. Comparison of the TFE dependence of the CD spectra of IGF-p and IGF-I suggest that the peptide model may contain a latent helical propensity analogous to that of the native protein.
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Despite the overall paucity of chemical shift dispersion, aromatic chemical shifts in IGF-p and IGF-(18–61) are distinct from random coil values. Dispersion is particularly marked between the respective ortho aromatic resonances of Tyr60 and Tyr24 and likewise between their respective meta resonances. The chemical shifts of Tyr60 (7.16 and 6.78 ppm in IGF-p at 4 °C and similarly 7.17 and 6.74 ppm in IGF-(18–61) at 40 °C) resemble those of native IGF-I (7.31 and 6.75), presumably due in each case to core packing (see below). Of particular interest are the non-negligible secondary shifts of corresponding Tyr60 ortho resonances: in IGF-p this shift (
) is 0.17 ppm relative to free tyrosine in 20% acetic acid. Corresponding values of are 0.21 ppm in IGF-(18–61) and 0.20 ppm in IGF-I. By contrast, the meta and ortho chemical shifts of Tyr24, which flexibly projects into solvent, in each case are near random coil values: 7.02 and 6.78 ppm in IGF-p, 6.94 and 6.73 ppm in IGF-(18–61), and 7.04 and 6.77 in native IGF-I.
Upfield methyl chemical shifts, a distinctive NMR signature of the native state, are attenuated in the one-disulfide species, presumably reflecting the absence of fixed ring-current shifts in molten or disordered conformations. Significant differences are observed, for example, between corresponding Leu14 methyl resonances, which are sensitive to the ring current of Phe23. In native IGF-I this interaction leads to a significant upfield secondary shift (chemical shifts 0.70 and 0.48 ppm in IGF-I at 40 °C relative to reference position 0.98 ppm of free leucine in 20% acetic acid). By contrast, in IGF-p the methyl resonances of Leu14 exhibit chemical shifts of 0.91 and 0.84 ppm at 4 °C; corresponding chemical shifts in IGF-(18–61) are 0.73 and 0.63 ppm at 40 °C. Progressive attenuation of Leu14 secondary shifts may reflect trends in the extent of long-range order (see "Discussion").
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N and dNN NOEs spanning residues 8–13 in PB and residues 53–60 in PAD; Figs. 5B and 6) are consistent with the location of helices in the native protein. A summary of nascent helix-related contacts is given in Fig. 6.
Despite these helical NOE signatures, rates of amide proton exchange in IGF-p are consistent with sequence-dependent rates predicted in a random coil at this pH and temperature. The absence of amide protection in D2O is in accord with the negligible CD-detectable helical structure. Explicit demonstration of a conformational equilibrium between an unfolded ensemble of states and transient native-like structures is provided by natural-abundance 1H-13C HSQC spectra (Fig. 7 and supplemental materials Fig. S2). The positions of cross-peaks in the H-C region, relative to residue-specific random coil values, reflect the influence of local secondary structure on chemical shifts. Although the 1H-13C HSQC "fingerprint" of IGF-I at 35 °C exhibits random coil shifts (supplemental materials Fig. S2, A) (has not completely been assigned), a trend is observed at lower temperatures wherein cross-peaks of residues within regions of nascent structure are broad and hence attenuated, whereas cross-peaks outside of these segments are intense. This trend is more pronounced in 20% deuteroacetic acid at 7 °C (Fig. 7) than in dilute HCl at the same pH and temperature (supplemental materials Fig. S2, B). Examples are provided by the broad H-C cross-peaks of Val11, Ala13, Val17, Cys18, Gly19, and Gly22 in PB and by Cys61 in PAD. In fact, only a minority of resonances at 7 °C (in the presence or absence of deuteroacetic acid) exhibits intensities similar to those at 35 °C as expected of a random coil. These anomalous features provide evidence for conformational exchange within a disordered ensemble. Addition of 20% deutero-TFE leads to further broadening of these conformationally sensitive resonances and a shift in the H-C cross-peak of Val11 toward a helical fingerprint position (supplemental materials Fig. S2, D). Traces through representative cross-peaks in the natural abundance 1H-13C HSQC spectra, assigned to regions of disorder or nascent structure, are provided in the supplemental materials (Fig. S2, E and F, respectively). The greater sensitivity of 1H-13C HSQC spectroscopy (relative to homonuclear TOCSY and NOESY spectroscopy) to conformational broadening is due to a combination of the breadth of the 13C chemical shift scale (
between substates) and the marked dependence of HSQC cross-peak amplitude on 13C T2 relaxation times.
