The development of fast-acting and highly stable insulin analogues is challenging. Insulin undergoes structural transitions essential for binding and activation of the insulin receptor (IR), but these conformational changes can also affect insulin stability. Previously, we substituted the insulin A6-A11 cystine with a rigid, non-reducible C=C linkage ("dicarba" linkage). A cis alkene permitted the conformational flexibility of the A-chain Nterminal helix necessary for high affinity IR binding

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... sulting in surprisingly rapid activity in vivo. Here, we show that, unlike the rapidly acting Lys B28 Pro B29 insulin analogue (KP insulin), cis dicarba insulin is not inherently monomeric. We also show that cis dicarba KP insulin lowers blood glucose levels even more rapidly than KP insulin, suggesting that an inability to oligomerize is not responsible for the observed rapid activity onset of cis dicarba analogues. While rapid-acting, neither dicarba species is stable, as assessed by fibrillation and thermodynamics assays. MALDI analyses and molecular dynamics simulations of cis dicarba insulin revealed a previously unidentified role of the A6-A11 linkage in insulin conformational dynamics. By controlling the conformational flexibility of the insulin B-chain helix, this linkage affects overall insulin structural stability. This effect is independent of its regulation of the A-chain N-terminal helix flexibility necessary for IR engagement. We conclude that high affinity IR binding, rapid in vivo activity, and insulin stability can be regulated by the specific conformational arrangement of the A6-A11 linkage. This detailed understanding of insulin's structural dynamics may aid in the future design of rapid-acting insulin analogues with improved stability. Since the discovery of insulin by Banting and Best, insulin therapy remains the primary treatment for type 1 (T1D) and late stage type 2 diabetes (T2D), being prescribed to effectively lower blood glucose levels (1-3). Long-acting (eg insulin glargine; Lantus ® , Sanofi) and rapid-acting insulin analogues (eg lispro insulin or KP insulin 4 ); Humalog ® , Eli Lilly) currently in clinical use were developed to mimic the physiological basalbolus insulin profile (recently reviewed in Mathieu et al. (3)). Although these analogues are undoubtedly life-savers for diabetic patients, their pharmacokinetic and pharmacodynamic performance remains suboptimal for some patients (3). Of high priority is the development of a new rapid-acting insulin analogue possessing faster onset of action and greater structural stability. To achieve this, a deeper understanding is required of the structural determinants of insulin necessary for receptor binding and function. Insulin is a small globular protein synthesized in the pancreatic b-cells and secreted as a two-chain polypeptide comprising a 21-residue A chain and a 30residue B chain. The secondary structure of insulin consists of three a-helices, two within the A chain (A1 to A8 and A12 to A18, respectively) and a single ahelix within the central segment of B chain (B9 to B19) (Fig. 1, A and B) (4). It is stored in pancreatic b-cells as 2Zn-coordinated hexamers that, when released into the blood stream, rapidly dissociate into active monomers (5). The monomeric form then adopts the active conformation, and in doing so reveals essential residues within the hydrophobic core for binding (6) but also primes the molecule to the formation of higher order oligomers (7-9). This is highlighted in the rapidacting KP insulin, an analogue possessing an inversion of Pro B28 Lys B29 in the B chain of native insulin to render the molecule essentially monomeric through disruption of the dimer interface ( Fig. 1A) (10,11). As http://www.jbc.org/cgi/a consequence, KP insulin is rapid acting, but concomitant exposure of core hydrophobic residues means that KP insulin readily forms fibrils (9, 12, 13) . This highlights the fine balance between the competing requirements for stability and conformational plasticity needed for optimal activity. High affinity binding to the insulin receptor (IR; a tyrosine kinase receptor (14) ) is achieved by interaction through two distinct binding surfaces (1 and 2) on the ligand (6,15-17). Insulin site 1 binding residues (also known as the "classical binding site") are also involved in insulin dimerization (18, 19) while site 2 binding residues overlap with the hexamerforming