Effect of polyols on the conformational stability and biological activity of a model protein lysozyme

The purpose of this study was to investigate the stabilizing action of polyols against various protein degradation mechanisms (eg, aggregation, deamidation, oxidation), using a model protein lysozyme. Differential scanning calorimeter (DSC) was used to measure the thermodynamic parameters, mid point transition temperature and calorimetric enthalpy, in order to evaluate conformational stability. Enzyme activity assay was used to corroborate the DSC results. Mannitol, sucrose, lactose, glycerol, and propylene glycol were used as polyols to stabilize lysozyme against aggregation, deamidation, and oxidation. Mannitol was found to stabilize lysozyme against aggregation, sucrose against deamidation both at neutral pH and at acidic pH, and lactose against oxidation. Stabilizers that provided greater conformational stability of lysozyme against various degradation mechanisms also protected specific enzyme activity to a greater extent. It was concluded that DSC and bioassay could be valuable tools for screening stabilizers in protein formulations.     

Effect of polyamines on mechanical and structural properties of Bombyx mori silk

Silkworm, Bombyx mori (B. mori) belongs to the Lepidoptera family. The silk produced from this insect, mulberry silk, gained lot of importance as a fabric. Silk is being exploited as a biomaterial due to its surprising strength and biocompatibility. Polyamines (PA) are important cell growth regulators. In the present work the effect of treatment of polyamines, putrescine (Put), spermidine (Spd), and spermine (Spm) on the quantity and quality of silk produced was assessed. Results showed that exogenous feeding of Spd at a concentration of 50 µM increased fiber length significantly. Analysis by Fourier transform infrared (FTIR) on the properties of silk obtained from Spd treated silkworms revealed an increase in percentage of absorption with no difference in peak positions of amide I and amide III groups. Scanning electron microscopy (SEM) revealed an increase in diameter of silk. Further, analysis at molecular level showed an increase in fibroin expression in Spd treated silk glands. However, the Spd treatment showed no significant difference with respect to fibroin to sericin ratio per unit weight of cocoon, silk tenacity, and percent elongation. Thus, the present results show that polyamine treatment would influence silk quality at structural, mechanical, and molecular level in the Bombyx mori, which can be exploited in silk biomaterial production.     

Dramatic changes in patterning gene expression during metamorphosis are associated with the formation of a feather-like antenna by the silk moth, Bombyx mori

Many moths use sex pheromones to find their mates in the dark. Their antennae are well developed with lateral branches to receive the pheromone efficiently. However, how these structures have evolved remains elusive, because the mechanism of development of these antennae has not been studied at a molecular level. To elucidate the developmental mechanism of this type of antenna, we observed morphogenesis, cell proliferation, cell death and antennal patterning gene expression in the branched antenna of the silk moth, Bombyx mori. Region-specific cell proliferation and almost ubiquitous apoptosis occur during early pupal stages and appear to shape the lateral branch cooperatively. Antennal patterning genes are expressed in a pattern largely conserved among insects with branchless antennae until the late 5th larval instar but most of them change their expression dramatically to a pattern prefiguring the lateral branch during metamorphosis. These findings imply that although antennal primordium is patterned by conserved mechanisms before metamorphosis, most of the antennal patterning genes are reused to form the lateral branch during metamorphosis. We propose that the acquisition of a new regulatory circuit of antennal patterning genes may have been an important event during evolution of the sensory antenna with lateral branches in the Lepidoptera.     

