In vitro insulin refolding: Characterization of the intermediates and the putative folding pathway

The in vitro refolding process of the double-chain insulin was studied based on the investigation of in vitro single-chain insulin refolding. Six major folding intermediates, named P1A, P2B, P3A, P4B, P5B, and P6B, were captured during the folding process. The refolding experiments indicate that all of these intermediates are on-pathway. Based on these intermediates and the formation of hypothetic transients, we propose a two-stage folding pathway of insulin. (1) At the early stage of the folding process, the reduced A chain and B chain individually formed the intermediates two A chain intermediates (P1A and P3A), and four B chain intermediates (P2B, P4B, P5B, and P6B). (2) In the subsequent folding process, transient Ⅰ was formed from P3A through thiol/disulfide exchange reaction; then, transients Ⅱ and Ⅲ, each containing two native disulfides, were formed through the recognition and interaction of transient Ⅰ with P4B or P6B and the thiol group's oxidation reaction mainly using GSSG as oxidative reagent; finally, transients Ⅱ and Ⅲ, through thiol/mixture disulfide exchange reaction, formed the third native disulfide of insulin to complete the folding.     

In vitro insulin refolding: Characterization of the intermediates and the putative folding pathway

The in vitro refolding process of the double-chain insulin was studied based on the investigation of in vitro single-chain insulin refolding. Six major folding intermediates, named P1A, P2B, P3A, P4B, P5B, and P6B, were captured during the folding process. The refolding experiments indicate that all of these intermediates are on-pathway. Based on these intermediates and the formation of hypothetic transients, we propose a two-stage folding pathway of insulin. (1) At the early stage of the folding process, the reduced A chain and B chain individually formed the intermediates: two A chain intermediates (P1A and P3A), and four B chain intermediates (P2B, P4B, P5B, and P6B). (2) In the subsequent folding process, transient I was formed from P3A through thiol/disulfide exchange reaction; then, transients II and III, each containing two native disulfides, were formed through the recognition and interaction of transient I with P4B or P6B and the thiol group's oxidation reaction mainly using GSSG as oxidative reagent; finally, transients II and III, through thiol/mixture disulfide exchange reaction, formed the third native disulfide of insulin to complete the folding.     

The relationship between the connecting peptide of recombined single chain insulin and its biological function

To investigate the relationship between the biological activity of recombined single chain insulin and the length of the connecting peptide, we designed and prepared three single chain insulin molecules, namely, PIP, [A]5PIP and [A]10PIP, by site-directed mutagenesis, in which B30 and A1 were linked through dipeptide A-K, heptapeptide A-A-A-A-A-A-K, and dodecapeptide A-A-A-A-A-A-A-A-A-A-A-K, respectively. Their receptor binding capacities were 0.14%, 14.3% and 11.1% of that of insulin respectively and their in vivo biological activities were in consistence with their receptor binding capacity; whereas their growth promoting activities were 17%, 116.3% and 38% of that of insulin. These results suggested the following conclusions. (i) The recombined single chain insulin could also possess the same metabolic and mitogenic function as insulin. (ii) The receptor binding capacity of recombined single chain insulin to insulin receptor was closely related to the length and amino acid composition of the connectin     

Insulin analogs with B24 or B25 phenylalanine replaced by biphenylalanine

B24 and B25 phenylalanines （Phe） play important roles in insulin structure and function. Insulin analogs with B24 Phe or B25 Phe replaced by biphenylalanine （Bip） were prepared by enzymatic semisynthesis. The biological activities were determined by receptor binding assay and in vivo mouse convulsion assay. The results showed that B25 Bip insulin has 139% receptor binding activity and 50% in vivo biological activity, whereas B24 Bip insulin is inactive, when compared with native insulin, suggesting that B24 Phe is crucial for insulin activity. The structures in solution were studied by circular dichroism and fluoremetry, and our results suggested that the insulin analogs with low activities tend to be more tightly packed. The association properties were studied by size exclusion chromatography. The Bip.amide replacement of B24 Phe in deshexapeptide insulin or B25 Phe in despentapeptide insulin will cause the monomeric B24 Phe-amide deshexapeptide insulin or B25 Phe-amide despentapeptide insulin to associate and form dimers, whereas the mutations of B24 Phe in insulin will make insulin dimers dissociate into insulin monomers.     