surfaces within a 2Zn-insulin hexamer ( Fig. 1A ) (6, 16, 20) . Recent crystallographic studies of an IR fragment comprised of the ectodomain and the aCT segment in complex with insulin through site 1 residues provided key insights into the mechanism of insulin:IR interaction (15, 21) . Key conformational changes in insulin required for effective IR binding were identified. Movement of the C-terminal segment (B24-B30) of the B chain from the hormone core is required for engagement with the IR aCT and L1 domains (22). It is likely that the end of the B chain opens in a zipper-like fashion (23) to allow accessibility to insulin site 1-binding residues that otherwise remain buried within the hydrophobic core (21, 22). Evident in our previous findings, insulin also undergoes further conformational change within the first A chain helix to enable binding. The helix rotates in order to avoid a steric clash between the first four residue sidechains and the aCT segment (24). In this conformation, residues A1 to A5 to form a single ahelical turn while residues A3 to A9 adopt a wider helix conformation, approximating an (i, i+5) p-helix (24). This conformation is also evident in the µIR: insulin crystal structure (PDB entries 4OGA) (21,24) and can be artificially achieved by a mutation of residue B26 that also promotes opening of the B chain (25,26). The correct folding and stabilization of the unique three-dimensional structure of mature insulin is supported by the three disulfide linkages: two interchain (Cys A7 -Cys B7 and Cys A20 -Cys B19 ) and one intrachain (Cys A6 -Cys A11 ) (Fig. 1, A and B) (5,27). Correct disulfide combination is essential for function (27-30). The Cys A7 -Cys B7 bond is surface exposed and holds the N-termini of the two chains together. Both Cys A6 -Cys A11 and Cys A20 -Cys B19 linkages are buried within the hydrophobic core. Once folded, the Cys A20 -Cys B19 disulfide is constrained in a fixed configuration and buried deeply within the core, suggesting its most likely function is to maintain structural stability. The intra-chain Cys A6 -Cys A11 cystine, although buried, is relatively flexible and can adopt several different configurations (30-32). In our recent study, we identified that the Cys A6 -Cys A11 linkage is an important modulator of the structural transitions of the N-terminal A chain helix required for insulin activity. Introduction of two isomeric fixed A6-A11 dicarba bridges in insulin did not perturb the overall structure but resulted in strikingly different receptor potencies, where the cis isomer permits high affinity IR binding and the trans isomer does not (Figs. 1C, 2A and (24)). Arising from our study of the cis dicarba insulin was the observation that this analogue promotes a more rapid lowering of blood glucose levels than native insulin. It is also less thermodynamically and chemically stable than native insulin (24). As these properties are reminiscent of the monomeric KP insulin (7,9,33), we investigate here whether the A6-A11 likage not only controls A chain flexibility but also influences the B chain conformation, leading to the monomeric state required for rapid receptor engagement. In this study, through a combination of biophysical analyses of cis dicarba insulin and the monomeric cis dicarba KP insulin counterpart, we show that, despite its rapid action in vivo and its accelerated fibril formation relative to native insulin, the cis dicarba insulin is not inherently monomeric. Limited proteolysis studies alluded to an unexpected conformational change in the B-chain helix. Such a link between the A6-A11 disulfide and the conformational dynamics of the B chain has not been previously described. These findings suggest a key role for the A6-A11 linkage, not only in regulating A chain flexibility that primes insulin for receptor engagement, but also in influencing the B chain conformation and regulating insulin's stability. RESULTS Chemical synthesis of dicarba insulins -We aimed to understand the mechanism by which the cis dicarba insulin was apparently rapid acting in vivo by directly comparing its in vitro and in vivo biological and biophysical activities with the monomeric c[Δ 4 A6,11]dicarba human lispro insulin (cis-and trans dicarba KP insulins). The cis-and trans-configured dicarba insulin A chains, in which a C=C dicarba bond replaces the A6-A11 intra-chain S-S bond (Fig. 1C) , were synthesised as previously described using a RCM and SPPS-catalysis