The enhancing effect of homocysteine thiolactone on insulin fibrillation and cytotoxicity of insulin fibril

In the current study both structural alteration and fibrillation of insulin were studied in the presence of homocysteine thiolactone (HCTL). The spectroscopic studies revealed that HCTL increases rate of insulin unfolding, giving rise to the appearance of solvent-exposed hydrophobic regions and induces a transition from α-helix into predominantly β-sheet structures. Thioflavin-T fluorescence studies revealed that HCTL markedly enhanced the quantity of insulin fibril formation in both agitating and non-agitating systems. Also gel electrophoresis results suggest that HCTL accelerates the process of formation of high molecular weight insulin aggregates. Moreover, insulin fibrils obtained in the presence of HCTL and those collected earlier in the pathway of insulin fibrillation displayed improved cytotoxicity against cancer cells. The enhancement of insulin fibril formation with elevated cytotoxic properties as occurred in the presence of HCTL, may suggest this homocysteine derivative as a possible contributing factor in the pathology of insulin fibrils.     

Human Valacyclovir Hydrolase/Biphenyl Hydrolase-Like Protein Is a Highly Efficient Homocysteine Thiolactonase

Homocysteinylation of lysine residues by homocysteine thiolactone (HCTL), a reactive homocysteine metabolite, results in protein aggregation and malfunction, and is a well-known risk factor for cardiovascular, autoimmune and neurological diseases. Human plasma paraoxonase-1 (PON1) and bleomycin hydrolase (Blmh) have been reported as the physiological HCTL detoxifying enzymes. However, the catalytic efficiency of HCTL hydrolysis by Blmh is low and not saturated at 20 mM HCTL. The catalytic efficiency of PON1 for HCTL hydrolysis is 100-fold lower than that of Blmh. A homocysteine thiolactonase (HCTLase) was purified from human liver and identified by mass spectrometry (MS) as the previously described human biphenyl hydrolase-like protein (BPHL). To further characterize this newly described HCTLase activity, BPHL was expressed in Escherichia coli and purified. The sequence of the recombinant BPHL (rBPHL) and hydrolytic products of the substrates HCTL and valacyclovir were verified by MS. We found that the catalytic efficiency (k_cat/K_m) of rBPHL for HCTL hydrolysis was 7.7 × 10⁴ M⁻¹s⁻¹, orders of magnitude higher than that of PON1 or Blmh, indicating a more significant physiological role for BPHL in detoxifying HCTL.     

Structural modifications of oxidized insulin B-chain induced by glycerol addition

Summary ¹H-NMR studies of the bovine insulin S-sulfonated B-chain are reported in H₂O/D₂O (9/1) and in glycerol-d ₅ (5 M) using two-dimensional NMR spectroscopy. The first results show that the oxidized insulin B-chain secondary structure differs from that of native insulin by a loss of the α-helix between the two disulfide bridges and that the glycerol favours the structuring of the peptide.     

Importance of the character and configuration of residues B24, B25, and B26 in insulin-receptor interactions

By use of isolated canine hepatocytes and insulin analogs prepared by trypsin-catalyzed semisynthesis, we have investigated the importance of the aromatic triplet PheB24-PheB25-TyrB26 of the COOH-terminal B-chain domain of insulin in directing the affinity of insulin-receptor interactions. Analysis of the receptor binding potencies of analogs bearing transpositions or replacements (by Tyr, D-Tyr or their corresponding 3,5-diiodo derivatives) in this region demonstrates a wide divergence in the acceptance both of configurational change (with [D-TyrB24,PheB26]insulin and [D-TyrB25,PheB26]insulin exhibiting 160 and 0.1% of the receptor binding potency of insulin, respectively) and of detailed side chain structure (with [TyrB24,PheB26]insulin and [TyrB25,PheB26]insulin exhibiting 2 and 80% of the receptor binding potency of insulin, respectively). Additional experiments addressed the solvent accessibilities of the 4 tyrosine residues of insulin and the insulin analogs at selected peptide concentrations by use of analytical radioiodination. Whereas two analogs ([TyrB25,PheB26]insulin and [D-TyrB24,PheB26]insulin) were found to undergo self aggregation, no strict correlation was found between the ability of an analog to aggregate and its potency for interaction with the insulin receptor. Related findings are discussed in terms of the interplay between side chain and main chain structure in the COOH-terminal domain of the insulin B-chain and the structural attributes of insulin that determine the affinity of insulin-receptor interactions.     