To investigate the dependence of the nascent structure on maintenance of the 18–61 disulfide bridge, IGF-p was reduced with 25 mM deuterated dithiothreitol in 25 mM ammonium bicarbonate (pH 8). Following lyophilization, the reduced sample was reconstituted in 20% deuteroacetic acid. Its natural abundance 1H-13C HSQC spectrum at 7 °C contained random coil PAD resonances without evidence of conformational broadening; the NOESY spectrum likewise contained few non-local contacts. PB cross-peaks were not observed in either spectrum, presumably due to aggregation or fibrillation of the reduced peptide.
13C-Edited NMR Studies Demonstrate Nascent Tertiary Structure—Comparison of the NOESY spectrum of IGF-p with spectra of native IGF-I (37, 38) and native insulin (50) indicates that PB contains a nascent native-like supersecondary structure: NOEs following the helix (Arg21 HN/Gly22 HN, Gly22 HN/Asp20-H,, and Phe23, H,
/Leu14 1,2-CH3) provide evidence of a -turn. Although the nascent helix in PAD also corresponds to a -helix in native IGF-I and insulin, some (i, i+3) NOEs in PAD are anomalous (supplemental materials Table S5). Such contacts, not characteristic of canonical -helices, presumably reflect the flexibility of the nascent helix. The disulfide bridge in IGF-p gives rise to characteristic inter-peptide NOEs between Cys18 and Cys61 (supplemental materials Table S5). The features are independent of the polypeptide concentration in the range 50 µM to 1 mM at 4–7 °C but are not observed at 25 °C.
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Relationship of IGF-p to Corresponding One-disulfide Intermediate—IGF-(18–61) at 40 °C yields a pattern of inter-residue NOEs similar to that of IGF-p at 4 °C (supplemental materials Fig. S3 and Table S5). Such similarity suggests that cooling IGF-p enhances intrinsic conformational preferences that in IGF-(18–61) are stabilized at higher temperature by flanking regions of the 70-residue polypeptide. Sequential assignment of the A and B-domains of IGF-(18–61) demonstrates corresponding helical segments and long-range NOEs (supplemental materials Fig. S3 and Table S3), including Phe23–Leu14 and Tyr60–Leu14. Significantly, the methyl chemical shifts of Leu14 are upfield of those in IGF-p (and downfield of those in native IGF-I), suggesting that the nascent core of the longer polypeptide is more ordered. Of particular interest, the non-random chemical shifts of the two aromatic rings in the nascent microdomain (Phe23 and Tyr60; canonical positions B24 and A19, respectively) are essentially identical in the two samples (Fig. 5D and supplemental materials Table S2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These concepts have been extensively investigated in studies of oxidative protein folding. Of interest in relation to disulfide pairing in the endoplasmic reticulum (62), oxidative folding is central to the biosynthesis of transmembrane and secretory proteins (63). In vitro the relative reactivities of thiol groups provide site-specific kinetic probes, enabling reaction intermediates to be trapped (64). The time course of formation and disappearance of free cysteines and specific pairing arrangements thus provides a chemical map of the oxidative folding pathway (65, 66). A paradigm has been provided by bovine pancreatic trypsin inhibitor (23, 24, 67). Like IGF-I and insulin, bovine pancreatic trypsin inhibitor is a small globular protein containing three disulfide bridges, and so its folding pathway provides a context for comparison. The subject of vigorous debate (36), refolding is characterized by a preferred sequence of one- and two-disulfide species. Both native and non-native pairings may be observed whose relative ratios are sensitive to experimental conditions (68). Unlike IGF-I intermediates (15, 35), bovine pancreatic trypsin inhibitor intermediates that are well populated at neutral pH exhibit native-like structures (23, 24, 67). Large kinetic barriers among such folded intermediates lead to a preferred final step: formation of an external disulfide bridge between solvent-exposed loops. Kinetic barriers are thus central to the logic of the overall disulfide pathway (27, 69). Given that a populated one-disulfide species exhibits a native-like fold (67), the kinetics of disulfide formation and rearrangement are unrelated to the logic of the initial conformational search from the unfolded state ensemble.
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Evidence for non-random initial folding trajectories is provided by the trapping of a unique one-disulfide intermediate containing the A20–B19 disulfide bridge (cystine-(18–61) in IGF-I; Refs. 15 and 43). Although several subsequent two-disulfide species have been characterized, this species appears to be an obligatory on-pathway intermediate.6 The initial conformational search leading to formation of this disulfide bridge is thus of central importance. Here, we have constructed and characterized a peptide model of this key intermediate. Although highly flexible, analysis of inter-molecular NOEs at low temperature indicates transient formation of a native-like microdomain surrounding the A20–B19 disulfide bridge. We thus view IGF-p as adopting an ensemble of disordered structures in equilibrium with native-like conformational substates (Fig. 9). We imagine that transient formation of a native-like subdomain provides an "internal template" constraining subsequent folding trajectories and possible disulfide pairings.