approach (24,34). The modified dicarba insulin A chains were then combined with requisite insulin B chains to provide cis and trans isomers of c[Δ 4 A6,11]-dicarba human insulin and c[Δ 4 A6,11]-dicarba KP insulin. The dicarba analogues were purified by RP-HPLC (see Figs. 1, D and E, and S1) before being subjected to biological and biophysical analyses. Cis dicarba insulin and cis dicarba KP insulin are equally potent to native insulin in receptor binding and activation - The binding affinities of the dicarba insulin analogues for the IR-B and IGF-1R were determined using competition binding assays (Figs. 2A, S2 and Table S1 ). Notably, the restrained A6-A11 cis and trans dicarba C=C bonds had the same effect when introduced into the monomeric KP insulin analogue as was previously seen with the cis and trans dicarba analogues of human insulin (24). Cis dicarba insulin and cis dicarba KP insulin were equipotent to native insulin in binding and activation of both the IR-B (Fig. 2, A and B ) and the IGF-1R (Fig. S1 , A and B) (Table S1), suggesting that the restrained cis dicarba by guest on July 23, 2018 http://www.jbc.org/ Downloaded from A6-A11 disulfide affects insulin B chain conformation 3 C=C bond allows both analogues to adopt a conformation that engages with both receptors in a manner similar to insulin. Conversely, trans dicarba insulin and trans dicarba KP insulin bind poorly to both the IR-B ( Fig. 2A ) and IGF-1R (Fig. S1A) (Table S1), indicating that the trans dicarba configuration restricts both analogues from forming a high affinity interaction with these receptors. The trans dicarba insulins were subsequently excluded in this and further activity assays due to their poor receptor binding affinities. The cis dicarba KP insulin promotes in vitro DNA synthesis and glucose uptake with equal potency to native insulin -Corresponding with its IR-B binding and activation potency, the cis dicarba KP insulin was equipotent with native insulin in promoting DNA synthesis in L6 rat skeletal myoblast overexpressing IR-A. This is in contrast to the cis dicarba insulin, which was 5 to 10-fold less potent than insulin in promoting mitogenic activity ( Fig. 2C and (24) ). The cis dicarba KP insulin was equipotent with cis dicarba insulin and insulin in promoting glucose uptake in cultured NIH3T3-L1 adipocytes (Fig. 2D ). There is a trend of lower activities for the cis dicarba insulin and cis dicarba KP insulin, however the effect was not significantly different. The cis dicarba insulin demonstrates identical selfassociation behaviour to native insulin -AUC was performed in order to determine whether the cis dicarba insulin is monomeric as suggested by its rapid action in vivo (Fig. 2, E and F, and (24)). In the presence of Zn 2+ , sedimentation equilibrium data for the cis dicarba insulin was fitted to a single species of apparent mass 34,500 ± 400 Da ( Fig. 3A) , consistent with the expected mass of a 2-Zn 2+ human insulin hexamer (34,726 Da). At higher concentrations the fit was not perfect (reduced χ 2 ~ 4), suggesting the presence of other high molecular weight species, consistent with previous observations for native mammalian insulins (35). In contrast, the shape of the equilibrium concentration distributions in the absence of Zn 2+ were clearly concentration dependent, implying reversible self-association (Fig. 3B) . Accordingly, these data could not be fit as a single species, in sharp contrast to those we have recently described for the strictly monomeric venom insulin of Conus geographus (Fig. S11) (36). Native insulin shows similar concentration dependence in the shape of its equilibrium concentration distributions (Fig. S11) , and also fails to fit to single-species models, consistent with its expected tendency for self-association. For an initial model-free assessment of the self-association of Zn 2+free cis dicarba and native insulin, the data were plotted as the square of the radial position scaled by rotor speed [w 2 (r 2 -r 0 2 )] vs the logarithm of the equilibrium concentration (Fig. 3C ). The slope of such a plot is proportional to the weight-average molecular weight of all species present at each point in the cell, and the observed non-linearity confirms the presence of multiple species in the sample. Over much of the accessible concentration range, the slopes of the cisdicarba and native insulin plots are similar, implying that the self-association behaviour of the two insulins are similar. At