A new B-chain mutant of insulin: comparison with the insulin crystal structure and role of sulfonate groups in the B-chain structure.

The solution structure of a new B-chain mutant of bovine insulin, in which the cysteines B7 and B19 are replaced by two serines, has been determined by circular dichroism, 2D-NMR and molecular modeling. This structure is compared with that of the oxidized B-chain of bovine insulin [Hawkins et al. (1995) Int. J. Peptide Protein Res.46, 424-433]. Circular dichroism spectroscopy showed in particular that a higher percentage of helical secondary structure for the B-chain mutant is estimated in trifluoroethanol solution in comparison with the oxidized B-chain. 2D-NMR experiments confirmed, among multiple conformations, that the B-chain mutant presents defined secondary structures such as a alpha-helix between residues B9 and B19, and a beta-turn between amino acids B20 and B23 in aqueous trifluoroethanol. The 3D structures, which are consistent with NMR data and were obtained using a simulated annealing protocol, showed that the tertiary structure of the B-chain mutant is better resolved and is more in agreement with the insulin crystal structure than the oxidized B-chain structure described by Hawkins et al. An explanation could be the presence of two sulfonate groups in the oxidized insulin B-chain. Either by their charges and/or their size, such chemical groups could play a destructuring effect and thus could favor peptide flexibility and conformational averaging. Thus, this study provides new insights on the folding of isolated B-chains.     

Comparative 2D NMR studies of human insulin and des-pentapeptide insulin: sequential resonance assignment and implications for protein dynamics and receptor recognition

The solution structure and dynamics of human insulin are investigated by 2D 1H NMR spectroscopy in reference to a previously analyzed analogue, des-pentapeptide(B26-B30) insulin (DPI; Hua, Q.X., & Weiss, M.A. (1990) Biochemistry 29, 10545-10555). This spectroscopic comparison is of interest since (i) the structure of the C-terminal region of the B-chain has not been determined in the monomeric state and (ii) the role of this region in binding to the insulin receptor has been the subject of long-standing speculation. The present NMR studies are conducted in the presence of an organic cosolvent (20% acetic acid), under which conditions both proteins are monomeric and stably folded. Complete sequential assignment of human insulin is obtained and leads to the following conclusions. (1) The secondary structure of the insulin monomer (three alpha-helices and B-chain beta-turn) is similar to that observed in the 2-Zn crystal state. (2) The folding of DPI is essentially the same as the corresponding portion of intact insulin, in accord with the similarities between their respective crystal structures. However, differences between insulin and DPI are observed in the extent of conformational broadening of amide resonances, indicating that the presence or absence of residues B26-B30 influences the overall dynamics of the protein on the millisecond time scale. (3) Residues B24-B28 adopt an extended configuration in the monomer and pack against the hydrophobic core as in crystallographic dimers; residues B29 and B30 are largely disordered. This configuration differs from that described in a more organic milieu (35% acetonitrile; Kline, A.D., & Justice, R.M., Jr. (1990) Biochemistry 29, 2906-2913), suggesting that the conformation of insulin in the latter study may have been influenced by solvent composition. (4) The insulin fold is shown to provide a model for collective motions in a protein with implications for the mechanism of protein-protein recognition. To our knowledge, this paper describes the first detailed analysis of a protein NMR spectrum under conditions of extensive conformational broadening. Such an analysis is made possible in the present case by comparative study of an analogue (DPI) with more tractable spectroscopic properties.     