The thermodynamic stability of insulin disulfides is not affected by the C-domain of insulin-like growth factor 1

Both Insulin and insulin-like growth factor 1 are members of insulin superfamily. They share homologous primary and tertiary structure as well as weakly overlapping biological activity. However, their folding behavior is different: insulin and its recombinant precursor (PIP) fold into one unique tertiary structure, while IGF-1 folds into two disulfides isomers with similar thermody-namic stability. To elucidate the molecular mechanism of their different folding behavior, we prepared a single-chain hybrid of insulin and IGF-1, [B10Glu]lns/IGF-1(C), and studied its folding behavior compared with that of PIP and IGF-1. We also separated a major non-native disulfides iso-mer of the hybrid and studied its refolding. The data showed that the C-domain of IGF-1 did not affect the folding thermodynamics of insulin, that is, the primary structure of the hybrid encoded only one thermodynamically stable disulfides linkage. However, the folding kinetics of insulin was affected by the C-domain of IGF-1.     

Microfilament-binding properties of N-terminal extension of the isoform of smooth muscle long myosin light chain kinase

Myosin light chain kinases （MLCK） phosphorylate the regulatory light chain of myosin II in thick filaments and bind to F-actin-containing thin filaments with high affinity. The ability of short myosin light chain kinase （S-MLCK） to bind F-actin is structurally attributed to the DFRXXL regions in its N-terminus. The long myosin light chain kinase （L-MLCK） has two additional DFRXXL motifs and six Ig-like modules in its N-terminal extension. The six Ig-like modules are capable of binding to stress fibers independently. Our results from the imaging analysis demonstrated that the first two intact Ig-like modules （2Ig） in N-terminal extension of L-MLCK is the minimal binding module required for microfilament binding. Binding assay confirmed that F-actin was able to bind 2Ig. Stoichiometries of 2Ig peptide were similar for myofilament or pure F-actin. The binding affinities were slightly lower than 5DFRXXL peptide as reported previously. Similar to DFRXXL peptides, the 2Ig peptide also caused efficient F-actin bundle formation in vitro. In the living cell, over-expression of 2Ig fragment increased ＂spike＂-like protrusion formation with over-bundled F-actin. Our results suggest that L-MLCK may act as a potent F-actin bundling protein via its DFRXXL region and the 2Ig region, implying that L-MLCK plays a role in cytoskeleton organization.     

Effect of different immunosuppressive drugs on calcineurin and its mutants

Several mutants in Loop7 region and near Loop7 region of calcineurin A (CN A) subunit have been constructed and purified using site-directed mutagenesis. Their phosphatase activity and the corresponding solution conformation were examined. Their phosphatase activities between wild-type CN and mutants were compared to identify the interaction of different immuno-suppressive drugs with CN. The results showed that the phosphatase activities of the mutants at Loop7 were much higher than the one of wild-type CN. Furthermore, circular dichroism spectra of the mutants revealed that their solution conformations gave rise in changes in native structure of the protein. Cyclophilin-CyclosporinA (CyP-CsA) significantly inhibited the phosphatase activity of wild-type CN, and had no effects on the phosphatase activity of mutants in Loop7 region, which indicates that the site-directed mutagenesis at Loop7 region made a significant change in the interaction between CyP-CsA and CN. Examination of the activities of these     

Protein photo-folding and quantum folding theory

The rates of protein folding with photon absorption or emission and the cross section of photon-protein inelastic scattering are calculated from quantum folding theory by use of a field-theoretical method.All protein photo-folding processes are compared with common protein folding without the interaction of photons(non-radiative folding).It is demonstrated that there exists a common factor(thermo-averaged overlap integral of the vibration wave function,TAOI) for protein folding and protein photo-folding.Based on this finding it is predicted that(i) the stimulated photo-folding rates and the photon-protein resonance Raman scattering sections show the same temperature dependence as protein folding;(ii) the spectral line of the electronic transition is broadened to a band that includes an abundant vibration spectrum without and with conformational transitions,and the width of each vibration spectral line is largely reduced.The particular form of the folding rate-temperature relation and the abundant spectral structure imply the existence of quantum tunneling between protein conformations in folding and photo-folding that demonstrates the quantum nature of the motion of the conformational-electronic system.     