家蚕前部丝腺特异表皮蛋白Bm11721的鉴定及表达

家蚕的丝腺是其丝蛋白合成和分泌的器官,根据其形态和功能的不同分为前部、中部和后部丝腺,前部丝腺不具有合成丝蛋白的能力,是丝蛋白构象发生转变的场所。剪切力在丝蛋白构象转变中起到重要的作用,其在家蚕前部丝腺主要由前部丝腺逐渐变细的管腔结构和富含几丁质及表皮蛋白的坚硬的内壁提供。鉴定家蚕前部丝腺新的几丁质结合蛋白,并调查其在家蚕幼虫不同组织的表达特征。通过几丁质亲和层析的方法在前部丝腺筛选并鉴定到一个新的具有几丁质结合功能的表皮蛋白Bm11721,其编码基因编号为BGIBMGA011721(Gen Bank Accession No.NM-001173285.1)。利用原核表达系统成功表达了该蛋白,通过Ni-NTA亲和层析的方法获得了Bm11721的重组蛋白并制备了多克隆抗体。组织表达分析发现无论是转录水平还是蛋白水平Bm11721均只在前部丝腺特异表达,且Bm11721蛋白在5龄期的前部丝腺中恒定表达。免疫荧光定位结果显示Bm11721蛋白定位在前部丝腺的内膜中,推测其可能与前部丝腺的机械硬度有关,为丝蛋白的构象转变提供剪切力。     

丝蛋白合成和分泌期家蚕丝腺中有氧氧化特性分析

【目的】有氧氧化中葡萄糖(Glc)、丙酮酸(PA)、乙酰Co A(AC)、还原型吡啶核苷酸(NADH)和腺苷三磷酸(ATP)摩尔数的理论比值为1∶2∶2∶10∶30~32,而己糖激酶(HK)、磷酸果糖激酶1(PFK1)、丙酮酸激酶(PK)、丙酮酸脱氢酶(PDH)、柠檬酸合酶(CS)、异柠檬酸脱氢酶(ICDHm)、α-酮戊二酸脱氢酶(α-KGDH)、NADH泛醌还原酶(NCR)、琥珀酸泛醌还原酶(SCR)、泛醌细胞色素C还原酶(CCR)、细胞色素C氧化酶(CCO)和ATP合酶(AS)活性的理论比值为1∶1∶2∶2∶2∶2∶2∶10∶2∶12∶12∶26~28。本研究旨在分析丝蛋白合成和分泌期家蚕Bombyx mori丝腺有氧氧化的特性。【方法】利用分光光度法和高效液相色谱法测定了上述生化指标的变化。【结果】丝蛋白合成和分泌期家蚕丝腺中检测不到Glc,产物含量以1/30 ATP,1/10 NADH,1/2 AC和1/2 PA的顺序递增;糖酵解途径相关酶活性,以PFK1活性最低;三羧酸循环相关酶活性以1/2 ICDHm,1/2α-KGDH和1/2 CS的顺序递增;氧化磷酸化相关酶包括1/26 AS,1/10 NCR,1/2 SCR,1/12 CCR和1/12 CCO的活性以1/26 AS活性最低;1/26 AS,1/2 ICDHm,1/2 PDH和PFK1的活性依次递增。NADH含量、ATP含量、PFK1活性、PDH活性和NCR活性在丝蛋白合成期升高,但在丝蛋白分泌期下降。【结论】据此推测,家蚕丝腺中PFK1,ICDHm和AS分别是糖酵解途径、三羧酸循环和氧化磷酸化的限速酶;糖酵解途径、丙酮酸脱氢、三羧酸循环和氧化磷酸化速率依次递减;有氧氧化速率在丝蛋白合成期升高,相反在丝蛋白分泌期降低。     

Thermal stability determinants of chicken egg-white lysozyme core mutants: hydrophobicity, packing volume, and conserved buried water molecules.

A series of 24 mutants was made in the buried core of chicken lysozyme at positions 40, 55, and 91. The midpoint temperature of thermal denaturation transition (Tm) values of these core constructs range from 60.9 to 77.3 degrees C, extending an earlier, more limited investigation on thermostability. The Tm values of variants containing conservative replacements for the wild type (WT) (Thr 40-Ile 55-Ser 91) triplet are linearly correlated with hydrophobicity (r = 0.81) and, to a lesser degree, with combined side-chain volume (r = 0.75). The X-ray structures of the S91A (1.9 A) and I55L/S91T/D101S (1.7 A) mutants are presented. The former amino acid change is found in duck and mammalian lysozymes, and the latter contains the most thermostable core triplet. A network of four conserved, buried water molecules is associated with the core. It is postulated that these water molecules significantly influence the mutational tolerance at the individual triplet positions. The pH dependence of Tm for the S91D mutant was compared with that of WT enzyme. The pKa of S91D is 1.2 units higher in the native than in the denatured state, corresponding to delta delta G298 = 1.7 kcal/mol. This is a low value for charge burial and likely reflects the moderating influence of the buried water molecules or a conformational change. Thermal and chemical denaturation and far UV CD spectroscopy were used to characterize the in vitro properties of I55T. This variant, which buries a hydroxyl group, has similar properties to those of the human amyloidogenic variant I56T.     

