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Protein Science (2002), 11:104-116.
Copyright © 2002 The Protein Society

A cavity-forming mutation in insulin induces segmental unfolding of a surrounding {alpha}-helix

Bin Xu1, Qing-Xin Hua1, Satoe H. Nakagawa2, Wenhua Jia1, Ying-Chi Chu3, Panayotis G. Katsoyannis3 and Michael A. Weiss1

1 Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA
2 Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA
3 Department of Pharmacology and Biological Chemistry, Mt. Sinai School of Medicine of New York University, New York, New York 10029, USA

Reprint requests to: Michael Weiss, Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA; email: weiss{at}biochemistry.cwru.edu; fax: (216) 368-3419.

(RECEIVED August 2, 2001; FINAL REVISION October 12, 2001; ACCEPTED October 12, 2001)

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1101/ps.32102.

1 Of insulin's three helices, the A1–A8 {alpha}-helix is least regular among crystal structures. In NMR studies, an A2–A8 {alpha}-helix is well defined, but largely lacks protected amide resonances in D2O solution (Hua and Weiss 1991; Hua et al. 1996a). Chemical shifts of the IleA2 side chain are less upfield than would be predicted by a ring-current model of TyrA19 (Jacoby et al. 1996), presumably because of variability in the details of A2–A19 packing. Back

2 Imprecision is defined by RMS deviations between members of an ensemble of models. Following alignment according to the main-chain atoms of residues A12–A20 and B9–B19, the A1–A8 segment shows a main-chain RMSD of 0.71 Å in DKP-insulin versus 1.51 Å in AlaA2-DKP-insulin. Back

3 CysA11 is somewhat better defined than CysA6. RMSD values of these side chains are variant ensemble, A6 1.93 Å and A11 1.39 Å; parent ensemble, A6 0.55 Å and A11 0.61 Å. Back

4 Motional narrowing is defined as the general narrowing of NMR resonances as a result of molecular motions in the laboratory frame. In the present context, motional narrowing refers to differential narrowing because of local or segmental mobility relative to baseline line widths determined by macromolecular tumbling ({tau}c). Back

5 This issue has been addressed in comparative crystallographic (Eriksson et al. 1993) and NMR studies (Mulder et al. 2000) of the L99A cavity mutant of T4 lysozyme (Eriksson et al. 1992, 1993). The L99A substitution is of particular interest because it creates a large internal cavity capable of binding ligands. The variant crystal structure is similar to that of wild type, including in its pattern of thermal B factors. In solution, however, studies of protein dynamics by heteronuclear NMR relaxation methods show that, whereas no change occurs on the picosecond-to-nanosecond time scales, conformational exchange is accentuated on the microsecond-to-millisecond time scale. Such cavity-associated motions involve an extensive portion of the protein surrounding the cavity (Mulder et al. 2000). Back

6 The Rf state, observed in T3Rf3 hexamers, differs from the R state at residues B1–B3. Whereas R6 hexamers contain a B1–B19 {alpha}-helix, the Rf state contains a B3–B19 or B4–B19 {alpha}-helix with frayed N-termini. Back


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 Electronic supplemental material
 References
 
To investigate the cooperativity of insulin's structure, a cavity-forming substitution was introduced within the hydrophobic core of an engineered monomer. The substitution, IleA2->Ala in the A1–A8 {alpha}-helix, does not impair disulfide pairing between chains. In accord with past studies of cavity-forming mutations in globular proteins, a decrement was observed in thermodynamic stability ({Delta}{Delta}Gu 0.4–1.2 kcal/mole). Unexpectedly, CD studies indicate an attenuated {alpha}-helix content, which is assigned by NMR spectroscopy to selective destabilization of the A1–A8 segment. The analog's solution structure is otherwise similar to that of native insulin, including the B chain's supersecondary structure and a major portion of the hydrophobic core. Our results show that (1) a cavity-forming mutation in a globular protein can lead to segmental unfolding, (2) tertiary packing of IleA2, a residue of low helical propensity, stabilizes the A1–A8 {alpha}-helix, and (3) folding of this segment is not required for native disulfide pairing or overall structure. We discuss these results in relation to a hierarchical pathway of protein folding and misfolding. The AlaA2 analog's low biological activity (0.5% relative to the parent monomer) highlights the importance of the A1–A8 {alpha}-helix in receptor recognition.

Keywords: Protein unfolding; insulin; cooperativity; protein structure; hormone insulin receptor; NMR spectroscopy

Abbreviations: CD, circular dichroism • DG/SA, distance-geometry simulated annealing • DKP-insulin, analog containing three substitutions in the B chain (see Table 1Go) • DQF-COSY, double-quantum filtered correlated spectroscopy • HPLC, high-performance liquid chromatography • IGF-I, single-chain insulin-like growth factor • NMR, nuclear magnetic resonance • NOE, nuclear Overhauser enhancement • NOESY, NOE spectroscopy • RMS, root mean square • RMSD, RMS deviations • rp-HPLC, reverse-phase HPLC • Amino acids are represented by standard one- and three-letter codes. "Native" elements of structure designate feature of crystal structures and may not correspond to the functional conformation in a receptor complex.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 Electronic supplemental material
 References
 
