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1.
Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα‐Cα distances, solvent exposure, and side‐chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C‐terminus of the B‐chain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild‐type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R‐state conformation and thus showing that the R‐state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function. 相似文献
2.
The nuclear magnetic resonance structure of the globular domain with residues 121-230 of a variant human prion protein with two disulfide bonds, hPrP(M166C/E221C), shows the same global fold as wild-type hPrP(121-230). It contains three alpha-helices of residues 144-154, 173-194 and 200-228, an anti-parallel beta-sheet of residues 128-131 and 161-164, and the disulfides Cys166-Cys221 and Cys179-Cys214. The engineered extra disulfide bond in the presumed "protein X"-binding site is accommodated with slight, strictly localized conformational changes. High compatibility of hPrP with insertion of a second disulfide bridge in the protein X epitope was further substantiated by model calculations with additional variant structures. The ease with which the hPrP structure can accommodate a variety of locations for a second disulfide bond within the presumed protein X-binding epitope suggests a functional role for the extensive perturbation by a natural second disulfide bond of the corresponding region in the human doppel protein. 相似文献
3.
The structure of insulin, a glucose homeostasis-controlling hormone, is highly conserved in all vertebrates and stabilized by three disulfide bonds. Recently, we designed a novel insulin analogue containing a fourth disulfide bond located between positions A10-B4. The N-terminus of insulin''s B-chain is flexible and can adapt multiple conformations. We examined how well disulfide bond predictions algorithms could identify disulfide bonds in this region of insulin. In order to identify stable insulin analogues with additional disulfide bonds, which could be expressed, the C β cut-off distance had to be increased in many instances and single X-ray structures as well as structures from MD simulations had to be used. The analogues that were identified by the algorithm without extensive adjustments of the prediction parameters were more thermally stable as assessed by DSC and CD and expressed in higher yields in comparison to analogues with additional disulfide bonds that were more difficult to predict. In contrast, addition of the fourth disulfide bond rendered all analogues resistant to fibrillation under stress conditions and all stable analogues bound to the insulin receptor with picomolar affinities. Thus activity and fibrillation propensity did not correlate with the results from the prediction algorithm. 相似文献
4.
Summary Protein engineering techniques were employed to graft the known anticoagulant Arg-Gly-Asp (RGD) motifcontaining sequences
onto the surface of a mutant, inactive insulin framework. To probe the effect of a disulfide bond on the resultant anticoagulant
activity, a native RGD-containing sequence from disintegrin dendroaspin, CFTPRGDMPGPYC, as well as a modified sequence, SFTPRGDMPGPYS
were each examined. The peptide was placed between the C-terminal of the B chain and the N-terminal of the A chain and connected
with B27 and A1 residues of the inactive insulin that lacks the characteristic intramolecular A6–11 disulfide bond within
the A chain. The two RGD-containing insulin genes were over-expressed in E. coli, and purified and designated as RGD-Cys-Ins and RGD-Ser-Ins, respectively. Their amino acid compositions and mass data were
in good agreement with those of expected. The RGD-Cys-Ins showed inhibition of platelet aggregation with an IC 50 of 3 μM, while the latter was 3.5-fold less active. The in vivo assay also indicated that the RGD-Cys-Ins had a higher activity in prolonging the bleeding time in mice than RGD-Ser-Ins.
Both RGD-Cys-Ins and RGD-Ser-Ins retained about 25% of the proinsulin immunoactivity and had almost no insulin receptor binding
activity. The results indicate the necessity for the RGD motif to be conformationally constrained for it to elicit a greater
anticoagulant activity. 相似文献
5.
Protein engineering techniques were employed to graft the known anticoagulant Arg-Gly-Asp (RGD) motif-containing sequences onto the surface of a mutant, inactive insulin framework. To probe the effect of a disulfide bond on the resultant anticoagulant activity, a native RGD-containing sequence from disintegrin dendroaspin, CFTPRGDMPGPYC, as well as a modified sequence, SFTPRGDMPGPYS, were each examined. The peptide was placed between the C-terminal of the B chain and the N-terminal of the A chainand connected with B27 and A1 residues of the inactive insulin that lacks the characteristic intramolecular A6-11 disulfide bondwithin the A chain. The two RGD-containing insulin genes were over-expressed in E. coli, and purified and designatedas RGD-Cys-Ins and RGD-Ser-Ins, respectively. Their amino acid compositions and mass data were in good agreement with those ofexpected. The RGD-Cys-Ins showed inhibition of platelet aggregation with an IC 50 of 3 M, while the latter was3.5-fold less active. The in vivo assay also indicatedthat the RGD-Cys-Ins had a higher activity in prolonging the bleeding time in mice than RGD-Ser-Ins. Both RGD-Cys-Ins and RGD-Ser-Ins retained about 25% of the proinsulin immunoactivity and had almost no insulin receptor binding activity. The results indicate the necessity for the RGD motif to be conformationally constrained for it to elicit a greater anticoagulant activity. 相似文献
6.