A thermodynamic coupling mechanism for the disaggregation of a model peptide substrate by chaperone secB

Molecular chaperones prevent protein aggregation in vivo and in vitro. In a few cases, multichaperone systems are capable of dissociating aggregated state(s) of substrate proteins, although little is known of the mechanism of the process. SecB is a cytosolic chaperone, which forms part of the precursor protein translocation machinery in Escherichia coli. We have investigated the interaction of the B-chain of insulin with chaperone SecB by light scattering, pyrene excimer fluorescence, and electron spin resonance spectroscopy. We show that SecB prevents aggregation of the B-chain of insulin. We show that SecB is capable of dissociating soluble B-chain aggregates as monitored by pyrene fluorescence spectroscopy. The kinetics of dissociation of the B-chain aggregate by SecB has been investigated to understand the mechanism of dissociation. The data suggests that SecB does not act as a catalyst in dissociation of the aggregate to individual B-chains, rather it binds the small population of free B-chains with high affinity, thereby shifting the equilibrium from the ensemble of the aggregate toward the individual B-chains. Thus SecB can rescue aggregated, partially folded/misfolded states of target proteins by a thermodynamic coupling mechanism when the free energy of binding to SecB is greater than the stability of the aggregate. Pyrene excimer fluorescence and ESR methods have been used to gain insights on the bound state conformation of the B-chain to chaperone SecB. The data suggests that the B-chain is bound to SecB in a flexible extended state in a hydrophobic cleft on SecB and that the binding site accommodates approximately 10 residues of substrate.     

胰岛素六聚体在水溶液中的构象柔性研究

用分子动力学（MD)模拟方法设计了两个模拟时间为600ps的对比计算机模拟实验,研究了R6态的胰岛素六聚体在水溶液中的构象柔性。通过对MD模拟所得到的轨迹的分析发现,包含锌离子和苯酚的胰岛素六聚体体系的构象柔性弱于不含锌离子和苯酚的胰岛素六聚体体系,对于不包含锌离子和苯酚的体系,胰岛素六聚体的构象柔性表现得较为突出,特别是在实验研究认为与胰岛素和受体结合位点有关的每个单体的B链羧端的β折叠部分,发生了快速而显著的构象变化,表现出了很大的构象柔性。这些模拟结果与实验观测结果相吻合。     

Influence of lβ-, dα- and dβ-Asp isomers of the Asp-76 residue on the properties of αA-crystallin 70–88 peptide

Proteins have been considered to consist exclusively of l-amino acids in living tissues. However, our previous studies showed that two specific aspartyl (Asp) residues in αA- and αB-crystallins from human eye lenses invert to the d-isomers to a high degree during aging. The reaction is also accompanied by isomerization into a form containing β-Asp (isoaspartate) residues. The appearance of d- and β-Asp in a protein potentially induces large changes to the higher order structure of the protein as well as to its function. However, it remains unclear whether the formation of the Asp isomer is the direct trigger of the change to the higher order structure and function. In this study, in order to clarify the effect of the inversion to d-isomers in a protein, we synthesized peptides corresponding to the 70–88 (KFVIFLDVKHFSPEDLTVK) fragment of human αA-crystallin and its corresponding diastereoisomers in which lα-Asp was replaced with lβ-Asp, dα-Asp, and dβ-Asp at position 76 and compared their biochemical properties with that of normal peptide. The peptides containing abnormal isomers (lβ-Asp, dα-Asp, and dβ-Asp residues, respectively) were more hydrophilic than the normal peptide (containing lα-Asp), lost β-sheet structure and changed to random structures. The normal peptide promoted the aggregation of insulin while the other three isomers suppressed the aggregation of insulin. This is the first evidence that a single substitution of an Asp isomer in a peptide induces a large change to the properties of the peptide.     