Insulin is a small globular protein containing two chains, designated A (21 residues) and B (30 residues). The hormone is stored in the pancreatic ß cell as a Zn2+-stabilized hexamer and functions in the bloodstream as a Zn2+-free monomer (Dodson and Steiner 1998). The structure of a monomer in solution resembles the crystallographic T-state (Fig. 1AGo), as defined in a variety of crystal forms (Blundell et al. 1971; Bentley et al. 1976; Bi et al. 1984; Dai et al. 1987; Baker et al. 1988; Badger et al. 1991; Ciszak and Smith 1994; Bao et al. 1997). Despite its small size, insulin contains representative features of larger proteins, including canonical elements of secondary structure and a well-ordered hydrophobic core. The present study focuses on the structural role of IleA2, an invariant side chain in the core (Blundell et al. 1971; Bentley et al. 1976; Derewenda et al. 1989; Badger et al. 1991; Kitagawa et al. 1984a,1984b; Nakagawa and Tager 1992). Adjoining side chains IleA2 and CysA6 (part of the internal A6–A11 disulfide bridge) anchor the A1–A8 {alpha}-helix through a network of long-range contacts involving LeuA16, TyrA19, LeuB6, LeuB11, and LeuB15 (Fig. 1CGo). Reorganization of the B chain on receptor binding is proposed to expose IleA2 to engage the insulin receptor (Derewenda et al. 1991; Hua et al. 1991; Nakagawa and Tager 1992). Conservation of IleA2 presumably reflects its dual role in structure and function. In this article we investigate the consequences of a cavity-forming mutation, AlaA2. Previous studies have shown that an AlaA2 substitution impairs insulin's activity by 200-fold (Kitagawa et al. 1984a; Nakagawa and Tager 1992) but have not addressed its structural consequences.


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  		Table 1. Design of insulin analogues
 


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  		Fig. 1. Structure of insulin. (A) Ribbon representation of crystal structure of native 2-Zn porcine insulin (T-state protomer; PDB entry 4INS). The A chain is shown in red and B chain in blue; disulfide bridges are shown in yellow (balls and sticks). The solution structure of DKP-insulin is similar to the crystallographic T-state (Hua et al. 1996). (B) Surface representation of T-state protomer (2-Zn molecule 1) showing "internal template" in contact with N-terminal A-chain {alpha}-helix (yellow ribbon; residues A1–A11). Polar or charged side chains are shown in red; nonpolar or aromatic side chains in blue. Images were generated using InsightII (Biosym, Inc.). (C) Stereo representation of side-chain environment of IleA2 in multiple crystal structures (T-state protomers; PDB entries 1APH, 1LPH, 1TRZ, 1ZNI, 2INS, 4INS). Structures are aligned with respect to main-chain atoms of residues A2–A8, A13–A20, and B9–B19.

 
Protein structures ordinarily show cooperativity and robustness to point mutations (Eriksson et al. 1992). Whereas cavity-forming mutations in globular proteins typically result in decreased thermodynamic stability (Sandberg and Terwilliger 1989; Milla et al. 1994; Xu et al. 1998, 2001), native-like structures are maintained through local conformational adjustments (Eriksson et al. 1992; Xu et al., 1998). The present study exploits a cavity-forming mutation in insulin to investigate the stability and modularity of the A1–A8 {alpha}-helix. Experimental design is influenced by an analogy proposed between the dynamics of the insulin monomer and a molten globule (MG; Hua et al. 1992, 1993). Mutagenesis studies of {alpha}-helices within equilibrium MG models have highlighted the limited cooperativity of individual {alpha}-helices (Pfeil 1981; Dolgikh et al. 1985; Pfeil et al. 1986; Griko et al. 1988; Schulman and Kim 1996; Schulman et al. 1997). Given the insulin monomer's molten features, would one or more of its helical segments lack cooperativity? Folding of insulin and insulin-like growth factors is characterized by stepwise stabilization of native structural elements with successive disulfide bond formation (Narhi et al. 1993; Hua et al. 1996b; Hober et al. 1997). In one branch (Hua et al. 1996a,1996b; Weiss et al. 2000; Qiao et al. 2001), the A1–A8 {alpha}-helix occurs as a late event, coupled to formation of the final disulfide bridge (cystine A6–A11) (Weiss et al. 2000). The preceding intermediate contains a native-like subdomain, which is proposed to provide a specific template to stabilize folding of the A1–A8 segment as an amphipathic {alpha}-helix (Hua et al. 1996a; Weiss et al. 2000).