low concentration (< 10 µM), the slope was consistent with that expected for monomeric insulin, suggesting that monomer dominated at these concentrations. The slope increased with increasing concentration, indicating that oligomeric species were dominant over much of the experimental concentration range. Only at the highest concentrations is there evidence of some divergence between the two curves, suggesting the possibility of some difference in the tendency of cis dicarba insulin to form higher-order oligomers. Attempts to fit the two datasets to a specific, consistent model of self-association were unsuccessful, due to numerical instabilities in relevant models (37), and perhaps also reflecting the putative subtle difference in higher-order oligomerisation. Nonetheless, under the conditions studied, the selfassociation behaviour of the cis dicarba insulin is qualitatively similar to that of native mammalian insulins. Like native insulin, cis dicarba insulin is only monomeric in the absence of Zn 2+ , and only at low concentration. Cis dicarba KP insulin lowers blood glucose levels more effectively and more rapidly compared to the cis dicarba insulin, KP insulin and native human insulin -Having established that the cis dicarba insulin is not monomeric we sought to compare the in vivo activities of this analogue with cis dicarba KP insulin, which we can assume is monomeric as per KP insulin. The cis dicarba KP insulin lowered blood glucose levels more effectively and more rapidly compared to native insulin and KP insulin when mice were treated with 0.75 IU/kg insulin or analogue under non-fasting conditions (Fig. 2, E and F) . Notably, the cis dicarba KP insulin was even more effective and rapid acting than the cis dicarba insulin. The glucose-lowering effect was most evident and significant in insulinresistant mice fed on high fat diet (Fig. 2F) . The difference in activities between native and cis dicarba KP insulin, which are both expected to be monomeric, implies that the improved activity of the cis dicarba analogues is not the result of a change in selfassociation. This is consistent with the above observation that native and cis dicarba insulin show similar self-association behaviour. Cis dicarba insulin and cis dicarba KP insulin more rapidly form fibrils than native human insulin -While the rapid action of the cis dicarba insulin compared to insulin is not due to this analogue being monomeric, it must have different biophysical properties to insulin that lead to this difference in biological activity. To explore this further we next investigated the ability to form fibrils in an AFM fibrillation assay. The cis dicarba insulin formed fibrils more rapidly than native insulin, with fibrils first detected after 2 h compared to 6 h for native insulin at the same temperature and concentration (60 °C and 1.16 mg/mL, respectively, Fig. 4) . Consistent with being monomeric, KP insulin also rapidly formed fibrils, with fibrils first detectable at 2 h. The cis dicarba KP insulin fibrillation is evident between t = 6 -8 h. Interestingly, the fibrils formed by both cis by guest on July 23, 2018 http://www.jbc.org/ Downloaded from A6-A11 disulfide affects insulin B chain conformation 4 dicarba insulin and cis dicarba KP insulin appeared shorter, thicker and of different morphology to those arising from native insulin and KP insulin ( Fig. 4 ; see cis dicarba insulin at t = 15 h and cis dicarba KP insulin at t = 8 h). These observations are consistent with the fact that thioflavin T (ThT), a dye commonly used to detect insulin fibrils, did not bind cis dicarba insulin fibrils (data not shown). ThT normally binds to insulin fibrils at two sites (between fibers and/or between protofilaments) (38). Our data suggest that while cis dicarba insulin is not inherently monomeric it is conformationally different to native insulin. Cis dicarba insulin and cis dicarba KP insulin are thermodynamically less stable than native insulin -Next, we compared the thermodynamic stability of the cis dicarba insulin with the cis dicarba KP insulin. Introduction of a dicarba A6-A11 bond into KP insulin also led to a decrease in thermal and chemical denaturation stabilities. The far-UV CD spectra (190 -260 nm) of Zn 2+ -free cis-and trans dicarba KP insulin exhibited lower helical content (34 % and 17 %, respectively) compared to KP insulin (44 %) (see Fig. 5A and Table 1 ). The [q] 222 values are directly proportional to the helical content