Two-dimensional NMR and photo-CIDNP studies of the insulin monomer: assignment of aromatic resonances with application to protein folding, structure, and dynamics

The aromatic 1H NMR resonances of the insulin monomer are assigned at 500 MHz by comparative studies of chemically modified and genetically altered variants, including a mutant insulin (PheB25----Leu) associated with diabetes mellitus. The two histidines, three phenylalanines, and four tyrosines are observed to be in distinct local environments; their assignment provides sensitive markers for studies of tertiary structure, protein dynamics, and protein folding. The environments of the tyrosine residues have also been investigated by photochemically induced dynamic nuclear polarization (photo-CIDNP) and analyzed in relation to packing constraints in the crystal structures of insulin. Dimerization involving specific B-chain interactions is observed with increasing protein concentration and is shown to depend on temperature, pH, and solvent composition. In the monomer large variations are observed in the line widths of amide resonances, suggesting intermediate exchange among conformational substates; such substates may relate to conformational changes observed in different crystal states and proposed to occur in the hormone-receptor complex. Additional evidence for multiple conformations in solution is provided by comparative studies of an insulin analogue containing a peptide bond between residues B29 and A1 (mini-proinsulin). This analogue forms dimers and higher-order oligomers under conditions in which native insulin is monomeric, suggesting that the B29-A1 peptide bond stabilizes a conformational substate favorable for dimerization. Such stabilization is not observed in corresponding studies of native proinsulin, in which a 35-residue connecting peptide joins residues B30 and A1; this extended tether is presumably too flexible to constrain the conformation of the B-chain. The differences between proinsulin and mini-proinsulin suggest a structural mechanism for the observation that the fully reduced B29-A1 analogue folds more efficiently than proinsulin to form the correct pattern of disulfide bonds. These results are discussed in relation to molecular mechanics calculations of insulin based on the available crystal structures.     

Aspirin-mediated acetylation induces structural alteration and aggregation of bovine pancreatic insulin

The simple aggregation of insulin under various chemical and physical stresses is still an important challenge for both pharmaceutical production and clinical formulation. In the storage form, this protein is subjected to various chemical modifications which alter its physicochemical and aggregation properties. Aspirin (acetylsalicylic acid) which is the most widely used medicine worldwide has been indicated to acetylate a large number of proteins both in vitro and in vivo. In this study, as insulin treated with aspirin at 37°C, a significant level of acetylation was observed by flourescamine and o-phthalaldehyde assay. Also, different spectroscopic techniques, gel electrophoresis, and microscopic assessment were applied to compare the structural variation and aggregation/fibrillation propensity among acetylated and non-acetylated insulin samples. The results of spectroscopic assessments elucidate that acetylation induces insulin unfolding which is accompanied with the exposure of protein hydrophobic patches, a transition from alpha-helix to beta-sheet and increased propensity of the protein for aggregation. The kinetic studies propose that acetylation increases aggregation rate of insulin under both thermal and chemical stresses. Also, gel electrophoresis and dynamic light scattering experiments suggest that acetylation induces insulin oligomerization. Additionally, the results of Thioflavin T fluorescence study, Congo red absorption assessment, and microscopic analysis suggest that acetylation with aspirin enhances the process of insulin fibrillation. Overall, the increased susceptibility of acetylated insulin for aggregation may reflect the fact that this type of modification has significant structural destabilizing effect which finally makes the protein more vulnerable for pathogenic aggregation/fibrillation.     