In this article we describe the synthesis and characterization of an IleA2->Ala analog. This large-to-small substitution occurs within an amphipathic {alpha}-helix unusually rich in ß-branched amino acids (sequence GIVEQCCT). The substitution is constructed with an engineered monomer (DKP-insulin; Weiss et al. 1991; DiMarchi et al. 1992; Shoelson et al. 1992; Ciszak et al. 1995) to facilitate spectroscopic studies. DKP-insulin contains three substitutions in the B chain (Table 1Go) that impair formation of dimers, trimers, and higher-order oligomers (DiMarchi et al. 1992, Shoelson et al. 1992). The engineered monomer, which shows enhanced potency (Shoelson et al. 1992) and thermodynamic stability (Weiss et al. 2001), provides a convenient template for analysis of substitutions in the A chain (Hua et al. 1996a; Weiss et al. 2000). A combination of CD and NMR is used to show that an AlaA2 substitution in DKP-insulin causes segmental destabilization of the A1–A8 segment. An otherwise native subdomain is retained. The thermodynamic stability of AlaA2-DKP-insulin is lower than that of DKP-insulin. Despite these perturbations, the variant A-chain pairs with the DKP-B chain with native fidelity and yield. The structure of the mutant insulin shows that a cavity-forming mutation in the core of a protein can lead to segmental unfolding in accord with a hierarchical perspective (Hober et al. 1992; Miller et al. 1993; Narhi et al. 1993; Hua et al. 1996a; Weiss et al. 2000). In particular, folding of the A1–A8 segment is not integral to insulin's overall structure or mechanism of disulfide pairing. To our knowledge, this is the first structure of an A2 insulin analog to be described. We speculate that insulin's limited cooperativity may facilitate segmental conformational change during the protein's complex "life cycle" of biosynthesis, assembly, and action (Derewenda et al. 1991; Hua et al. 1991; Dodson and Steiner 1998; Lipkind and Steiner 1999).


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 Electronic supplemental material
 References
 
AlaA2-DKP-insulin was prepared by total chemical synthesis (see Materials and Methods). In contrast to studies of certain B-chain substitutions (Wang et al. 1991; Hu et al. 1993), the yield of insulin chain combination was similar to that obtained in the synthesis of native or DKP-insulin (Hua et al. 1996a). The analog's affinity for the human insulin receptor is 150-fold lower than that of native insulin and 250-fold lower than that of DKP-insulin; such a marked impairment is in accord with previous studies (Kitagawa et al. 1984a,1984b; Nakagawa and Tager 1992). CD spectra indicate a reduction in {alpha}-helix content relative to DKP-insulin (Fig. 2BGo and Table 2Go); the value of mean residue ellipticity at 222 nm is -7510 deg•cm•mole-1, significantly less negative than the parent value of -9580 deg•cm•mole-1. AlaA2-DKP-insulin also shows decreased thermal and thermodynamic stabilities (asterisks in Fig. 2Go, C and D). Analysis of native and variant guanidine-denaturation transitions by a two-state model (Brems et al. 1990; Sosnick et al. 2000) implies free energies of unfolding ({Delta}Gu) of 4.9 kcal/mole (DKP-insulin) and 3.7 kcal/mole (AlaA2-DKP-insulin) at 4°C. Because the variant m value is also lower than the native m value, such fitting may underestimate the variant's stability and so overestimate the thermodynamic perturbation (Pace and Shaw 2000). An upper bound on the analog's {Delta}Gu value of 4.5 kcal/mole may be calculated using the native m value and variant Cmid value (see Materials and Methods for justification; Luo and Baldwin 2001). The analog's decrement in stability ({Delta}{Delta}Gu) is thus estimated to lie between 0.4 and 1.2 kcal/mole (Table 2Go).



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  		Fig. 2. Model of partial insulin fold (A) and CD studies (B–D). (A) Cylinder model of segmental unfolding of AlaA2-DKP-insulin. (B) Far-UV spectra of DKP-insulin [native (filled squares)], and AlaA2-DKP-insulin [variant (open squares)] indicate attenuated helix content in variant. (C) Thermal unfolding of AlaA2-DKP-insulin (open squares), monitored by ellipticity at 222 nm, precedes (asterisk) that of DKP-insulin (filled squares). (D) Guanidine unfolding transitions of AlaA2-DKP-insulin (open squares) and DKP-insulin (filled squares) show that the variant shows decreased thermodynamic stability as inferred by a two-state model ({Delta}{Delta}Gu 0.4–1.2 kcal/mole; see Table 2Go for {Delta}Gu values).

 

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  		Table 2. Spectroscopic and thermodynamic studies
 
1H-NMR studies of AlaA2-DKP-insulin were conducted at neutral pH in aqueous solution and in 20% deuterioacetic acid (Weiss et al. 1991), as described in previous studies of DKP-insulin (see Materials and Methods; Hua et al. 1996a). Complete sequential assignment was in each case obtained; patterns of NOEs and chemical shifts are similar under the two conditions (electronic supplemental material). Differences in chemical shifts (relative to those of DKP-insulin) are in general small and not localized (Table 3Go). Analysis of secondary shifts (defined as differences between observed chemical shifts and tabulated random-coil values) indicates an overall correlation (Fig. 3Go and electronic supplemental material). Perturbations in A1–A5 H{alpha} resonances trend downfield, that is, away from helix-related values and toward random-coil values. The magnitude of such changes is small. Baseline chemical shift dispersion in the native A1–A8 {alpha}-helix of DKP-insulin is less marked than would be predicted based on crystal structures (Hua et al. 1996a; Jacoby et al. 1996), presumably because of protein dynamics1.