The richness of the NOESY spectrum of IGF-p is remarkable in light of its uninformative CD spectrum. This apparent contradiction presumably reflects the differing physical mechanisms of these probes: whereas CD detects the mean handedness of the ensemble of main chain conformations, NOEs can arise through the transient proximity of protons in a flexible polypeptide. Because the CD spectrum excludes formation of well ordered -helices, we interpret non-random NOE patterns as evidence of nascent conformational preferences rather than fixed structure. Despite such NOEs, chemical shift dispersion is limited. We presume that conformational fluctuations lead to averaging of ring-current shifts and other sources of chemical shift anisotropy that could otherwise lead to non-random secondary shifts (70).7 The low apparent helix content implied by the CD spectrum of IGF-p is also in accord with the absence of protected amide resonances in D2O within NOE-defined nascent helical segments. Evidence for conformational fluctuations in a related analog 1GF-(18–61) has previously been obtained from tyrosine fluorescence studies (15).
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An unusual feature of IGF-I refolding is its bifurcation to yield two products (14, 29). Designated native IGF-I and IGF-swap (14), the two products share the 18–61 disulfide bridge but differ by interchange of Cys46 and Cys47; respective pairing schemes are (18–61, 6–48, and 47–52) and (18–61: 6–47 and 48–52). These isomers exhibit near equal thermodynamic stabilities. Proinsulin and single chain insulin analogs by contrast refold to form a unique native state; alternative pairing schemes are metastable, and if present, rearrange to the native state. That the number of products is precisely two demonstrates that a non-random folding pathway is encoded but must be "ambiguous" following formation of cystine-(18–61). Studies of chimeric PIP analogs indicate that the respective B-domains of proinsulin and IGF-I are responsible for determining the relative stability of the swapped isomer (72). Specification of native IGF-I disulfide pairing in vivo is attributed to specific IGF-1-binding proteins, present in equimolar proportions. Such partner proteins bind native IGF-I but not IGF-swap, thus favoring formation of the native isomer (73).
Application to Insulin and Proinsulin—Insulin chain combination provides a peptide model for the folding of proinsulin (31). Although the mechanism of disulfide pairing is not well characterized, evidence for the kinetic importance of an homologous A20–B19-related microdomain is provided by the contrasting effects of N- and C-terminal A chain substitutions. Whereas diverse substitutions in the N-terminal segment of the A chain are well tolerated (39), substitutions in the C-terminal -helix can profoundly impair efficiency of chain combination (74). Because the A20–B19-related microdomain is proposed to function as a specific folding nucleus, destabilization of this substructure would also destabilize the transition state. An example of blocked chain combination is provided by mutations of LeuA16 (Leu57 in IGF-I). This side chain packs against the A20–B19 disulfide bridge and is proposed to assist in orienting CysA20 and CysB19 for proper disulfide pairing. Off-nucleus contacts involving the N-terminal segment of the A chain are optional in the proposed polarized transition state and so may be bypassed. Unstable N-terminal analogs are thus readily prepared. Substitutions in B-domain residues involved in the A20–B19-related microdomain are also associated with impaired chain combination (75) and decreased efficiency of PIP expression in yeast (43).
The robustness of insulin chain combination to drastic changes in the sequence and structure of the N-terminal A chain segment demonstrates asymmetric encoding of folding information. A polarized kinetic model rationalizes why it is possible to achieve efficient biosynthetic expression and folding of PIP variants containing pairwise substitution of either "off nucleus" cystine (A6–A11 or A7–B7) but not of cystine A20–B19 (76). Likewise, syntheses of two-disulfide insulin analogs lacking either cystine A6–A11 or A7–B7 were accomplished with high yield (36, 39). Efficient pairing of such analogs seems remarkable in light of their marked instabilities. In transfected mammalian cells a variant proinsulin analog lacking cysteines A6 and A11 exhibits an efficiency of folding and secretion similar to that of wild-type proinsulin (39). Expression and secretion are impaired on pairwise substitution of either A7–B7 or A20–B19, presumably due to misfolding and/or degradation.
Populated one- and two-disulfide intermediates in the folding of proinsulin have not been characterized. This is due in part to a technical barrier. Whereas the refolding of reduced IGF-I can be investigated near neutral pH (14, 29), studies of proinsulin are restricted to basic conditions (pH 9.5–10.5) due to pH-dependent aggregation of the unfolded polypeptide. Under these conditions the protein rapidly oxidizes to form an assortment of three-disulfide isomers, which then rearrange to the native state (31). The relevance of this phenomenology to in vitro folding at neutral pH or physiological folding in the ER (pH 6) is unclear. Because aggregation of reduced proinsulin at neutral pH can be circumvented by the addition of protein-disulfide isomerase (and in particular of its chaperonin domain; Ref. 77), it would be of future interest to investigate the kinetic refolding pathway of proinsulin in the presence of this ER foldase.
