The characteristics and motion model of insulin monomer.

The extensive conformational comparisons among the determined structures of the different species and crystal forms of insulin and the varied insulin derivatives were performed by using the least-squares superimposition technique and the graphics technique. The results of the investigation showed that the structure of molecule I in 2Zn insulin was closer to that of the natural monomer; the conformational difference between two molecules of a dimer came out during dimerization and it was further improved and stabilized during the hexamerization and packing of hexamers in crystal; through the hinge peptides, such as A10, B4, B8, B24, B20 and B23, there was a flexible relative motion among the structural segments in the insulin molecule, and the residues at the B-chain C-terminal might have a shift of more than 10A; the mobility for each residue side-chain was very different due to the different surroundings.     

Structural changes in the monomeric despentapeptide (B30-B26) insulin crystal

Insulin modified by the removal of its 5 B chain C terminal residues is monomeric but remains substantially potent. The crystal structures of the beef and insulin (dpi) with two molecules in the asymmetric unit has been determined by x-ray analysis. The 3-dimensional structure ofdpi proves to be generally similar to that of native molecule in 2Zn insulin. More detailed comparison reveals that the slight differences in the two independent molecules of beefdpi are distributed uniformly throughout the structure in contrast to insulin in 2Zn insulin, where the structural changes are concentrated in specific regions. The loss of symmetry in thedpi crystal appears to be the inability of the A9 serine to pack effectively in the C2 cell. The efficient packing of the sheepdpi molecule whose crystal structure has also been determined and where A9 is glycine supports this conclusion.     

THE POSSIBLE MECHANISM OF BINDING INTERACTION OF INSULIN MOLECULE WITH ITS RECEPTOR

Detailed structural comparisons and investigation of DPI, 2Zn insulin and some other derivatives of insulin were performed by the least-squares superimposition technique and the graphics technique. It is pointed out in this paper that the binding interaction with the receptor molecule should take place mainly on an amphipathic surface of the insulin molecule. In the middle, there is a hydrophobic surface with an area of about 150 consisting of many hydrophobic residues; while the polar or charged groups distributing around the hydro. phobic surface construct a hydrophilic zone. The hydrophobic surface is usually covered by the extended B-chain C-terminal peptides with great mobility and protected from the solvent molecules. The angle between the amphipathic surface and the surface of dimerization is about 20 degrees. The results from the detailed structural comparison between A1-(L-Trp) insulin and A1-(D-Trp) insulin have provided a very good explanation to their great difference in biological activity,     

MOLECULAR CLOSE-PACKING METHOD AND ITS APPLICATION TO CRYSTAL STRUCTURE DETERMINATION OF DESHEXAPEPTIDE (B25--B30) INSULIN

Based on the molecule-packing theory, we defined a molecule-packing function express-ing the compatibility of packing among the symmetry-related molecules in a unit cell. Acomputer program imitating the close-packing of molecules in the objective crystal latticeand giving the function value of each rotation and translation of the molecule in the unitcell was performed, and it therefore made the close-packing of molecules expressquantitatively. This method not only could judge a correct solution from several peaks ofthe rotation or translation function but it may also independently, quantitatively and quicklysolve some specific problems of rotation and translation. Using known structure of despenta-peptide (B26--B30) insulin as an example, the effectiveness of this method and its programwas inspected, and this method was successfully applied to solving the translation problem ofthe unknown structure of deshexapeptide (B25--B30) insulin. The molecular close-packingmethod proved by the results of R--search     

新颖的双胰岛素取代钴(III)卟啉的制备和表征

A novel insulin-disubstituted Co(Ⅲ)protoporphyrin IX,CoPI,where I is insulin and CoP is Co(Ⅲ)protopor-phyrin IX,was prepared by covalently coupling the propionate groups on the porphyrin ring to the ε-amino groupsof B29-Lys on insulin via amide linkages.The FTIR spectra in the amide I region(1600—1700 cm~(-1))and circulardichroism study show that CoPI has a conformation similar to the insulin at pH 6.9,whereas it exhibits significantconformational changes in structure as compared with the insulin self at pH 8.2.The pH absorption titration indi-cates that the alkaline conditions(pH≥8.0)are required for the formation of complexes between the free CoP andthe insulin.The thermodynamic and kinetic data reveal that free CoP is bound to either the zinc-insulin or free insu-lin with a dissociation constant of(2.0±0.3)×10~(-5)or(2.2±0.3)×10~(-5)mol/L.     