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  		Table 3. Chemical-shift differences (pH 7 and 25°C) a
 


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  		Fig. 3. Correlation of 1H-NMR secondary shifts (|{Delta}{delta}|) between AlaA2-DKP-insulin (ordinate) and DKP-insulin (abscissa) by proton class. Such differences reflect sites of change in proton magnetic environments: (A) amide, (B) H{alpha}, (C) Hß, and (D) aromatic and methyl resonances. Shifts were observed at pH 7.0 and 25°C. The A1–A11 segment is shown in red, the remainder of the A chain in green, and the B chain in black. Outlying points are assigned as shown. Tables of chemical shifts and secondary shifts are provided as supplemental material (Table S1 in electronic supplemental material). Secondary shifts are defined as difference between observed chemical shifts and tabulated random-coil values (Wüthrich 1986).

 
Comparison of native and variant NOESY spectra indicates segmental perturbations in the A1–A8 region; native-like spectra features are otherwise retained. Whereas NOEs between amide protons (dNN contacts) in the A12–A19 and B9–B19 segments are similar to those observed in DKP-insulin, dNN contacts in the A1–A8 segment are weak or absent (Fig. 4AGo and Table 4Go). Analysis of helix-related (i, i + 3) contacts (d{alpha}N(i,i+3) and d{alpha}ß(i,i+3); shown in Wüthrich format in Fig. 4CGo) shows retention of native-like A12–A19 and B9–B19 helices; such contacts are absent in the A1–A8 segment. NMR-defined secondary structure is thus in accord with attenuated CD ellipticity at 222 nm as a consequence of segmental destabilization of the A1–A8 {alpha}-helix (Fig. 2AGo). Native-like contacts between side chains in the B9–B19 {alpha}-helix (LeuB11, ValB12, and LeuB15) and the B-chain's C-terminal ß-strand (PheB24 and TyrB26) are retained (Fig. 4BGo), indicating maintenance of the B chain's supersecondary structure. Whereas IleA2 in DKP-insulin shows prominent NOEs to the aromatic resonances of TyrA19, however, no long-range NOEs are observed involving the methyl resonance of AlaA2 (dashed line in Fig. 4BGo; see also Table 4Go). Native-like long-range NOEs elsewhere in the molecule are retained. These include contacts between the side chains of IleA10 and HisB5 (not shown), LeuA13 and PheB1, and TyrA19 and LeuB15 (Fig. 4BGo and Table 4Go).



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  		Fig. 4. NOESY spectra of AlaA2-DKP-insulin (A and B) and summary of sequential assignments (C). (A) Amide and aromatic region in H2O shows dNN connectivities in A chains (red labels) and B chains (black labels). Location of key unobserved peaks are marked by asterisks at predicted locations (open squares). (B) NOEs between aromatic and aliphatic resonances in D2O include long-range NOEs involving side chains of TyrA19, PheB1, PheB24, and TyrB26 and methyl groups of LeuB15, LeuB11, LeuA16, ValB12, LeuB17, and ThrB27. (C) Sequential and medium-range NOEs in AlaA2-DKP-insulin are summarized in Wüthrich format. Relative strength of NOEs is schematically represented by thickness of line. Dashed lines indicate possible cross peaks obscured by resonance overlap.

 

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  		Table 4. Segmental and long-range NOEs
 
The solution structure of AlaA2-DKP-insulin was calculated based on 416 NOE-derived distances, dihedral and hydrogen-bond restraints (Table 5Go). DG/SA ensembles of DKP-insulin and AlaA2-DKP-insulin are shown in Figure 5Go, A and B, respectively. Whereas the variant protein retains the overall insulin motif, the A1–A8 helix is not well defined. Disorder in the A1–A3 segment is associated with imprecision2in the B26–B30 segment as a result of loss of interchain contacts. Excepting the variable position of AlaA2 and the imprecise convergence of the location of the internal A6–A11 disulfide bridge3, the remainder of the analog's hydrophobic core (Fig. 5DGo) is similar to that of DKP-insulin (Fig. 5CGo). The side chains of cystine A6–A11, LeuA16, TyrA19, LeuB11, and LeuB15 remain largely inaccessible to solvent in the AlaA2 structure. It is possible that fluctuations in the analog's structure can lead to transient exposure of these core side chains. The TyrA19 side chain has similar average solvent accessibility value in the AlaA2-DKP ensemble as in the DKP ensemble, but is more variable within the ensemble of AlaA2-DKP (20% ± 7% versus 22% ± 3%). No residual cavity is consistently observed in the ensemble. Imprecision of the A1–A8 segment of AlaA2-DKP-insulin and adjoining regions of the B chain reflects absence of restraints. In 20% acetic acid, motional narrowing4 is observed among resonances in the A1–A8 segment, which indicates that the variant segment is in fast exchange among a range of configurations. Motional narrowing is not observed in aqueous solution at neutral pH. Rather, several resonances in the A1–A8 segment are broader than those of analogous spin systems in the well-ordered subdomain. It is likely that line widths at neutral pH are influenced by conformational exchange on a millisecond time scale, leading to differential broadening.