Cholera toxin (CT) produced by Vibrio cholerae and heat-labile enterotoxin (LT-I), produced by enterotoxigenic Escherichia coli, are AB5 heterohexamers with an ADP-ribosylating A subunit and a GM1 receptor binding B pentamer. These toxins are among the most potent mucosal adjuvants known and, hence, are of interest both for the development of anti-diarrheal vaccines against cholera or enterotoxigenic Escherichia coli diarrhea and also for vaccines in general. However, the A subunits of CT and LT-I are known to be relatively temperature sensitive. To improve the thermostability of LT-I an additional disulfide bond was introduced in the A1 subunit by means of the double mutation N40C and G166C. The crystal structure of this double mutant of LT-I has been determined to 2.0 A resolution. The protein structure of the N40C/G166C double mutant is very similar to the native structure except for a few local shifts near the new disulfide bond. The introduction of this additional disulfide bond increases the thermal stability of the A subunit of LT-I by 6 degrees C. The enhancement in thermostability could make this disulfide bond variant of LT-I of considerable interest for the design of enterotoxin-based vaccines. 相似文献
7.
The disulfide bond pattern of catrocollastatin-C was determined by N-terminal sequencing and mass spectrometry. The N-terminal disintegrin-like domain is a compact structure including eight disulfide bonds, seven of them in the same pattern as the disintegrin bitistatin. The protein has two extra cysteine residues (XIII and XVI) that form an additional disulfide bond that is characteristically found in the disintegrin-like domains of cellular metalloproteinases (ADAMs) and PIII snake venom Zn-metalloproteinases (SVMPs). The C-terminal cysteine-rich domain of catrocollastatin-C contains five disulfide bonds between nearest-neighbor cysteines and a long range disulfide bridge between CysV and CysX. These results provide structural evidence for a redefinition of the disintegrin-like and cysteine-rich domain boundaries. An evolutionary pathway for ADAMs, PIII, and PII SVMPs based on disulfide bond engineering is also proposed. 相似文献
9.
The potential for engineering stable proteins with multiple amino acid substitutions was explored. Eleven lysine, five methionine, two tryptophan, one glycine, and three threonine substitutions were simultaneously made in barley chymotrypsin inhibitor-2 (CI-2) to substantially improve the essential amino acid content of the protein. These substitutions were chosen based on the three-dimensional structure of CI-2 and an alignment of homologous sequences. The initial engineered protein folded into a wild-type-like structure, but had a free energy of unfolding of only 2.2 kcal/mol, considerably less than the wild-type value of 7.5 kcal/mol. Restoration of the lysine mutation at position 67 to the wild-type arginine increased the free energy of unfolding to 3.1 kcal/mol. Subsequent cysteine substitutions at positions 22 and 82 resulted in disulfide bond formation and a protein with nearly wild-type thermodynamic stability (7.0 kcal/mol). None of the engineered proteins retained inhibitory activity against chymotrypsin or elastase, and all had substantially reduced inhibitory activity against subtilisin. The proteolytic stabilities of the proteins correlated with their thermodynamic stabilities. Reduction of the disulfide bond resulted in substantial loss of both thermodynamic and proteolytic stabilities, confirming that the disulfide bond, and not merely the cysteine substitutions, was responsible for the increased stability. We conclude that it is possible to replace over a third of the residues in CI-2 with minimal disruption of stability and structural integrity. 相似文献
10.
We report the synthesis of bombyxin-IV, a disulfide-linked, heterodimeric, insulin superfamily peptide from the silkworm, Bombyx mori. The two chains (A- and B-chains) were synthesized separately by the solid-phase method using fluoren-9-ylmethoxycarbonyl (Fmoc) group as a protecting group for -amino group. Three disulfide bonds were bridged step by step (A6–A11, A20–B22, and A7–B10) in a good yield. Synthetic bombyxin-IV was identical with natural one with regard to the retention time on a reversed-phase column and the molecular weight measured by mass spectrometry. Circular dichroism (CD) spectrum of the synthetic bombyxin-IV was very similar to that of the natural one. The specific activity of synthetic bombyxin-IV is equal to that of natural one (0.1 ng/ Samia unit). These results suggest that the synthetic bombyxin-IV has the tertiary structure identical with the natural peptide. Our method developed for synthesis of bombyxin-IV would be generally applicable to the synthesis of insulin-like heterodimeric peptides. 相似文献
11.