Spectral Studies on the Conformational Transitions of Bovine Insulin During Denaturant-induced Unfolding

Transitions among various molecule states and conformational changes of bovine insulin were investigated under different denaturing conditions by means of fluorescence phase diagrams,fluorescence quenching,1-anilinonaphthalene-8-sulfonate（ANS） binding assay and circular dichroism（CD） spectra.In both guanidine hydrochloride（GuHCl）-and urea-denatured procedures,the spatial structure of insulin molecules changed from ordered states to relative unordered ones with the increasing of denaturant concentration.The GuHCl-denatured process followed a four-state model,for there were two intermediates existed in 2.0 and 6.0 mol/L GuHC1,respectively.Intermediate I1 is more compact than the normal protein.And intermediate I2 has lost most of the secondary structures.When GuHCl concentration was above 6.0 mol/L,the fluorophores originally existed in the internal of insulin molecules would expose to the surface.However,the urea-denatured process followed a three-state model,only one intermediate existed in 2.5 mol/L urea.During the urea-denatured procedure,the fluorophores originally existed in theinternal of insulin molecules didn＇t expose to the surface.     

STUDIES ON THE CRYSTAL STRUCTURE OF Al-(D-TRYPTOPHAN) INSULIN

We have determined the crystal structure of Al-(D-Trp) insulin and discovered that it belongs to the trigonal system with space group R3. The parameters of the unit cell are a=b=78.6, c=50.0. A set of data for half a sphere reciprocal space to a spacing of 2.2 were collected. The model was adjusted and refined by using a step-by-step approach and a stereochemically-restrained least squares program, assisted by manual revision based on the difference Fourier maps, to a final R-factor of 0.218. The main and side chains of both Al-D-Trp residues in the asymmetric unit are well ordered. The packing of Al-(D-Trp) insulin in the unit cell, the conformational differences with other insulin structures and its structure and function relationship bave also been discussed.     

[B10Glu,B24-Asp-B25]人胰岛素类似物的制备、鉴定与性质研究

采用PCR方法,将人胰岛素分子B链B10位His突变为Glu,在B24和B25位之间插入Asp,构建了[B10Glu,B24-Asp-B25]胰岛素原基因.利用通用型质粒pBV220构建表达载体,在大肠杆菌DH5α中表达,表达蛋白为包含体形式,约占菌体总蛋白的20%～30%.经过复性和凝胶过滤得到胰岛素原融合蛋白.用胰蛋白酶和羧肽酶B酶切,经DEAE离子交换和RP-HPLC纯化得胰岛素突变体类似物.用凝胶过滤法测定了蛋白质分子自身的缔合性质,用圆二色谱测定了构象变化.放射性免疫活性及受体结合活性测定结果表明,突变体分子缔合性明显下降,放免活性和受体结合活性分别约为人胰岛素的73.6%和146%.     

STUDIES ON THE CRYSTAL STRUCTURE OF Al-(L-TRYPTOPHAN) INSULIN AT 2.1RESOLUTION

In order to study the biological effect of alterations to the N-terminus of the insulin A-chain, we have determined the crystal structure of Al-(L-Trp) insulin and discovered that it belongs to the trigohal system with space group R3. The parameters oof the unit cell are a=b=80.3A, c=37.5A. The model was adjusted and refined by using a stereochemically-restrained least squares program, assisted by manual revision of the model based on the difference Fourier map, to a final R-factor of 0.195. The main and side chains of both Al-(L-Trp) residues in the asymmetric unit are well ordered. It was found that the Al-Trp residue of molecule I occupied two distinct positions. We have proposed from the results of the three-dimensional structure that the 4-zinc insulin hexameric form is a stored state of insulin molecules in a conformation of low activity. The structural details of the insulin molecule and its structure and function relationship have also been discussed.     