Mutational Analysis of the Absolutely Conserved B8Gly： Consequence on Foldability and Activity of Insulin

B8Gly is absolutely conserved in insulins during evolution. Moreover, its corresponding position is always occupied by a Gly residue in other members of insulin superfamily. Previous work showed that Ala replacement of B8Gly significantly decreased both the activity and the foldability of insulin. However, the effects of substitution are complicated, and different replacements sometimes cause significantly different results. To analyze the effects of B8 replacement by different amino acids, three new insulin/single-chain insulin mutants with B8Gly replaced by Ser, Thr or Leu were prepared by protein engineering, and both their foldability and activity were analyzed. In general, replacement of B8Gly by other amino acids causes significant detriment to the foldability of single-chain insulin： the conformations of the three B8 mutants are essentially different from that of wild-type molecules as revealed by circular dichroism; their disulfide stabilities in redox buffer are significantly decreased; their in vitro refolding efficiencies are decreased approximately two folds; the structural stabilities of the mutants with Set or Thr substitution are decreased significantly, while Leu substitution has little effect as measured by equilibrium guanidine denaturation. As far as biological activity is concerned, Ser replacement of B8Gly has only a moderate effect： its insulin receptor-binding activity is 23% of native insulin. But Ttir or Leu replacement produces significant detriment： the receptorbinding potencies of the two mutants are less than 0.2% of native insulin. The present results suggest that Gly is likely the only applicable natural amino acid for the B8 position of insulin where both foldability and activity are concerned.     

Crystal structure of (L-Arg)-BO bovine insulin at 0.21 nm resolution

The crystal structure of (L-Arg)-B0 bovine insulin has been determined, using data to 0.21 nm and atomic parameters of 2Zn porcine insulin as a starting model, by the difference Fourier method, the restrained least square method and X-PLOR package, interspersed with careful review of the electron density, to a final R-factor of 0.182 and r.m.s. deviation of 0.002 2nm for the bond lengths and 4.3° for the bond angles. The electron densities of additional (L-Arg)-B0 residues to B-chain N-terminus of two monomers in each asymmetric unit are very dear. The crystallographic micro-environment of the N-terminus of the B-chain is different from that of rhombohedral 2-zinc insulin.     

The folding nucleus of the insulin superfamily: a flexible peptide model foreshadows the native state

Oxidative folding of insulin-like growth factor I (IGF-I) and single-chain insulin analogs proceeds via one- and two-disulfide intermediates. A predominant one-disulfide intermediate in each case contains the canonical A20-B19 disulfide bridge (cystines 18-61 in IGF-I and 19-85 in human proinsulin). Here, we describe a disulfide-linked peptide model of this on-pathway intermediate. One peptide fragment (19 amino acids) spans IGF-I residues 7-25 (canonical positions B8-B26 in the insulin superfamily); the other (18 amino acids) spans IGF-I residues 53-70 (positions A12-A21 and D1-D8). Containing only half of the IGF-I sequence, the disulfide-linked polypeptide (designated IGF-p) is not well ordered. Nascent helical elements corresponding to native alpha-helices are nonetheless observed at 4 degrees C. Furthermore, (13)C-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 Cys(A20) and Cys(B19). 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.     

Role of disulfide bonds in the structure and activity of human insulin

Insulin contains two inter-chain disulfide bonds between the A and B chains (A7-B7 and A20-B19), and one intra-chain linkage in the A chain (A6-A11). To investigate the role of each disulfide bond in the structure, function and stability of the molecule, three des mutants of human insulin, each lacking one of the three disulfide bonds, were prepared by enzymatic conversion of refolded mini-proinsulins. Structural and biological studies of the three des mutants revealed that all three disulfide bonds are essential for the receptor binding activity of insulin, whereas the different disulfide bonds make different contributions to the overall structure of insulin. Deletion of the A20-B19 disulfide bond had the most substantial influence on the structure as indicated by loss of ordered secondary structure, increased susceptibility to proteolysis, and markedly reduced compactness. Deletion of the A6-A11 disulfide bond caused the least perturbation to the structure. In addition, different refolding efficiencies between the three des mutants suggest that the disulfide bonds are formed sequentially in the order A20-B19, A7-B7 and A6-A11 in the folding pathway of proinsulin.     

A protein caught in a kinetic trap: structures and stabilities of insulin disulfide isomers