of the proteins. As seen with the cis dicarba insulin (24), the cis dicarba KP insulin (Fig. 5B ) exhibited smaller [q] 222 negative magnitudes measured at 20 °C consistent with their lower initial helical content (Table 1) . Denaturation induced by increasing temperature (20 -70°C) (Fig. 5B ) or GdnHCl concentrations (0 -8M) (Fig. 5C ) was monitored at wavelength 222 nm using CD. The relative thermodynamic stabilities of KP insulins were determined by comparing the relative change of ellipticity with increasing temperature at 1 °C intervals, i.e. by comparing the slope of temperature denaturation curves. Native and KP insulins exhibited similar thermal denaturation curves, whereas the cis dicarba KP insulin exhibited relatively small changes in ellipicity due to its significantly lower starting helical content ( Fig. 5B and Table 1 ). The cis dicarba KP insulin is also considerably less stable upon GdnHCl denaturation, with DG°u = 1.71 kcal mol -1 , as was seen with the dicarba insulin isomers (Fig. 5C , Table 1 and (24) ). The effect of introduction of the cis dicarba bond on thermodynamic stability is striking and much greater than the introduction of the KP mutation (Fig. 5, B and C, and (24) ). The observed reduction in stability is thus independent of being monomeric. The cis dicarba A6-A11 linkage promotes a structural change in the B chain helix, which leads to instability -In order to explain why the cis dicarba peptides are less thermodynamically stable, we performed a limited proteolysis study that allowed us to detect structural differences between the cis dicarba peptides, native insulin and KP insulin. This involved RP-HPLC separation of fragments generated by chymotrypsin proteolysis and subsequent mass spectrometry. Firstly, we observed that cis-and trans dicarba insulin isomers eluted later (15.8 and 14.6 min, respectively) than native insulin (13.3 min) when separated by RP-HPLC (Fig. 1D) . Similarly, the cis-and trans KP isomers also eluted with delayed retention times (16.3 and 15.1 min, respectively) compared to KP insulin (13.5 min) (Fig. 1E) . The relative delay in the retention times of dicarba insulins is an indication of an apparent increase in hydrophobicity compared to the native forms, with cis dicarba insulins being more surface hydrophobic. This was the first indication that the cis A6-A11 dicarba linkage induces a more open conformation of insulin than in the native hormone, and that it is likely that hydrophobic residues of the core are more exposed. The enzymatic stability of dicarba insulin analogues was investigated through limited proteolysis by chymotrypsin under non-reducing conditions. The rate and kinetics of proteolysis were monitored through RP-HPLC as described in the Experimental Procedures. Our results show that cis dicarba insulin is significantly more rapidly cleaved by chymotrypsin compared to native insulin (Fig. 6A) . At a protein: enzyme ratio of 86: 0.078 µM, the native insulin remained almost completely undigested after 3 h of proteolysis (> 95 % undigested peptide remaining), whereas almost no intact cis dicarba insulin remained. The monomeric KP insulin is more susceptible to proteolysis than insulin with ~ 65% undigested peptide remaining after 3 h (~ 30 % reduction compared to native insulin). However, the cis dicarba KP insulin is rapidly cleaved with only ~ 30% of undigested peptide remaining at 3 h (~ 30% reduction compared to KP insulin). As different batches of chymotrypsin were used in the cis dicarba insulin and cis dicarba KP insulin experiments (Fig. 6A vs Fig. S4A ) we cannot determine if the difference in cleavage rates between the two cis dicarba analogues is significant. Clearly both are much more rapidly cleaved than native insulin and KP insulin, respectively. Our RP-HPLC chromatograms of a chymotrypsincleaved insulin ( Fig. 6B ; also see Fig. S3E ) and the cis dicarba insulin (Fig. 6C ; also see Fig. S3F ) were comparable to previously reported data, which identified 4 non-reduced metabolites (termed A, B, C, D) post-cleavage (39). Through positive ion mode of MALDI mass analysis of the entire digest, we detected native insulin metabolites A, C and D following the chymotrypsin digest (protein: enzyme ratio of 86: 0.078 µM; t = 60 min) but metabolite B was not detected (see chromatograms in Figs. 6B and S3E, MALDI analysis in Fig. S5 and schematic diagram in Fig. S7 ). By analysis of individual RP-HPLC fractions we could assign the different