Comparison of the Chaperoning Action of Glycerol and β-Casein on Aggregation of Proteins in the Presence of Crowding Agent

β-Casein is one of the major components of the milk micelles of most mammals and has been shown to exhibit in vitro chaperone-like activity. Glycerol is a chemical chaperone belonging to the polyol family, which increases protein stability and inhibits protein aggregation. These prompted us to compare the chaperone-like activity of β-casein and glycerol. In this study, the effect of β-casein and glycerol on folding of the target proteins (ovotransferrin, insulin and α-lactalbumin) in the presence of dextran, as a macromolecular crowding agent, is examined using visible absorption spectroscopy, intrinsic fluorescence spectroscopy, 1-anilino-8-naphthalene sulfonic acid fluorescence binding and near CD spectroscopy. In the presence of dextran, the rate and extent of aggregation of target proteins was enhanced and β-casein was less effective in preventing the aggregation and precipitation of target proteins. These data support the hypothesis that β-casein interacts more effectively with slowly aggregating rather than rapidly aggregating target proteins. It is proposed that dextran-induced changes to protein conformation and the rate of intermolecular association are in a kinetic competition with the chaperoning action of β-casein; however our results demonstrated the higher activity of glycerol, as a chemical chaperone, than β-casein on the folding of target proteins, especially in the presence of dextran. This is likely due to the stabilizing effect of glycerol on protein structure and environment. The implications for the in vivo functions of β-casein and glycerol, based on their exhibiting such in vitro chaperone-like activities, are discussed.     

Controlling the aggregation and rate of release in order to improve insulin formulation: molecular dynamics study of full-length insulin amyloid oligomer models

Insulin is a hormone that regulates the physiological glucose level in human blood. Insulin injections are used to treat diabetic patients. The amyloid aggregation of insulin may cause problems during the production, storage, and delivery of insulin formulations. Several modifications to the C-terminus of the B chain have been suggested in order to improve the insulin formulation. The central fragments of the A and B chains (LYQLENY and LVEALYL) have recently been identified as β-sheet-forming regions, and their microcrystalline structures have been used to build a high-resolution amyloid fibril model of insulin. Here we report on a molecular dynamics (MD) study of single-layer oligomers of the full-length insulin which aimed to identify the structural elements that are important for amyloid stability, and to suggest single glycine mutants in the β-sheet region that may improve the formulation. Structural stability, aggregation behavior and the thermodynamics of association were studied for the wild-type and mutant aggregates. A comparison of the oligomers of different sizes revealed that adding strands enhances the internal stability of the wild-type aggregates. We call this “dynamic cooperativity”. The secondary structure content and clustering analysis of the MD trajectories show that the largest aggregates retain the fibril conformation, while the monomers and dimers lose their conformations. The degree of structural similarity between the oligomers in the simulation and the fibril conformation is proposed as a possible explanation for the experimentally observed shortening of the nucleation lag phase of insulin with oligomer seeding. Decomposing the free energy into electrostatic, van der Waals and solvation components demonstrated that electrostatic interactions contribute unfavorably to the binding, while the van der Waals and especially solvation effects are favorable for it. A per-atom decomposition allowed us to identify the residues that contribute most to the binding free energy. Residues in the β-sheet regions of chains A and B were found to be the key residues as they provided the largest favorable contributions to single-layer association. The positive ∆∆G _mut values of 37.3 to 1.4 kcal mol⁻¹ of the mutants in the β-sheet region indicate that they have a lower tendency to aggregate than the wild type. The information obtained by identifying the parts of insulin molecules that are crucial to aggregate formation and stability can be used to design new analogs that can better control the blood glucose level. The results of our simulation may help in the rational design of new insulin analogs with a decreased propensity for self-association, thus avoiding injection amyloidosis. They may also be used to design new fast-acting and delayed-release insulin formulations.     