Proinsulin contains six cysteines whose specific pairing (A6-A11, A7-B7, and A20-B19) is a defining feature of the insulin fold. Pairing information is contained within A and B domains as demonstrated by studies of insulin chain recombination. Two insulin isomers containing non-native disulfide bridges ([A7-A11,A6-B7,A20-B19] and [A6-A7,A11-B7,A20-B19]), previously prepared by directed chemical synthesis, are metastable and biologically active. Remarkably, the same two isomers are preferentially formed from native insulin or proinsulin following disulfide reassortment in guanidine hydrochloride. The absence of other disulfide isomers suggests that the observed species exhibit greater relative stability and/or kinetic accessibility. The structure of the first isomer ([A7-A11,A6-B7,A20-B19], insulin-swap) has been described [Hua, Q. X., Gozani, S. N., Chance, R. E., Hoffmann, J. A., Frank, B. H., and Weiss, M. A. (1995) Nat. Struct. Biol. 2, 129-138]. Here, we demonstrate that the second isomer (insulin-swap2) is less ordered than the first. Nativelike elements of structure are retained in the B chain, whereas the A chain is largely disordered. Thermodynamic studies of guanidine denaturation demonstrate the instability of the isomers relative to native insulin (DeltaDeltaG(u) > 3 kcal/mol). In contrast, insulin-like growth factor I (IGF-I) and the corresponding isomer IGF-swap, formed as alternative products of a bifurcating folding pathway, exhibit similar cooperative unfolding transitions. The insulin isomers are similar in structure and stability to two-disulfide analogues whose partial folds provide models of oxidative folding intermediates. Each exhibits a nativelike B chain and less-ordered A chain. This general asymmetry is consistent with a hierarchical disulfide pathway in which nascent structure in the B chain provides a template for folding of the A chain. Structures of metastable disulfide isomers provide probes of the topography of an energy landscape.     

Peptide models of four possible insulin folding intermediates with two disulfides

The single-chain insulin (PIP) can spontaneously fold into native structure through preferred kinetic intermediates. During refolding, pairing of the first disulfide A20-B19 is highly specific, whereas pairing of the second disulfide is likely random because two two-disulfide intermediates have been trapped. To get more details of pairing property of the second disulfide, four model peptides of possible folding intermediates with two disulfides were prepared by protein engineering, and their properties were analyzed. The four model peptides were named [A20-B19, A7-B7]PIP, [A20-B19, A6-B7]PIP, [A20-B19, A6-A11]PIP, and [A20-B19, A7-A11]PIP according to their remaining disulfides. The four model peptides all adopt partially folded structure with moderate conformational differences. In redox buffer, the disulfides of the model peptides are more easily reduced than those of the wild-type PIP. During in vitro refolding, the reduced model peptides share similar relative folding rates but different folding yields: The refolding efficiency of the reduced [A20-B19, A7-A11]PIP is about threefold lower than that of the other three peptides. The present results indicate that the folding intermediates corresponding to the present model peptides all adopt partially folded conformation, and can be formed during PIP refolding, but the chance of forming the intermediate with disulfide [A20-B19, A7-A11] is much lower than that of forming the other three intermediates.     

In vitro refolding/unfolding pathways of amphioxus insulin-like peptide: implications for folding behavior of insulin family proteins

Amphioxus insulin-like peptide (AILP) belongs to the insulin superfamily and is proposed as the common ancestor of insulin and insulin-like growth factor 1. Herein, the studies on oxidative refolding and reductive unfolding of AILP are reported. During the refolding process, four major intermediates, P1, P2, P3, and P4, were captured, which were almost identical to those intermediates, U1, U2, U3, and U4, captured during the AILP unfolding process. P4 (U4) has the native disulfide A20-B19; P1 (U1), P2 (U2), and P3 (U3) have two disulfide bonds, which include A20-B19. Based on the analysis of the time course distribution and properties of the intermediates, we proposed that fully reduced AILP refolded through 1SS, 2SS, and 3SS intermediate stages to the native form; native AILP unfolded through 2SS and 1SS intermediate stages to the full reduced form. A schematic flow chart of major oxidative refolding and reductive unfolding pathways of AILP was proposed. Implication for the folding behavior of insulin family proteins was discussed. There may be seen three common folding features in the insulin superfamily: 1) A20-B19 disulfide is most important and formed during the initial stage of folding process; 2) the second disulfide is nonspecifically formed, which then rearranged to native disulfide; 3) in vitro refolding and unfolding pathways may share some common folding intermediates but flow in opposite directions. Furthermore, although swap AILP is a thermodynamically stable final product, a refolding study of swap AILP demonstrated that it is also a productive intermediate of native AILP during refolding.     

Inactive conformation of an insulin despite its wild-type sequence.

The peptide group between residues B24 and B25 of insulin was replaced by an ester bond. This modification only in the backbone was meant to eliminate a structurally important H-bond between the amide proton of B25 and the carbonyl oxygen of A19, and consequently to enhance detachment of the C-terminal B-chain from the body of the molecule, exposing the underlying A-chain. According to a model derived from the effects of side-chain substitutions, main-chain shortening, and crosslinking, this conformational change is prerequisite for receptor binding. Contrary to the expectation that increased flexibility would increase receptor binding and activity, depsi-insulin ([B24-B25 CO-O]insulin) has turned out be only 3-4% potent. In search of an explanation for this observation, the solution structure of depsi-insulin was determined by two-dimensional 1H-NMR spectroscopy. It was found that the loss of the B25-A19 H-bond does not entail detachment of the C-terminal B-chain. On the contrary, it is overcompensated by a gain in hydrophobic interaction achieved by insertion of the Phe B25 side chain into the molecule's core. This is possible because of increased rotational freedom in the backbone owing to the ester bond. Distortion of the B20-B23 turn and an altered direction of the distal B-chain are consequences that also affect self-association. The exceptional position of the B25 side chain is thus the key feature of the depsi-insulin structure. Being buried in the interior, it is not available for guiding the interaction with the receptor, a crucial role attributed to it by the model. This seems to be the main reason why the structure of depsi-insulin represents an inactive conformation.     