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  		Table 5. DG/SA restraints and statistical parameters
 


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  		Fig. 5. Structures of DKP-insulin and AlaA2-DKP-insulin. (A) Solution structure of DKP-insulin (Hua et al. 1996a) is similar to the crystallographic T-state (Baker et al. 1988). (B) Solution structure of AlaA2-DKP-insulin has disordered N-terminal A-chain segment (A1–A8) and more divergent C-terminal B-chain tail (B26–B30), whereas remainder of molecule is essentially unchanged. (C) Environment of IleA2 in DKP-insulin ensemble is similar to that in collection of crystal structures (see Fig. 1CGo). (D) Environment of AlaA2 in AlaA2-DKP-insulin. AlaA2 and ValA3 are disordered, whereas the A6–A11 disulfide bridge is less-well converged; the remainder of the hydrophobic core is similar to that of DKP-insulin. ß-Carbons of AlaA2 side chain are shown as black balls. Structures in panels A–D are aligned with respect to main-chain atoms of residues A13–A20 and B9–B19.

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 Electronic supplemental material
 References
 
The present study used a combination of CD and NMR spectroscopy to characterize the response of an engineered insulin monomer to a potential cavity in its hydrophobic core. Whereas CD provides an overall estimate of the extent of order in an ensemble, NMR enables specific sites of order and disorder to be distinguished. These complementary probes in part circumvent the "absence of evidence" dilemma posed by the variable precision of polypeptide segments in a distance-geometry model (Liu et al. 1992; Clore et al. 1993; Tjandra et al. 2000). Thus, whereas imprecision of the A1–A8 segment in the DG/SA ensemble of AlaA2-DKP-insulin reflects the absence of selected NOE restraints ("absence of evidence"; Table 4Go), its physical reality is corroborated by attenuation of the analog's helix-specific CD signature ("evidence of absence"; Table 2Go). In the future, it would be of interest to characterize the dynamics of AlaA2-DKP-insulin on multiple time scales by heteronuclear relaxation methods (Barbato et al. 1992; Peng and Wagner 1992; Mulder et al. 2000) and analysis of residual dipolar couplings (Tjandra and Bax 1997; Tjandra et al. 1997; Tjandra et al. 2000; Goto et al. 2001). The extent and time scale of structural fluctuations in AlaA2-DKP-insulin are presently not well characterized.

To our knowledge, segmental unfolding of portions of a protein surrounding a putative cavity has not previously been observed as a response to a large-to-small substitution (for review, see Matthews 1995). Its occurrence here may reflect the small size of the insulin monomer and the key role played by the side chain of IleA2 in tethering the A1–A8 {alpha}-helix to the hydrophobic core. Our results are in accord with a prescient early study of A2 and A19 analogs that proposed that van der Waals interactions between the side chains of IleA2 and TyrA19 are essential to insulin's structure and function (Kitagawa et al. 1984b). The economy of insulin's design leaves little "redundancy" within its network of tertiary contacts. The likelihood of structural changes among mutant insulins highlights the need for structural studies when interpreting relative binding activities. Insulin's putative receptor-binding surface as defined by alanine scanning mutagenesis (Kristensen et al. 1997) does not resolve structural effects from the role of individual side chains in receptor recognition. The very low activity of AlaA2-DKP-insulin, for example, is likely to reflect both segmental unfolding and loss of a specific contact between the IleA2 and the insulin receptor. Packing of the IleA2 side chain in the hormone-receptor interface (Pullen et al. 1976; Baker et al. 1988) is proposed to require a conformational change in the B chain (Derewenda et al. 1991; Hua et al. 1991; Nakagawa and Tager 1992). Mutations in the B chain may affect activity by hindering or facilitating this putative change (Kobayashi et al. 1982; Mirmira and Tager 1989; Hua et al. 1991; Mirmira et al. 1991; Kurapkat et al. 1997). In contrast, insulin's A1–A8 {alpha}-helix is thought to function as a preformed recognition element (Weiss et al. 2000). Because insulin's active structure is not well understood, detailed interpretation of relative activities will require crystallographic analysis of a hormone-receptor complex.

Cavity-forming mutations in globular proteins
Loss-of-volume substitutions in cores of globular proteins often create novel cavities or crevices (Eriksson et al. 1992, 1993). Although the size of packing defects depends on the extent of local conformational adjustments, impaired packing interactions are associated with decreased thermodynamic stability (Eriksson et al. 1992). The extent of destabilization varies from case to case. Structural studies of T4 phage lysozyme variants have indicated a correlation between cavity size and thermodynamic impairment (Eriksson et al. 1992; Xu et al. 1998). Extensive thermodynamic studies of alanine substitutions have been described in T4 phage lysozyme (Xu et al. 1998), Arc repressor (Milla et al. 1994), gene 5 protein (Terwilliger 1995), and human growth hormone (Wells 1994). In relation to such studies, AlaA2-DKP-insulin shows a typical thermodynamic decrement (0.4–1.2 kcal/mole relative to DKP-insulin; Table 2Go). Because the partial fold of AlaA2-DKP-insulin presumably represents its ground state, however, creation of a crevice in a hypothetical native-like structure (estimated to be 75 Å3 if a native-like A1–A8 {alpha}-helix would have been maintained) must impose a larger thermodynamic penalty than that associated with partial unfolding. The relationship between cavity volume and stability proposed by Matthews and coworkers (Eriksson et al. 1992) would have predicted a corresponding free-energy decrement of 1.8–2.5 kcal/mole.