In vitro production of Bombyx mori silk fibroin by organ culture of the posterior silk glands; isotope labeling and fluorination of the silk fibroin

An in vitro silk fibroin production system has been developed by culture of posterior silk glands from Bombyx mori. A large amount of the silk fibroin was produced continuously and effectively with a rotation culture procedure. Modified Grace's insect medium was used, and oxygen bubbling in the medium was performed. In addition, half of the medium was replaced with fresh medium every 6 h. The production yield of silk fibroin produced after 100 h culture was 81 mg/g wet weight of posterior silk gland. This culture system was used successfully for efficient (15)N isotope labeling of silk fibroin, which is required for (15)N solid state nuclear magnetic resonance (NMR) analysis of silk fibroin. Moreover, the introduction of fluorinated amino acids into silk fibroin was also carried out using this culture system. (c) 1993 John Wiley & Sons, Inc.     

The dissolution and characterization of Bombyx mori silk fibroin in calcium nitrate-methanol solution and the regeneration of films

Bombyx mori silk fibroin from the silkworm was found to be soluble in a calcium nitrate-methanol system. Fibroin dissolves in 75% w/v Ca(NO₃)₂/MeOH solution at a temperature of 67°C. The viscometric behavior of the fibroin-salt solution was analyzed and the fibroin's secondary structures were determined via ¹³C solution nmr. Fourier transform ir, solid state ¹³C-nmr, x-ray diffraction, differential scanning calorimetry, scanning electron microscopy (SEM), and polarizing microscopy were used to characterize regenerated films and fibers. A compositional phase diagram of fibroin in the salt solution was constructed. Viscosity data indicate that there is aggregation of fibroin chains within the salt solution. The extremely high value of intrinsic viscosity of 8.7 dL/g at 298 K may be due to aggregation. Aggregation may be caused by the complexing of calcium ions with the fibroin chains at their amide linkages. The energy required for viscous flow for the fibroin solution (ΔH_vis = 9.03 kcal/mol) is greater than that of the solvent (ΔH_vis = 7.01 kcal/mol). Chain entanglements may be hindering the free motion of chains thus increasing the energy required for the viscous flow. ¹³C-nmr shows that fibroin chains exist in two independent conformational environments. While most of the molecule is in a random coil conformation, there is evidence of some order within the chains of fibroin. In as-cast regenerated films, the fibroin chains are in a random coil/α-helix conformation with some β-sheet content. Crystallinity induced by immersion of thin films in methanol is evidenced via x-ray diffraction, which shows lattice spacings at 4.042 Å. Thin films have a fibrillar morphology that is clearly shown under the SEM and the polarizing microscope. Fibers were hand pulled from the concentrated fibroin-salt solutions and coagulated with acetone and methanol. A microscopic analysis was done using the polarizer. © 1997 John Wiley & Sons, Inc. Biopoly 42: 61–74, 1997     

Sexual dimorphism in neuronal projections from the antennae of silk moths (Bombyx mori,Antheraea polyphemus) and the gypsy moth (Lymantria dispar)

Summary The antennal lobe of both sexes of the silk moth Bombyx mori contains 55–60 ventrally located antennal glomeruli; in addition, that of the male contains a dorsal macroglomerular complex (MGC). A group of identifiable glomeruli consisting of two lateral large glomeruli (LLG) and four medial small glomeruli (MSG) is present in both sexes, but the LLG are greatly enlarged in the female. A MGC is also present in the male gypsy moth Lymantria dispar and male giant silk moth Antheraea polyphemus. The MGC in all of these species is organized into 3–4 distinct levels of glomeruli. Antennal sensory fibers were stained by cobalt backfills in B. mori, A. polyphemus, and L. dispar. Most fibers stained from cut long hairs (sensilla trichodea) projected to MGC in males and LLG in both sexes of B. mori. The distribution of fibers in the MGC of B. mori was topographically biased in that a majority of fibers from anterior branches projected medially in MGC while most fibers from posterior branches projected laterally or anteriorly. Terminal arborizations of single fibers were each restricted to a single glomerular level of the MGC. Fibers projecting to the posterior antennal center were frequently stained in cut-hair and control preparations, apparently by uptake of cobalt through intact sensilla on flagellar branches.     