masses to individual peaks ( Fig. S5 and S6 ; tables in bottom panel). Metabolite A eluted » 1 min after the undigested peptide (Fig. 6, B and C) , as was seen for all digested insulins (native and dicarba analogues) (labelled as 2 (grey) in Figs. S3 and S4, E -G) . Peptides equivalent to metabolite C and metabolite D eluted at t R » 3.5 min and » 9.5 min respectively for all insulins (see Fig. 6 , B, C and F; labelled as 3 (Metabolite C; orange) and 4 (Metabolite D; green) in Figs. S3 and S4, E -G) . by guest on July 23, 2018 http://www.jbc.org/ Downloaded from A6-A11 disulfide affects insulin B chain conformation 5 Close comparison of the RP-HPLC chromatograms revealed the presence of an apparently unique metabolite in chymotrypsin digests of cis dicarba insulins (Metabolite E in Fig. 6C ; Metabolite 5, cis dicarba KP insulin Fig. S4F ) that was not detected in native insulin (Fig. 6B ) or KP insulin (Fig. S4E) . Metabolite E was detected in proteolytic samples of cis dicarba insulin as early as t = 1 h (kinetics of cleavage shown in Fig. S3C and F) . Analysis from MALDI data in negative ion mode identified the cis dicarba insulin metabolite E from a fractionated and reduced sample (Fig. 6D) . As shown in the simplified schematic diagram, Figure 6F (orange box), the new metabolite is a full-length twochain peptide (with intact A6-A11 dicarba bond and A7-B7 and A19-B20 disulfide bonds) with a single cleavage at the C-terminal end of Tyr B16 of B chain (indicated in cis dicarba insulin crystal structure in Fig. 6E, blue circled 1) . This new metabolite E was not identified in native insulin (Figs. 6B, S3 and S5) or KP insulin (Fig. S4 ) cleaved with chymotrypsin, suggesting that chymotrypsin is able to access Tyr B16 in cis dicarba insulin more readily than in insulin or KP insulin. In order to understand why only cis dicarba insulin is cleaved at this new site, we superimposed insulin structures on the active site of chymotrypsin, ensuring that the tyrosine residue that is recognised by the enzyme was localised in the appropriate binding pocket (Fig. 7) . Significant steric interactions between the insulin structure and chymotrypsin (Fig. 7B ) revealed that it would be necessary for the B chain helix of insulin to bend significantly to allow Tyr B16 to engage with the chymotrypsin enzyme. Such bending behaviour is evident in our previously reported MD simulations of insulin and its cis dicarba analogue (24). It is significant that this bending motion occurs more frequently and persists longer in cis dicarba insulin (see below). Hydrogen bonds between Val B12 and Tyr B16 , Glu B13 and Leu B17 , and Tyr B16 and Gly B20 must all be broken, allowing the Ca-Ca distances between Glu B13 and Tyr B16 and between Tyr B16 and Gly B20 to increase from their unperturbed values of ~ 5.5 Å to ³ 7 Å (Fig. 7A ). This allows Tyr B16 to occupy the chymotrypsin binding pocket, with the two neighbouring loops of the helix wrapping around the binding pocket walls (compare Fig. 7B vs 7C) . These changes in the B chain helix structure are concomitant with the twisting of Cys B7 , generally resulting in a decrease of the Ca-Ca distance across the A7-B7 disulfide linkage and an increase in its torsional strain. The effects of the bulging in the B chain helix are thereby transmitted to the N-terminal helix of the A chain. This results in the N-terminal end of this latter helix being tilted away from the B chain helix, although it is important to note that this conformation of the A chain is also seen in the absence of B helix bulging, and also in circumstances when Cys B7 is not twisted. The propensity of the B chain helices of both insulin and the cis dicarba insulin to undergo bulging can be assessed through the MD simulation data. In Figure 8 , this bulging is monitored via the Glu B13 -Tyr B16 Ca-Ca distance (green). The corresponding Cys A7 -Cys B7 Ca-Ca distances (purple) and the strain in the A7-B7 disulfide linkages (blue) are also shown. It is clear that, while bending of the B chain helix can occur in both insulin and the cis dicarba insulin, it occurs far more frequently in the cis dicarba analogue (Fig. 8, A and B , outlined in black boxes), with the bulging events being more prolonged and showing increased spreading of the loops of the helix (Fig. 7A) . As noted previously, the A chain N-terminal helix is far more labile in the cis dicarba insulin than in insulin (24). It appears that in the more stable insulin structure, the