Insulin: a small protein with a long journey

Insulin is a hormone that is essential for regulating energy storage and glucose metabolism in the body. Insulin in liver, muscle, and fat tissues stimulates the cell to take up glucose from blood and store it as glycogen in liver and muscle. Failure of insulin control causes diabetes mellitus (DM). Insulin is the unique medicine to treat some forms of DM. The population of diabetics has dramatically increased over the past two decades, due to high absorption of carbohydrates (or fats and proteins), lack of physical exercise, and development of new diagnostic techniques. At present, the two largest developing countries (India and China) and the largest developed country (United States) represent the top three countries in terms of diabetic population. Insulin is a small protein, but contains almost all structural features typical of proteins: α-helix, β-sheet, β-turn, high order assembly, allosteric T®R-transition, and conformational changes in amyloidal fibrillation. More than ten years’ efforts on studying insulin disulfide intermediates by NMR have enabled us to decipher the whole picture of insulin folding coupled to disulfide pairing, especially at the initial stage that forms the nascent peptide. Two structural switches are also known to regulate insulin binding to receptors and progress has been made to identify the residues involved in binding. However, resolving the complex structure of insulin and its receptor remains a challenge in insulin research. Nevertheless, the accumulated knowledge of insulin structure has allowed us to specifically design a new ultra-stable and active single-chain insulin analog (SCI-57), and provides a novel way to design super-stable, fast-acting and cheaper insulin formulations for DM patients. Continuing this long journey of insulin study will benefit basic research in proteins and in pharmaceutical therapy.     

Multivariate Analysis of Phenol in Freeze-Dried and Spray-Dried Insulin Formulations by NIR and FTIR

Dehydration is a commonly used method to stabilise protein formulations. Upon dehydration, there is a significant risk the composition of the formulation will change especially if the protein formulation contains volatile compounds. Phenol is often used as excipient in insulin formulations, stabilising the insulin hexamer by changing the secondary structure. We have previously shown that it is possible to maintain this structural change after drying. The aim of this study was to evaluate the residual phenol content in spray-dried and freeze-dried insulin formulations by Fourier transform infrared (FTIR) spectroscopy and near infrared (NIR) spectroscopy using multivariate data analysis. A principal component analysis (PCA) and partial least squares (PLS) projections were used to analyse spectral data. After drying, there was a difference between the two drying methods in the phenol/insulin ratio and the water content of the dried samples. The spray-dried samples contained more water and less phenol compared with the freeze-dried samples. For the FTIR spectra, the best model used one PLS component to describe the phenol/insulin ratio in the powders, and was based on the second derivative pre-treated spectra in the 850–650 cm⁻¹ region. The best PLS model based on the NIR spectra utilised three PLS components to describe the phenol/insulin ratio and was based on the standard normal variate transformed spectra in the 6,200–5,800 cm⁻¹ region. The root mean square error of cross validation was 0.69% and 0.60% (w/w) for the models based on the FTIR and NIR spectra, respectively. In general, both methods were suitable for phenol quantification in dried phenol/insulin samples.     

Study on preparation and unique properties of a novel insulin analogue with N-terminal Arg-4, Pro-3, Lys-2, Pro-1extension at insulin B-chain

A novel insulin analog, PIns, with N-terminal Arg-4, Pro-3, Lys-2, Pro-1extension at human regular insulin B-chain was acquired through gene engineering. Preproinsulin for PIns was cloned and expressed using a bacterial expression system at a high level (72.1%) as fusion protein carrying a modified thioredoxin N-terminal region (1–21) linked to N-terminus of proinsulin by a lysine residue. Purified fusion protein was refolded and converted into PIns by a single enzymatic reaction. After PIns was purified, the homogeneity of it was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectronic focusing electrophoresis, amino acid composition analysis and mass spectrometry methods. A decreased tendency of self-association of PIns as compared with regular insulin was demonstrated by the size exclusion HPLC analysis. When subcutaneously administrated into normal rats, the PIns showed a faster rate of onset of action and a shorter duration of action compared with regular insulin, similar to the pharmacokinetic characteristics of insulin Lispro. These results showed that PIns is a rapid insulin analog. Furthermore, the N-terminal Arg-4, Pro-3, Lys-2, Pro-1extension at insulin B-chain can be excised by DPPIV and recombinant peptidase with DPPIV-like activities. It is suggested that PIns serves as an artificial insulin precursor and can be transformed to regular insulin in vivo due to the truncation of N-terminal sequence of PIns B-chain by DPPIV.     