The mode of disulfide linkages in bombyxin-IV, an insulin superfamily peptide consisting of A- and B-chains, was determined as A6–A11, A7–B10, and A20–B22. An intermolecular bond of A20–B22 was identified by sequencing and mass spectrometric analysis of the fragments generated by thermolysin digestion of natural bombyxin-IV. The mode of the remaining two bridges was determined by chemical and selective synthesis of three possible disulfide bond isomers of bombyxin-IV. A- and B-chains were synthesized by solid-phase method, and three disulfide bonds were bridged stepwise and in a fully controlled manner. Retention time on reversed-phase high-performance liquid chromatography (HPLC), thermolysin digests, and biological activity of the synthetic [A6–A11, A7–B10, A20–B22-cystine]-bombyxin-IV revealed that it was identical with the natural bombyxin-IV. Two other isomers with respect to disulfide bond arrangement, [A6–A7, A11–B10, A20–B22-cystine]- and [A6–B10, A7–A11, A20–B22-cystine]-bombyxin-IVs, were distinguishable from the natural one by use of HPLC, thermolysin digestion, and bioassay. 相似文献
12.
Aspergillus oryzae protyrosinase (pro-TY) has a unique feature that the proenzyme is activated under conditions of acidic pH. The pro-TY was inactive at pH 7.0. The latent enzyme was activated at pH 3.0, and was slightly activated by sodium dodecyl sulfate (SDS). The molecular masses of the pro-TY and acid-activated tyrosinase (acid-TY) were 266 and 165 kDa, respectively, as estimated by gel-filtration chromatography. The CD spectra showed that the tertiary and/or quaternary structure was changed after the acid activation. On the basis of these results, we deduce that the intersubunit polar interaction is disrupted at pH 3.0, and that the tetrameric pro-TY dissociates to dimers. Tryptophan fluorescence spectra and binding assay of 8-anilino-1-naphthalene sulfonic acid (ANS) suggested that hydrophobic amino acid residues of the active site were exposed to solvent after acid treatment. It was likely that Cys108 formed an intermolecular disulfide bond between the subunits of dimeric acid-TY. The dimerization of acid-TY involving the intermolecular disulfide bond is essential for the activity. 相似文献
13.
Recombinant human monoclonal antibodies have become important protein-based therapeutics for the treatment of various diseases. The antibody structure is complex, consisting of beta-sheet rich domains stabilized by multiple disulfide bridges. The dimerization of the C(H)3 domain in the constant region of the heavy chain plays a pivotal role in the assembly of an antibody. This domain contains a single buried, highly conserved disulfide bond. This disulfide bond was not required for dimerization, since a recombinant human C(H)3 domain, even in the reduced state, existed as a dimer. Spectroscopic analyses showed that the secondary and tertiary structures of reduced and oxidized C(H)3 dimer were similar, but differences were observed. The reduced C(H)3 dimer was less stable than the oxidized form to denaturation by guanidinium chloride (GdmCl), pH, or heat. Equilibrium sedimentation revealed that the reduced dimer dissociated at lower GdmCl concentration than the oxidized form. This implies that the disulfide bond shifts the monomer-dimer equilibrium. Interestingly, the dimer-monomer dissociation transition occurred at lower GdmCl concentration than the unfolding transition. Thus, disulfide bond formation in the human C(H)3 domain is important for stability and dimerization. Here we show the importance of the role played by the disulfide bond and how it affects the stability and monomer-dimer equilibrium of the human C(H)3 domain. Hence, these results may have implications for the stability of the intact antibody. 相似文献
14.