A6-A11 disulfide linkage rapidly dampens the bending of the B chain helix and restores its helical structure. In contrast, this conformational lability of aAN in the cis dicarba insulin allows the bulge in the B chain helix to form more readily and to persist long enough to allow engagement with and digestion by chymotrypsin. It is important to note that there does not appear to be any correlation between bulging of the B chain and the unwinding motion of the A chain necessary for complexation of the insulin receptor (Fig. S9) . A second important consequence of enhanced B chain bulging for the cis dicarba insulin is that the hydrophobic core of the hormone is opened up, resulting in increased exposure to solvent (Fig. 8, red) . This is consistent with the longer observed elution time of the cis analogue in the RP-HPLC experiments (Fig. 1D ). In summary, the limited proteolytic-MS integrated analyses revealed that the kinetics of dicarba insulins proteolysis are different from those of native insulin, leading to the formation of new metabolites. This supports the notion that installation of the intra-chain dicarba bridge enhances structural perturbation near Tyr B16 to permit access of chymotrypsin to this site. DISCUSSION For the last decade, insulin analogue design has focused on improving insulin efficacy and stability. Ideally, we require new rapid-acting insulin analogues that perfectly mimic the normal rapid onset of bolus insulin action. Desirably, insulin analogues would also be physically and chemically stable during pump delivery or at sites of injection. The 'bottleneck' to creating the perfect insulin arises from our incomplete understanding of the relationship between insulin's structure and function, particularly with respect to the fine balance between activity and stability. Previously, we reported the chemical synthesis of two A6-A11 dicarba insulin analogues (cis-and trans dicarba insulins). Using these stereoisomers, we obtained remarkable insight into the previously unexplored function of the insulin A6-A11 disulfide bond in modulating insulin activity. Unique to these insulin analogues, only cis dicarba insulin is biologically active while trans dicarba insulin is inactive. We proposed that the underlying cause of this difference lies in the structural dynamics of the A6-A11 linkage, and that this dictates insulin's ability to transition into its active conformation. We demonstrated that the configuration of the A6-A11 by guest on July 23, 2018 http://www.jbc.org/ Downloaded from A6-A11 disulfide affects insulin B chain conformation 6 linkage could modulate insulin's ability to engage with the receptor through its influence on the conformational flexibility of the N-terminal A chain helix (24). In the current study, we seek to explain why in vivo the cis dicarba insulin promotes more rapid lowering of blood glucose than native insulin. Taken together with our observation of reduced thermal and chemical denaturation stabilities we hypothesised the rapid action might be attributed to the cis dicarba insulin being monomeric. To address this, we undertook biophysical analyses of the cis dicarba insulin in comparison to the cis isomer of dicarba KP insulin, which we assume is monomeric as per KP insulin. We first compared receptor binding and biological activity of the dicarba KP insulins with the cis-and trans dicarba insulins and native insulin. Consistent with the unique biological characteristics of cis-and trans dicarba isomers of native insulin, the cis dicarba KP insulin was also equipotent to native insulin (receptor binding, receptor activation, DNA synthesis in myoblasts and glucose uptake by adipocytes) while the trans dicarba KP insulin was inactive. This confirms the functional role of the native A6-A11 cystine bridge. Even in the context of a disrupted dimer interface induced by the B chain C-terminal KP mutation, the A6-A11 bond still influences the ability of insulin to engage with the receptor. Next, we determined that the cis dicarba insulin is not inherently monomeric. The AUC results clearly showed that the distribution of cis dicarba insulin into monomeric and dimeric forms under zinc free conditions was similar to that of native insulin. This was surprising and prompted us to further explore the biophysical differences between the cis dicarba insulin and native insulin that might account for the more rapid action of the cis dicarba analogue. Further evidence of