New Aspartic Proteinase of Ulysses Retrotransposon from Drosophila virilis

This work is focused on the investigation of a proteinase of Ulysses mobile genetic element from Drosophila virilis. The primary structure of this proteinase is suggested based on comparative analysis of amino acid sequences of aspartic proteinases from retroviruses and retrotransposons. The corresponding cDNA fragment has been cloned and expressed in E. coli. The protein accumulated in inclusion bodies. The recombinant protein (12 kD) was subjected to refolding and purified by affinity chromatography on pepstatin-agarose. Proteolytic activity of the protein was determined using oligopeptide substrates melittin and insulin B-chain. It was found that the maximum of the proteolytic activity is displayed at pH 5.5 as for the majority of aspartic proteinases. We observed that hydrolysis of B-chain of insulin was totally inhibited by pepstatin A in the micromolar concentration range. The molecular weight of the monomer of the Ulysses proteinase was determined by MALDI-TOF mass-spectrometry.     

Secondary structure of food proteins by Fourier transform spectroscopy in the mid-infrared region

Fourier transform spectroscopy in the mid-infrared (400–5,000 cm⁻¹) (FT-IR) is being recognized as a powerful tool for analyzing chemical composition of food, with special concern to molecular architecture of food proteins. Unlike other spectroscopic techniques, it provides high-quality spectra with very small amount of protein, in various environments irrespective of the molecular mass. The fraction of peptide bonds in α-helical, β-pleated sheet, turns and aperiodic conformations can be accurately estimated by analysis of the amide I band (1,600–1,700 cm⁻¹) in the mid-IR region. In addition, FT-IR measurement of secondary structure highlights the mechanism of protein aggregation and stability, making this technique of strategic importance in the food proteomic field. Examples of applications of FT-IR spectroscopy in the study of structural features of food proteins critical of nutritional and technological performance are discussed.     

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.     

Mechanism of zinc(II)-promoted amyloid formation: zinc(II) binding facilitates the transition from the partially α-helical conformer to aggregates of amyloid β protein(1–28)

The amyloidoses are a group of disorders characterized by aberrant protein folding and assembly, leading to the deposition of insoluble protein fibrils (amyloid), which provokes cell dysfunction and later cell death. One of the physiologically relevant environmental factors able to affect the conformation and hence the aggregation properties of amyloidogenic proteins/peptides is metal ions. Zn(II) promotes aggregation of most amyloidogenic peptides/proteins in vitro, including amyloid β protein (Aβ), but the underlying mechanism is not known. To better understand this mechanism the present study focused on the partially α-helical conformer, supposed to be an intermediate in Aβ aggregation. This partially α-helical conformer is stabilized by 10–20% 2,2,2-trifluoroethanol (TFE): therefore, the influence of Zn binding on the aggregation of the amylidogenic model peptide Aβ(1–28) (Aβ28) was investigated at different TFE concentrations. The results showed a synergistic effect of Zn(II) and 10% TFE, i.e., that either Zn or 10% TFE accelerated Aβ28 aggregation on its own, but with them together an at least 10 times promotion of Aβ28 aggregation was observed. Further studies by thioflavin T fluorescence spectroscopy, transmission electron microscopy, and circular dichroism (CD) spectroscopy suggested that the aggregates of Zn-Aβ28 formed in 10%TFE contain a β-sheet secondary structure and are more of the amyloid type. CD spectroscopy indicated that Zn binding disrupted partially the α-helical structure of Aβ28 in TFE. Thus, we propose that the promotion of Aβ28 aggregation by Zn is based on the transformation of the partially α-helical conformer (intermediate) towards the β-sheet amyloid structure by a destabilization of the α-helix in the intermediate. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.