The physical origins of the AlaA2 analog's thermodynamic decrement are likely to be complex, the net consequence of simultaneous and compensating changes in configurational entropy and enthalpy on the one hand, and solvation entropy and enthalpy on the other. Unfavorable contributions to the observed {Delta}{Delta}Gu presumably include loss of van der Waals interactions in the perturbed hydrophobic core. Favorable contributions presumably arise from changes in configurational entropy and from the substitution of an amino acid of low intrinsic {alpha}-helical propensity (the native isoleucine) by an amino acid of high intrinsic {alpha}-helical propensity (alanine; O'Neil and DeGrado 1990; Chakrabartty et al. 1994). Changes in solvation free energy (Eisenberg and McLachlan 1986) are difficult to estimate in the context of segmental instability.

Studies of model globular proteins have illuminated how surrounding structure can adjust to large-to-small substitutions. In general, such changes are modest and localized to the neighborhood of the substitution. The extent of change appears to depend on how rigid or extensive is the network of surrounding interactions (Xu et al. 1998). The largest structural change characterized in a series of T4 lysozyme variants (main-chain RMSD 0.3 Å) involves a hinge-bending displacement leading to closure of the predicted cavity (Xu et al. 1998). This substitution (L84A) occurs at a side chain that, like IleA2 in insulin, is close to the protein surface and engaged in a tertiary contact of limited size. It is possible that crystallization of such mutant proteins can in itself enhance order, as indicated in principle by laser Raman spectroscopy: line widths of globular proteins in solution are broader than in a single crystal5 (Altose et al. 2001).

Possible implications for folding, misfolding, and function
Studies of the oxidative refolding of a single-chain insulin precursor (analogous to proinsulin; Qiao et al. 2001) and IGF-Is (Hober et al. 1992; Miller et al. 1993; Hua et al. 1996b) indicate a nonrandom pathway of disulfide pairing. Analysis of equilibrium models obtained by pairwise substitution of cystines by alanine or serine has indicated that successive disulfide pairing is accompanied by stepwise stabilization of native-like structural elements (Narhi et al. 1993; Hua et al. 1996b; Hober et al. 1997; Weiss et al. 2000). Because insulin's isolated A and B chains contain sufficient information to specify the folding of proinsulin (Wang and Tsou 1991), structures of two-chain analogs have been investigated as peptide models of protein-folding intermediates (Oas and Kim 1988). Of particular interest are the partial folds of insulin analogs lacking the A6–A11 disulfide bridge (des-[A6–A11]Ser-DKP-insulin and des-[A6–A11]Ala-DKP-insulin; Hua et al. 1996b; Weiss et al. 2000). These analogs are remarkable for segmental unfolding of the A1–A11 segment. Although the extent of disorder and thermodynamic instability are more marked than those observed here, in each case the response of the molecule is segmental and characterized by detachment of the A2 side chain from the hydrophobic core. Structures of the disulfide analogs indicate that one face of the corresponding intermediate's hydrophobic core provides an internal template for the A1–A8 coil->helix transition (Hua et al. 1996a), that is, as an "internal template" to align the A6 and A11 thiolates for specific disulfide pairing (Fig. 1BGo). The structure of this template, as inferred from the crystal structure of the native T state, is shown in Figure 6Go. The A1–A4 portion of the {alpha}-helix (yellow ribbon in Fig. 6AGo) occupies a deep groove in the surface of the monomer. The floor of the groove is occupied by invariant side chains TyrA19 and LeuB15. The internal-template hypothesis raised the possibility that—even with the three native cystines intact—the A1–A8 {alpha}-helix is not integral to the structure of the insulin monomer (Weiss et al. 2000). The structure of AlaA2-DKP-insulin supports this hypothesis.



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  		Fig. 6. Structure of the A1–A8 {alpha}-helix with respect to its nonpolar template. (A) Stick representation of crystallographic T-state protomer (Baker et al. 1988) showing A1–A5 (yellow ribbon) and underlying nonpolar template, largely defined by the side chains of TyrA19 and LeuB15 (green, shown in space-filling representation) as well as of LeuB11 and TyrB26. Image was generated using InsightII (Biosym, Inc.). (B) Surface representation of template (green) generated by GRASP (Nicholls et al. 1991). For clarity, residues A1–A5 were removed to highlight the underlying nonpolar groove. In oxidative folding of proinsulin and insulin-related growth factors, this groove is proposed to provide a template to direct folding of the A1–A8 {alpha}-helix as a late event (Hua et al. 1996a,b; Weiss et al. 2000).