Studies on the crystal structure of (D-Ala)-B0 porcine insulin at 1.9 A resolution

The crystal structure of (D-Ala)-B0 porcine insulin has been determined, using data to 1.9 A and atomic parameters of 2 Zn porcine insulin as a starting model, and through the use of the difference method and the restrained least square method, to a final R-factor of 0.211 and r.m.s. deviation of 0.057 A for the bond lengths. The electron densities of B0 residues were very clear. Introduction of B0 residues into the molecules had reduced the thermal vibration of the N-terminus of B-chain for both molecules I and II and made the molecules pack closer in the crystal. The obvious differences between the crystal structures of 2 Zn and (D-Ala)-B0 porcine insulin were the conformations of partial polar groups around the possible receptor binding surface and the assembly mode of two helixes of A-chain in molecule I. In the local environment of the N-terminus of B-chain there were great differences between the crystal structures of (D-Ala)-B0 porcine insulin, (Trp)-B1 porcine insulin and Des B1(Phe) bovine insulin. In this paper the structure-immunoactivity relationships of insulin molecule have also been discussed briefly.     

Changes of conformation and aggregation state induced by binding of lanthanide ions to insulin

To clarify the mechanism of lanthanide ions (Ln3+) on the across-membrane transport of insulin and subsequent reducing blood glucose, the interactions of Ln3+with Zn-insulin and Zn-free insulin are investigated by spectroscopic methods. The results reveal that the binding of Ln3+ to insulin can induce its structure changes from secondary to quaternary structure, depending on the Ln3+ concentration. In the lower concentration, it triggers the conformational changes of insulin monomer in the binding region with insulin receptor (B(24-30)). It would affect insulin-insulin receptor interaction. Moreover, Ln3+ binding promotes the assembly of insulin monomer from dimer to polymer. The potency of Ln3+ in inducing insulin's aggregation is stronger than that of Zn2+. Furthermore, the aggregation can be reversed partly by EDTA-treatment, indicating that it is not due to denaturation. Similar to Zn2+ effect, Ln3+ can stabilize insulin hexamer in a certain range of concentration, but is stronger than the former.     

Stepwise conformational transition of crystalline disodium adenosine 5′-triphosphate with relative humidity as studied by high resolution solid state 13C and 31P NMR

Stepwise conformational transition of disodium adenosine 5′-triphosphate (Na₂ATP) crystals as a function of relative humidity (RH), was examined by means of high resolution solid state NMR spectroscopy. The presence of two types of molecules, A and B, with altered conformations in an asymmetric unit, is evident from both ³¹P and ¹³C NMR spectra, irrespective of the three different crystalline forms, mono-, di- and trihydrates, in which, cell parameters changed linearly with RH. Hydration-dependent conformational transition from the monohydrate (RH 0–10%) through dihydrate (20–40%) to trihydrate (60–90%), was well monitored by stepwise upfield displacements of the C1′ ¹³C signals of ribose moiety (molecule B), although, the corresponding peak for the molecule A, is almost unchanged. This means that, adaptation of stepwise structural transitions to the linear expansion of cell parameters is almost entirely ascribed to the conformational readjustment of the molecule B, together with varying proportion of two types of hydrate forms, at the RH range of 20–40%. We consider that, small clusters or thin layers are formed in the beginning of the transition and subsequently, the number of them, rather than the size or thickness increases, because the phosphorous spin–lattice relaxation times, T₁^P, for the three phosphorous nuclei, including those of mixed state, of the two hydrate forms, turned out to be similar due to sufficient fast spin diffusion rates among them. Since only one direction of the cell parameters is changed, as determined by the previous X-ray diffraction study, we conclude that thin layer type micro crystals might be produced during the stepwise transition. Further, it was found that, these molecules acquired motional flexibility mainly at the bound water molecules, together with increased relative humidity, as determined by a variety of relaxation parameters.     