A comparison of the dynamic behavior of monomeric and dimeric insulin shows structural rearrangements in the active monomer

Molecular dynamics (MD) simulations (5-10ns in length) and normal mode analyses were performed for the monomer and dimer of native porcine insulin in aqueous solution; both starting structures were obtained from an insulin hexamer. Several simulations were done to confirm that the results obtained are meaningful. The insulin dimer is very stable during the simulation and remains very close to the starting X-ray structure; the RMS fluctuations calculated from the MD simulation agree with the experimental B-factors. Correlated motions were found within each of the two monomers; they can be explained by persistent non-bonded interactions and disulfide bridges. The correlated motions between residues B24 and B26 of the two monomers are due to non-bonded interactions between the side-chains and backbone atoms. For the isolated monomer in solution, the A chain and the helix of the B chain are found to be stable during 5ns and 10ns MD simulations. However, the N-terminal and the C-terminal parts of the B chain are very flexible. The C-terminal part of the B chain moves away from the X-ray conformation after 0.5-2.5ns and exposes the N-terminal residues of the A chain that are thought to be important for the binding of insulin to its receptor. Our results thus support the hypothesis that, when monomeric insulin is released from the hexamer (or the dimer in our study), the C-terminal end of the monomer (residues B25-B30) is rearranged to allow binding to the insulin receptor. The greater flexibility of the C-terminal part of the beta chain in the B24 (Phe-->Gly) mutant is in accord with the NMR results. The details of the backbone and side-chain motions are presented. The transition between the starting conformation and the more dynamic structure of the monomers is characterized by displacements of the backbone of Phe B25 and Tyr B26; of these, Phe B25 has been implicated in insulin activation.     

A peptide model of insulin folding intermediate with one disulfide

Insulin folds into a unique three-dimensional structure stabilized by three disulfide bonds. Our previous work suggested that during in vitro refolding of a recombinant single-chain insulin (PIP) there exists a critical folding intermediate containing the single disulfide A20-B19. However, the intermediate cannot be trapped during refolding because once this disulfide is formed, the remaining folding process is very quick. To circumvent this difficulty, a model peptide ([A20-B19]PIP) containing the single disulfide A20-B19 was prepared by protein engineering. The model peptide can be secreted from transformed yeast cells, but its secretion yield decreases 2-3 magnitudes compared with that of the wild-type PIP. The physicochemical property analysis suggested that the model peptide adopts a partially folded conformation. In vitro, the fully reduced model peptide can quickly and efficiently form the disulfide A20-B19, which suggested that formation of the disulfide A20-B19 is kinetically preferred. In redox buffer, the model peptide is reduced gradually as the reduction potential is increased, while the disulfides of the wild-type PIP are reduced in a cooperative manner. By analysis of the model peptide, it is possible to deduce the properties of the critical folding intermediate with the single disulfide A20-B19.     

<Emphasis Type="Italic">In vitro</Emphasis> insulin refolding: Characterization of the intermediates and the putative folding pathway

The in vitro refolding process of the double-chain insulin was studied based on the investigation of in vitro single-chain insulin refolding. Six major folding intermediates, named P1A, P2B, P3A, P4B, P5B, and P6B, were captured during the folding process. The refolding experiments indicate that all of these intermediates are on-pathway. Based on these intermediates and the formation of hypothetic transients, we propose a two-stage folding pathway of insulin. (1) At the early stage of the folding process, the reduced A chain and B chain individually formed the intermediates: two A chain intermediates (P1A and P3A), and four B chain intermediates (P2B, P4B, P5B, and P6B). (2) In the subsequent folding process, transient I was formed from P3A through thiol/disulfide exchange reaction; then, transients II and III, each containing two native disulfides, were formed through the recognition and interaction of transient I with P4B or P6B and the thiol group’s oxidation reaction mainly using GSSG as oxidative reagent; finally, transients II and III, through thiol/mixture disulfide exchange reaction, formed the third native disulfide of insulin to complete the folding.