structural differences between the dicarba insulins and native insulin was provided by our fibrillation assays. The qualitative differences in fibrillation rate and fibril conformation between the cis dicarba insulin and native insulin are additional indicators of conformational differences in their structures. It is well established that the movement of the B chain C-terminus away from the B chain helix and hydrophobic core promotes fibrillation, as is seen with KP insulin (Fig. 4 and (8,33,40,41) ). The AUC results show that the cis dicarba insulin is not inherently monomeric, suggesting that the dimer interface is not disrupted, and hence this is not the source of the increased fibrillation rate of the cis dicarba insulin. Another key feature of fibril formation is the transition of the A chain N-terminal helix to a bsheet, a process which requires displacement of the A chain away from the B chain helix and from the hormone core (9,33). Therefore, we postulated that cis dicarba insulin's increased rate of fibrillation compared to native insulin was likely to be connected to its increased flexibility and altered helicity (more plike) at the N-terminus of the A chain. However, upon further investigation using limited chymotrypsin proteolysis we were able to detect an unexpected difference in the structure of the B chain between the cis dicarba insulin and native insulin. The initial cleavage in the cis dicarba insulin occurs at the C-terminal end of Tyr B16 of B chain. This site is not the first site of cleavage in native insulin (the product was not detected), suggesting that the enzyme is unable to readily access this site in fully intact native insulin; cleavage at Tyr B16 only occurs after the molecule has been cleaved at other sites. In the cis dicarba insulin, initial cleavage at Tyr B16 indicates that chymotrypsin can readily access this site, implying that the Cterminal end of the B chain has a tendency to be in a non-native, partially open, or bulged, conformation, thereby allowing enzyme access. Interestingly, this bulged conformation does not affect the ability of the cis dicarba insulin to bind the insulin receptor. Previous mutation studies at Tyr B16 (e.g. to His or Ala) highlighted the importance of this residue in receptor binding as well as being involved in the dimer interface. Interestingly, the Tyr B16 Ala insulin mutant behaves as a monomer on size exclusion chromatography (42). Previously, MD simulations have captured insulin in both "open" and "wide open (receptor bound)" states (23). In that study, the open state referred to a zipper-like opening from the end of the B chain. This state was also observed in our MD investigations of native insulin and its cis dicarba analogue. However, here we additionally observe that simulations of the cis dicarba insulin show a significantly enhanced propensity for outward bulging of the B chain helix. This opens up the helix loop between Val B12 and Leu B17 , exposing Tyr B16 for chymotrypsin cleavage (see Fig. 7) . Hence, the cis dicarba linkage causes two fundamental changes to the dynamics of the insulin structure. On the one hand, it increases the mobility of the A chain N-terminal helix, enhancing its ability to engage favourably with the insulin receptor. On the other, it decreases the stability of the overall structure. This is seen in both the destabilisation of the B chain helix, increasing its susceptibility to chymotrypsin digestion, and in an increase in the solvent exposure of the hydrophobic core of insulin, leaving the hormone vulnerable to degradation via the formation of fibrils. Recently Wade et al reported that the dicarba substitution of the intra-A-chain disulfide bond of the insulin-like peptide H2 relaxin resulted in significantly reduced stability to enzyme degradation in plasma, despite maintaining the ability to bind relaxin's cognate receptor RXFP1. The mechanisms underlying the instability were not explored. Similar to dicarba insulins, dicarba H2 relaxins also displayed structural differences to the native peptide (43). We postulate that the role of intra-A-chain disulfide bond in regulating peptide stability is conserved across the insulin-relaxin superfamily. However, further investigation is required to confirm this. The rapid action of both cis dicarba insulin and cis dicarba KP insulin in lowering blood glucose (in comparison with native insulin) is most likely by guest on July 23, 2018 http://www.jbc.org/ Downloaded from