Investigation of mechanical properties of insulin crystals by atomic force microscopy

Mechanical properties of protein crystals and aggregates depend on the conformational and structural properties of individual protein molecules as well as on the packing density and structure within solid materials. An atomic force microscopy (AFM)-based approach is developed to measure the elastic modulus of small protein crystals by nanoindentation and is applied to measure the elasticity of insulin crystals. The top face of the crystals deposited on mica substrates is identified as the (001) face. Insulin crystals exhibit a nearly elastic response during the compression cycle. The elastic modulus measured on the top face has asymmetric distribution with a significant width. This width is related to the uncertainty in the deflection sensitivity. A model that takes into account the distribution of the sensitivity values is used to correct the elastic modulus. Measurements performed in aqueous buffer on several crystals at different locations with three different AFM probes give a mean elastic modulus of 164 +/- 10 MPa. This value is close to the static elastic moduli of other protein crystals measured by different techniques that are usually measured in the range from 100 MPa to 1 GPa. The measured modulus of insulin crystals falls between the elastic modulus values of insulin amyloid fibrils measured previously at two orthogonal directions (a modulus of 14 MPa was measured by compressing the fibril in the direction perpendicular to the fibril axis, and a modulus of 3.3 GPa was measured in the direction along the fibril axis). This comparison indicates the heterogeneous structure of fibrils in the direction perpendicular to the fibril axis, with a packing density of the amyloid fibril core that is higher than the average packing density in insulin crystals. The mechanical wear of insulin crystals is detected during AFM measurements. In nanoindentation experiments on insulin crystal, the compressive load by the AFM tip ( approximately 1 nN, corresponding to a pressure of around 5 MPa) occasionally removes protein molecules from the top or the second top layer of insulin crystal in a sequential manner. The molecular model of this surface damage is proposed. In addition, the removal of the multiple layers of molecules is observed during the AC-mode imaging in aqueous buffer. The number of removed layers depends on the scan size.     

Crystal structural studies of destripeptide (B28-B30) insulin

Single crystals of destripeptide (B28-B30) insulin (DTRI) in three forms were obtained by hanging-drop vapor diffusion method. Form 1 belongs to P21 space group with cell parameters a-4.77 nm, b=6.19 nm, c=6.12 nm, β=110.3°. Form 2 belongs to P4122 or P4322 space group with cell parameters a= 6.45 nm, c=12.07 nm. Form 3 belongs to P212121 space group with cell parameters a=4.98 nm, b=5.16 nm, c=10.06 nm. The structure of form 1 crystal was determined by molecular replacement method and refined at 0.23 nm resolution. The R-factor of the final model is 18.8% with r.m.s. deviations of 0.001 5 nm and 3.3?for the bond lengths and the bond angles, respectively. Studies on the crystal structure show that the removal of B28 Pro has brought DTRI structural changes which made it dissociate more easily than native insulin although DTRI can still form a hexamer.     

Preliminary crystallographic analysis of deshexapeptide (B25-B30) insulin

Satisfactory single crystals of deshexapeptide (B25-B30) insulin for X-ray crystal structure analysis have been grown in citrate buffer by the method of hanging-drop gas phase diffusion. The crystal belongs to the monoclinic system with space group C2. The unit cell constants are a = 42.6 A, b = 37.9 A, c = 27.2 A, beta = 125.4 degrees and there is only one molecule of deshexapeptide insulin in an asymmetric unit.     

The ABC of Insulin: The Organic Chemistry of a Small Protein

Insulin is a small protein crucial for regulating the blood glucose level in all animals. Since 1922 it has been used for the treatment of patients with diabetes. Despite consisting of just 51 amino acids, insulin contains 17 of the proteinogenic amino acids, A- and B-chains, three disulfide bridges, and it folds with 3 α-helices and a short β-sheet segment. Insulin associates into dimers and further into hexamers with stabilization by Zn²⁺ and phenolic ligands. Selective chemical modification of proteins is at the forefront of developments in chemical biology and biopharmaceuticals. Insulin's structure has made it amenable to organic and inorganic chemical reactions. This Review provides a synthetic organic chemistry perspective on this small protein. It gives an overview of key chemical and physico-chemical aspects of the insulin molecule, with a focus on chemoselective reactions. This includes N-acylations at the N-termini or at Lys^B29 by pH control, introduction of protecting groups on insulin, binding of metal ions, ligands to control the nano-scale assembly of insulin, and more.     