 
Modularity of the A1–A8 segment implies that, regardless of its local state of organization, native-like structure is elsewhere maintained. This finding extends to the A-chain observations well-established in studies of the B chain. Crystallographic studies of insulin hexamers have shown, for example, that insulin's overall structure is compatible with alternative B1–B8 configurations: either extended strand (as in the T-state; Baker et al. 1988) or {alpha}-helix6 (the R/Rf state; Bentley et al. 1976; Smith et al. 1984). Further, the monomer's globular {alpha}-helical domain is unaffected by truncation or detachment of the B chain's C-terminal ß-strand (B24-B30; Bao et al. 1997). A degree of modularity of the A1–A8 {alpha}-helix was previously inferred from comparison of crystal structures (Chothia et al. 1983). The T->R transition is accompanied by an approximately 20° rotation of this helix, which in turn requires multiple adjustments in core packing. The concerted series of conformational changes among insulin hexamers, including variable packing of the A1–A8 {alpha}-helix, has been analyzed as a model for the transmission of conformational change in proteins (Chothia et al. 1983).

The biological "life cycle" of insulin is likely to require a series of conformational changes. Although the A1–A8 segment appears to function in receptor binding as a recognition {alpha}-helix (Pullen et al. 1976; Baker et al. 1988; Hua et al. 1996a; Weiss et al. 2000), nonhelical configurations may be important in prohormone processing. Cleavage of the junction of the C-peptide-portion (ArgA0) of proinsulin and GlyA1 (Duguay et al. 1997) is effected by a subtilisin-related converting enzyme conserved among neuroendocrine processing proteases (Lipkind and Steiner 1999). Such enzymes are thought to accept extended strands—but not {alpha}-helices—into their active sites (Lipkind and Steiner 1999). In such a complex, the side chain of IleA2 is itself proposed to dock within a conserved pocket of the converting enzyme rather than within proinsulin's hydrophobic core (Lipkind and Steiner 1999). Accordingly, we suggest that the structure of AlaA2-DKP-insulin provides a model of how fluctuations in the structure of proinsulin may enable its docking with the converting enzyme. Selective unfolding of the A1–A8 segment may also facilitate partial exposure of hydrophobic surfaces, leading to aberrant aggregation as intermediates in insulin fibrillation (Brange et al. 1997; Kelly et al. 1997; Bouchard et al. 2000; Nettleton et al. 2000; Sipe and Cohen 2000; Nielsen et al. 2001). This process would be analogous to the ordered assembly of ß-sheet-rich structures encountered in pathological amyloidogenesis (Orpiszewski and Benson 1999). Because insulin fibrillation is accelerated by partial thermal unfolding, it is of future interest to investigate the structure and dynamics of the A1–A8 segment in native insulin at elevated temperatures and effects of the IleA2->Ala substitution on rates of fibrillation.


   Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
Electronic supplemental material
 References
 
Materials
4-Methylbenzhydrylamine resin (0.6 mmole of amine/g; Star Biochemicals, Inc.) was used as solid support for synthesis of the A-chain analog; (N-butoxy-carbonyl, O-benzyl)-threonine-PAM resin, (0.56 mmole/g; Bachem, Inc.) was used as solid support for synthesis of the B-chain analog. tert-Butoxycarbonyl-amino acids and derivatives were obtained from Bachem and Peninsula Laboratories; N,N`-dicyclohexylcarbodiimide and N-hydroxybenzotriazole (recrystallized from 95% ethanol) from Fluka. Chromatography resins were preswollen microgranular carboxymethylcellulose (CM-cellulose; Whatman CM52), DE53 cellulose (Whatman) and Cellex E (Ecteola cellulose; Sigma); solvents were HPLC grade.

Peptide synthesis
The general protocol for solid-phase synthesis is as described (Barany and Merrifield 1980). The C-terminal Asn in the synthesis of the A chain was incorporated into solid support by coupling tert-butoxycarbonyl aspartic acid {alpha}-benzyl ester with 4-methylbenzhydrylamine resin. After the final deprotection, the Asp residue was converted to an Asn residue.

(i) Synthetic A-chain S-sulfonate
Peptidyl resin (0.82 g), after deblocking, sulfitolysis, and chromatographic purification (Hu et al. 1993; Hua et al. 1996a), yielded ~244 mg of purified S-sulfonated AlaA2 A-chain variant.

(ii) Synthetic B-chain S-sulfonate
After deblocking, sulfitolysis, and chromatographic purification, 610 mg of peptidyl resin yielded ~125 mg of purified S-sulfonated B chain. Amino-acid analyses were in agreement with expected values.

Peptide purification
Crude S-sulfonated A chain was purified by chromatography on a Cellex E column (1.5 x 47 cm) as described (Hu et al. 1993; Hua et al. 1996a), dialyzed against distilled water, and lyophilized to yield the purified AlaA2 A-chain S-sulfonate. Crude S-sulfonated [AspB10, LysB28, ProB29] B chain was likewise purified on a cellulose DE53 column (1.5 x 47 cm), dialyzed, and lyophilized to yield the DKP B-chain S-sulfonate.

Chain recombination
Chain recombination used S-sulfonated A- and B chains (approximately 2:1 by weight) in 0.1 M glycine (pH 10.6) in the presence of dithiothreitol (Chance et al. 1981). The DKP-insulin analog was isolated from the combination mixture as described (Hu et al. 1993; Hua et al. 1996a) and purified on a 0.9 x 23 cm CM-cellulose chromatography and rp-HPLC on a Vydac 218 TP column (0.46 x 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. Rechromatography of this material on reverse-phase HPLC (rp-HPLC) under the same conditions gave a single sharp peak. Amino acid analysis and mass spectrometry gave expected values.