A distinguishing feature of camel ( Camelus dromedarius) VHH domains are noncanonical disulfide bonds between CDR1 and CDR3. The disulfide bond may provide an evolutionary advantage, as one of the cysteines in the bond is germline encoded. It has been hypothesized that this additional disulfide bond may play a role in binding affinity by reducing the entropic penalty associated with immobilization of a long CDR3 loop upon antigen binding. To examine the role of a noncanonical disulfide bond on antigen binding and the biophysical properties of a VHH domain, we have used the VHH R303, which binds the Listeria virulence factor InlB as a model. Using site directed mutagenesis, we produced a double mutant of R303 (C33A/C102A) to remove the extra disulfide bond of the VHH R303. Antigen binding was not affected by loss of the disulfide bond, however the mutant VHH displayed reduced thermal stability ( Tm = 12°C lower than wild‐type), and a loss of the ability to fold reversibly due to heat induced aggregation. X‐ray structures of the mutant alone and in complex with InlB showed no major changes in the structure. B‐factor analysis of the structures suggested that the loss of the disulfide bond elicited no major change on the flexibility of the CDR loops, and revealed no evidence of loop immobilization upon antigen binding. These results suggest that the noncanonical disulfide bond found in camel VHH may have evolved to stabilize the biophysical properties of the domain, rather than playing a significant role in antigen binding. 相似文献
15.
The Cys 2-Cys 10 disulfide bond in ribonuclease T1 was broken by substituting Cys 2 and Cys 10 by Ser and Asn, respectively, as present in ribonuclease F1. This C2S/C10N variant resembles the wild-type protein in structure and in catalytic activity. Minor structural changes were observed by 2-dimensional NMR in the local environment of the substituted amino acids only. The thermodynamic stability of ribonuclease T1 is strongly reduced by breaking the Cys 2-Cys 10 bond, and the free energy of denaturation is decreased by about 10 kJ/mol. The folding mechanism is not affected, and the trans to cis isomerizations of Pro 39 and Pro 55 are still the rate-limiting steps of the folding process. The differences in the time courses of unfolding and refolding are correlated with the decrease in stability: the folding kinetics of the wild-type protein and the C2S/C10N variant become indistinguishable when they are compared under conditions of identical stability. Apparently, the Cys 2-Cys 10 disulfide bond is important for the stability but not for the folding mechanism of ribonuclease T1. The breaking of this bond has the same effect on stability and folding kinetics as adding 1 M guanidinium chloride to the wild-type protein. 相似文献
16.
Recombinant human interleukin-1 receptor antagonist (IL-1ra) in aqueous solutions unfolds and aggregates when subjected to hydrostatic pressures greater than about 180 MPa. This study examined the mechanism and thermodynamics of pressure-induced unfolding and aggregation of IL-1ra. The activation free energy for growth of aggregates (DeltaG-/+(aggregation)) was found to be 37 +/- 3 kJ/mol, whereas the activation volume (DeltaV-/+(aggregation)) was -120 +/- 20 mL/mol. These values compare closely with equilibrium values for denaturation: The free energy for denaturation, DeltaG(denaturation), was 20 +/- 5 kJ/mol, whereas the partial specific volume change for denaturation, DeltaV(denaturation), was -110 +/- 30 mL/mol. When IL-1ra begins to denature at pressures near 140 MPa, cysteines that are normally buried in the native state become exposed. Under oxidizing conditions, this results in the formation of covalently cross-linked aggregates containing nonnative, intermolecular disulfide bonds. The apparent activation free energy for nucleation of aggregates, DeltaG-/+(nuc), was 42 +/- 4 kJ/mol, and the activation volume for nucleation, DeltaV-/+(nuc),was -175 +/- 37 mL/mol, suggesting that a highly solvent-exposed conformation is needed for nucleation. We hypothesize that the large specific volume of IL-1ra, 0.752 +/- 0.004 mL/g, coupled with its relatively low conformational stability, leads to its susceptibility to denaturation at relatively low pressures. The positive partial specific adiabatic compressibility of IL-1ra, 4.5 +/- 0.7 +/- 10(-12) cm2/dyn, suggests that a significant component of the DeltaV(denaturation) is attributable to the elimination of solvent-free cavities. Lastly, we propose that hydrostatic pressure is a useful variable to conduct accelerated formulation studies of therapeutic proteins. 相似文献
17.