IGF-p is related to a one-disulfide analog of a mini-proinsulin (43). As in IGF-(18–61) (15), this PIP analog was engineered by pairwise substitution of cystine A6–A11 by Ala and A7–B7 by Ser. Its efficiency of folding and secretion in S. cerevisiae is reduced by 102–103-fold relative to the wild-type PIP, suggesting failure of quality control in the ER. In vitro the one-disulfide variant exhibits a marked attenuation of CD-detected helix content and non-cooperative reduction properties. Although efficient in vitro formation of the A20–B19 disulfide bridge was observed and ascribed to a kinetically preferred pathway (43), interpretation of these results remains unclear as no control studies were described. In particular, because the variant polypeptide contained only two cysteines, assessment of efficiency and consideration of kinetic guidance await future comparison of the rates of oxidative pairing of (a) the same polypeptide in denaturant, (b) a variant polypeptide containing non-conservative substitutions in the A20–B19-related microdomain, and (c) intrinsically unfolded model polypeptides engineered to contain two cysteines with the same spacing as in the PIP analog.
Concluding Remarks—A major challenge is posed by the problem of proinsulin folding in -cells. The subtlety of this problem is highlighted by the findings of Arvan and colleagues (78) that substitutions well tolerated in vitro can be associated with disulfide mispairing in cell culture (78). Furthermore, mini-proinsulin analogs, although highly efficient in refolding assays in vitro (79), can quantitatively misfold in mammalian cell culture to yield a metastable disulfide isomer. This isomer passes ER quality control and is efficiently secreted (79). These observations strongly suggest that folding of proinsulin in vivo is under kinetic control. By implication, determinants of structure and stability in vitro may or may not correlate with determinants of kinetic guidance in vivo.
The conformational repertoire of IGF-p foreshadows the native state of IGF-I. An analogous bias toward native-like topologies within an unfolded state ensemble has been proposed by Shortle and co-workers (80) to accelerate folding and provide a barrier against misfolding (80). Although this proposal is controversial (81, 82), our results establish that in the presence of a disulfide bridge a flexible peptide model of a protein-folding intermediate can exhibit nascent native-like NOEs even in the absence of stable secondary structure. Accordingly, we envisage that the A20–B19-related microdomain of proinsulin functions in the -cell as a specific folding nucleus to accelerate the initial conformational search leading to formation of canonical cystine A20–B19 (cystine–(18–61) in IGF-1). In the future this hypothesis can be addressed through mutagenesis of residues in proinsulin predicted to be either engaged in or peripheral to the A20–B19-related microdomain. The existence of a polarized transition state would imply that destabilization of this microdomain, but not of flanking structural elements, would block folding, induce ER stress, and activate the unfolded protein response. The relationship between the folding of proinsulin and ER stress is of central interest in relation to -cell exhaustion in Type II diabetes mellitus (83, 84).
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5 and Tables S1–S5.
1 To whom correspondence should be addressed: 10900 Euclid Ave., Cleveland, OH 44106-4935. Tel.: 216-368-5991; Fax: 216-368-3419; E-mail: maw21{at}case.edu.
2 The abbreviations used are: IGF, insulin-like growth factor; CD, circular dichroism; ER, endoplasmic reticulum; Fmoc, N-(9-fluorenyl)methoxycarbonyl; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser enhancement spectroscopy; RP-HPLC, reverse-phase high-performance liquid chromatography; PIP, porcine insulin precursor; t-BOC, N-tert-butoxycarbonyl; TOCSY, total correlation spectroscopy; HSQC, heteronuclear single-quantum coherence; TFE, trifluoroethanol.
3 Whereas formation of nascent helical segments is envisaged in transition state of -helical domains (18), studies of refolding of -sheet proteins suggest that hydrophobic collapse can precede stabilization of secondary structure (82).
4 In PIP at pH 10 cystine A20–B19 is formed by parallel branches: either before or after formation of A6–A11 (30). Local pairing of A6–A11 is thought to be peripheral to the initial conformational search (36).
5 Deconvolution of CD spectra suggest that native PIP exhibits a helix content of 48%, consistent with crystal structures of related analogs (83), the one-disulfide PIP analog exhibits a helix content of about 9% (43), similar to that observed on removal of cystine A7–B7 (84).
6 If disulfide pairing were random, as in the presence of a denaturant, for example, a combinatorial calculation suggests that a polypeptide sequence containing six cysteines (such as the insulin-related superfamily) could form any of 30 different one-disulfide species.
7 The methyl resonances of Leu14, expected to be shifted upfield by the ring current of Phe23, exhibit near random coil chemical shifts of 0.91 and 0.84 ppm in IGF-p, in contrast to upfield positions 0.70 and 0.48 ppm in native IGF-I.
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