Effects of cysteic acid groups on the gas-phase reactivity and dissociation of [M + 4H]4+ ions from insulin chain B

Gas-phase ion/molecule reactions and collision-induced dissociation (CID) were conducted on [M + 4H]4+ of insulin chain B. This Fourier transform mass spectrometry work involved ions from the oxidized peptide (with two cysteic acid residues) and its reduced form (with two cysteine residues). Kinetic behavior during deprotonation and hydrogen/deuterium exchange reactions indicates that insulin B (ox) ions have two distinct structural types. In contrast, insulin B (red) ions have only one major reacting population, which has a more compact structure than the oxidized ions. No significant differences in fragmentation patterns for the two insulin B (ox) populations were observed when CID was performed as a function of deprotonating reaction time. However, markedly different fragmentation was found between [M + 4H]4+ of insulin B (ox) and (red). Therefore, the presence of cysteic acid groups in insulin B (ox) significantly impacts dissociation and presumably structure. This suggests that some insulin B (ox) ions are zwitterionic, with the five basic sites protonated and one cysteic acid group deprotonated. Molecular dynamics calculations revealed several viable structures that are consistent with the experimental results. For example, the most stable form of the reduced ion, which is unprotonated at the His10, is very compact and has lost the alpha-helix of native insulin. Low energy structures for the oxidized ions include a zwitterion with an intraionic interaction between anionic Cyx7 and cationic His10, as well as a nonzwitterionic conformer that lacks a proton at Phe1; both structures retain the alpha-helix. These structures may account for the two experimentally observed isomers, although others are possible. In addition, experiments on oxidized insulin B were conducted from methanolic solution, which may denature the conformation, and pure aqueous solution, which may leave a native conformation. These differences in solvent composition had no effect on the gas-phase results.     

毛细管区带电泳监测牛胰岛素的去折叠过程

用毛细管区带电泳监测了二硫苏糖醇作用下二硫键还原引起的牛胰岛素去折叠全过程,同时用离线的基质辅助激光解吸／电离飞行时间的质谱配合确证。从毛细管电泳谱图能直接观察胰岛素去折叠过程中发生的变化,获得蛋白质去折叠信息。结果表明,毛细管区带电泳作为监测蛋白质构象变化的一种有效手段,方法简便、快速、灵敏度高、样品消耗量少。     

胰岛素B链螺旋片段构象能量的研究

本文作为胰岛素及其化学修饰物分子构象能量系列研究工作的组成部分,以2Zn-、(D-Ala)~(B0)-和(L-Leu)~(B1)-猪胰岛素晶体结构为依据,报告分子B9-B19螺旋(化学序列为S·H·L·V·G·A·L·Y·L·V·C)六种X射线晶体学构象能量的研究结果。构象二面角的定义和计算遵循IUPAC-IUB规则。构象总能量(包括静电能EES,非键能ENB和扭角势能ETOR三项)的计算使用程序ECEPP/2,键长和键角取标准值(由     

载有胰岛素的可生物降解微球的制备与表征

用乙交酯与丙交酯的无规共聚物(PLGA)和聚乙二醇单甲醚-聚丙交酯两嵌段共聚物(MPEG-PLA)的合金作为囊材料,包裹胰岛素固体粉末,包裹率分析表明,固体粉末法对胰岛素的包裹率高于双乳液法.所得微球球形很好,尺寸在1～3μm范围,剖面具有核壳结构,胰岛素以晶粒的形式包裹在高分子壳层中.两种高分子在凝聚过程中发生相分离,在壳层中有分层现象.测定微球的体外释放行为,由聚合物合金制备的微球的暴释现象得到了缓解,两种聚合物的配比不同,其暴释缓解的程度也不一样.