There is an increasing interest in the generation of Fc-fusion molecules to exploit the effector functions of Fc and the fusion partner, towards improving the therapeutic potential. The Fc-fusion molecules have unique structural and functional attributes that impart various advantages. However, the manufacturing of Fc-fusion molecules possesses certain challenges in the biopharmaceutical development. The fusion of unnaturally occurring two or more domains in a construct can pose problems for proper folding and are prone to aggregation and degradation. Reshuffling of disulfide bridges represents a posttranslational event that affects folding. This can play a critical role in the correct structure of a molecule and leads to structural heterogeneity in biotherapeutics; it may also impact the in vivo biological activities, safety, and efficacy of the biopharmaceutical. Our work presents an investigation case of a doublet band, as observed only in nonreducing sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) for a bi-specific, N- and C-terminal Fc-fusion molecule. Other characterization and orthogonal methods from the analytical panel did not indicate the presence of two distinct species, including the orthogonal CE-SDS (Caliper Lab Chip GXII). Therefore, it was necessary to determine if the phenomenon was an analytical artifact or a real variant of our Fc-fusion molecule. With the comprehensive mass spectrometry-based characterization, we were able to determine that the doublet band was related to the reshuffling of one disulfide bridge in one of the fused domains. Our work illustrates the application of nonreducing peptide mapping by mass spectrometry to characterize and identify disulfide variants in a complex N- and C-terminal Fc-fusion molecule, and further adoption to monitor the disulfide structural variants in the intermediate process samples to drive the manufacturing of a consistent product with the desired quality attributes. 相似文献
18.
ScFv‐h3D6 is a single chain variable fragment that precludes Aβ peptide‐induced cytotoxicity by withdrawing Aβ oligomers from the amyloid pathway to the worm‐like pathway. Production of scFv molecules is not a straightforward procedure because of the occurrence of disulfide scrambled conformations generated in the refolding process. Here, we separately removed the disulfide bond of each domain and solved the scrambling problem; and then, we intended to compensate the loss of thermodynamic stability by adding three C‐terminal elongation mutations, previously described to stabilize the native fold of scFv‐h3D6. Such stabilization occurred through stabilization of the intermediate state in the folding pathway and destabilization of a different, β‐rich, intermediate state driving to worm‐like fibrils. Elimination of the disulfide bridge of the less stable domain, VL, deeply compromised the yield and increased the aggregation tendency, but elimination of the disulfide bridge of the more stable domain, VH, solved the scrambling problem and doubled the production yield. Notably, it also changed the aggregation pathway from the protective worm‐like morphology to an amyloid one. This was so because a partially unfolded intermediate driving to amyloid aggregation was present, instead of the β‐rich intermediate driving to worm‐like fibrils. When combining with the elongation mutants, stabilization of the partially unfolded intermediate driving to amyloid fibrils was the only effect observed. Therefore, the same mutations drove to completely different scenarios depending on the presence of disulfide bridges and this illustrates the relevance of such linkages in the stability of different intermediate states for folding and misfolding. 相似文献
19.
Urea-induced denaturations of RNase T1 and reduced and carboxyamidated RNase T1 (RTCAM) as a function of temperature were analyzed using the linear extrapolation method, and denaturation m values, deltaCp, deltaH, deltaS, and deltaG quantities were determined. Because both deltaCp and m values are believed to reflect the protein surface area newly exposed on denaturation, the prediction is that the ratio of m values for RNase T1 and RTCAM should equal the deltaCp ratio for the two proteins. This is not the case, for it is found that the m value of RTCAM is 1.5 times that of RNase T1, while the denaturation deltaCp's for the two proteins are identical. The paradox of why the two parameters, m and deltaCp, are not equivalent in their behavior is of importance in the interpretations of their respective molecular-level meanings. It is found that the measured denaturation deltaCp's are consistent with deltaCp's calculated on the basis of empirical relationships between the change in surface area on denaturation (deltaASA), and that the measured m value of RNase T1 agrees with m calculated from empirical data relating m to deltaASA. However, the measured m of RTCAM is so much out of line with its calculated m as to call into question the validity of always equating m with surface area newly exposed on denaturation. 相似文献
20.
Introduction: In several biomedical research fields, the cross-linking of peptides and proteins has an important impact on health and wellbeing. It is therefore of crucial importance to study this class of post-translational modifications in detail. The huge potential of mass spectrometric technologies in the mapping of these protein-protein cross-links is however overshadowed by the challenges that the field has to overcome. Areas covered: In this review, we summarize the different pitfalls and challenges that the protein-protein cross-linking field is confronted with when using mass spectrometry approaches. We additionally focus on native disulfide bridges as an example and provide some examples of cross-links that are important in the biomedical field. Expert commentary: The current flow of methodological improvements, mainly from the chemical cross-linking field, has delivered a significant contribution to deciphering native and insult-induced cross-links. Although an automated data analysis of proteome-wide peptide cross-linking is currently only possible in chemical cross-linking experiments, the field is well on the way towards a more automated analysis of native and insult-induced cross-links in raw mass spectrometry data that will boost its potential in biomedical applications. 相似文献
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