Biological assays
For insulin receptor-binding studies, plasma membranes were partially purified from human placenta. [125I]-insulin was purchased from Dupont NEN. Assays were performed at 4°C as described (Marshall et al. 1974; Cara et al. 1990; Weiss et al. 2001) with relative activity defined as the ratio of human insulin to analog required to displace 50% of specifically bound 125I-insulin.

Spectroscopy
1H-NMR spectra were obtained at 600 MHz at 25°C in 50 mM potassium phosphate (pH 7) and in 20% deuteroacetic acid (pH 1.9) as described (Hua and Weiss 1991; Hua et al. 1996a); the protein concentration was 1.5 mM. No significant differences were observed in the pattern of interresidue NOEs and chemical shifts at pH 7 or pH 1.9 for AlaA2-DKP, comparing with those for DKP-insulin (Hua et al. 1996a). The range of pH conditions enables resonances overlapping in one spectrum to be resolved in another. Acidic pH facilitates analysis of amide resonances, some of which are attenuated at pH 7 as a result of base-catalyzed solvent exchange. Resonance assignment was based on 2D NOESY (mixing times 100 and 200 ms), total correlated spectroscopy (TOCSY) (mixing times 30 and 55 ms), and double-quantum filtered correlated spectroscopy (DQF-COSY) spectra. Spectra in H2O were obtained using pulse-field gradients and laminar shaped pulses (Hua et al. 1996a). CD spectra were obtained using an Aviv spectropolarimeter equipped with thermistor temperature control and automated titration unit for guanidine denaturation studies. CD samples contained 25–50 µM insulin or analog in 50 mM potassium phosphate (pH 7); samples were diluted to 5 µM for equilibrium denaturation studies.

Thermodynamic modeling
Guanidine denaturation data were fitted by nonlinear least squares to a two-state model as described (Sosnick et al. 2000; Weiss et al. 2000). In brief, CD data {theta}(x), where x indicates the concentration of denaturant, were fitted by a nonlinear least-squares program according to


where x is the concentration of guanidine and where {theta}A and {theta}B are baseline values in the native and unfolded states as approximated by pre- and post-transition lines {theta}A(x) = {theta}AH2O + mAx and {theta}B(x) &equals {theta}BH2O + mBx. Fitting the original CD data and base lines simultaneously circumvents artifacts associated with linear plots of {Delta}G as a function of denaturant according to {Delta}GO(x) = {Delta}GH2OO + mOx (for review, see Sosnick et al. 2000). Nonetheless, the m value obtained in fitting the variant unfolding curve is significantly lower than the m value obtained in fitting the wild-type unfolding curve (DKP-insulin; see Table 2Go). This situation can be associated with an underestimate of the AlaA2 analog's stability. The analog's lower m value may reflect its greater exposed hydrophobic surface in the absence of denaturant or existence of a native-state ensemble containing a distribution of incompletely folded forms of differing stability. Analysis of unfolding curves in this setting has recently been considered by Luo and Baldwin in a study of an equilibrium MG (apomyoglobin; Luo and Baldwin 2001). In brief, the apparent Cmid is interpreted as a mean value

where each species in the ensemble makes a fractional contribution to the weight-averaged unfolding curve. If the distribution of stabilities in the mutant ensemble is small relative to its mean decrement relative to the wild type, then the variant's stability may be estimated in a linear-extrapolation model by the product of the wild-type slope and <Cm>. This approximation fails if only a small fraction of the variant ensemble contributes to the experimental probe of foldedness. Because insulin does not show a detectable change in tyrosine fluorescence on unfolding, concordance of fluorescent and codeleted unfolding curves could not be evaluated as a criterion of apparent two-state MG unfolding as proposed by Luo and Baldwin (2001).

Molecular modeling
The volume and location of inferred protein cavities were obtained using the program SURFNET (Laskowski 1995). Analysis of polar and nonpolar protein surfaces was obtained using the Connolly surface feature implemented in InsightII (Biosym, Inc.).


   Electronic supplemental material
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Electronic supplemental material
References
 
Supplemental information contains one figure depicting changes in 1H-NMR secondary shifts between AlaA2-DKP-insulin and DKP-insulin by proton class in 20% acetic acid at pH 1.9 and 25°. Tables S1 and S2 provide chemical shifts of assigned 1H NMR resonances of AlaA2-DKP for conditions corresponding to Figures 3Go and S1, respectively. Table S3 is a list of DG/SA restraints for AlaA2-DKP. The atomic coordinates for AlaA2-DKP-insulin have been deposited in the RCSB Protein Data Bank (PDB number 1K3M).


   Acknowledgments
 
This work was supported in part by grants from the National Institute of Health to P.G.K. and M.A.W. and by the Diabetes Research and Training Center at the University of Chicago. We thank P. DeMeyts, G.G. Dodson, E. Dodson, D.F. Steiner, J. Whittingham, and the late H.S. Tager for helpful discussion.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.


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 Discussion
 Materials and methods
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