Effect of 1-methyl-3-octyleimmidazolium chloride on the stability and activity of lysozyme: a spectroscopic and molecular dynamics studies

Herein, the binding of 1-methyl-3-octylimidazolium chloride [OMIM][Cl] ionic liquid with hen egg white lysozyme (HEWL) has been studied using fluorescence, time resolved fluorescence, UV–visible and circular dichroism (CD) spectroscopy, in combination with computational study. The fluorescence results revealed that [OMIM][Cl] quenches the fluorophore of HEWL through static quenching mechanism. The calculated thermodynamic parameters show that [OMIM][Cl] bind with HEWL through hydrophobic interactions. In addition, the negative value of Gibbs energy change (?G) indicates that the binding process was spontaneous. Furthermore, UV–vis and CD results indicate that [OMIM][Cl] induce the conformational change in HEWL and increase its enzymatic activity. Additionally, molecular docking results showed that [OMIM][Cl] binds at the active site of HEWL where both the fluorophore residues (Trp108 and Trp62) and the catalytic residues (Glu35 and Asp52) reside. Molecular dynamic simulation results show the reduction of intra-molecular hydrogen bond of HEWL when it binds with [OMIM][Cl].     

Modifications of stability and function of lysozyme

Approaches to improving the functionality of lysozyme are presented. Lysozyme was variously modified and the stabilities of the derivatives were determined by thermal denaturation experiments. Contributions of salt bridge(s), hydrophobic interactions(s), and cross-linkage(s) were evaluated. The stabilities against proteolysis were also considered. For the latter stability, it might be important to depress the rate of unfolding, i.e., to stabilize the native conformation. As a rule, salt bridges and hydrophobic interactions stabilize the native conformation and cross-linkages destabilize the denatured conformation. However, cross-linkages are apt to introduce strains in the native conformation and only suitable lengths of cross-linkages can stabilize the protein. The stabilization was shown to be generally effective in improving the functionality of proteins. Catalytic groups in lysozyme (Glu-35 and Asp-52) were variously modified and finally converted to the respective amides. The participation of these groups in the catalytic function was confirmed. The specificity of lysozyme was modified. Asp-101, which lies on the top of the active site cleft of lysozyme, was variously modified and the effects on the hydrolysis patterns of a hexamer of N-acetylglucosamine were analyzed. Some approaches to endowing lysozyme with altered functions are also presented. In order to give higher esterase activity to lysozyme, the complementarity of enzyme and substrate was investigated by modifying substrate and the active site cleft of lysozyme. An attempt was made to convert lysozyme into a transaminase by introducing pyridoxamine to the active site cleft of lysozyme. Finally, we have started to apply genetic engineering to this kind of investigation and would like to see how far we can go with protein engineering to improve the nature of proteins.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Raman study of poly(alanine-glycine)-based peptides containing tyrosine, valine, and serine as model for the semicrystalline domains of Bombyx mori silk fibroin

For a deeper insight into the structure of Bombyx mori silk fibroin, some model peptides containing tyrosine (Y), valine (V), and serine (S) in the basic (AG)n sequence were synthesized by the solid-phase method and analyzed by Raman spectroscopy in order to clarify their conformation and to evaluate the formation and/or disruption of the ordered structure typical of B. mori silk fibroin upon incorporation of Y, V, and S residues into the basic (AG)n sequence. The Raman results indicated that the silk I structure remains stable only when the Y residue is positioned near the chain terminus; otherwise, a silk I --> silk II conformational transition occurs. The peptides AGVGAGYGAGVGAGYGAGVGAGYG(AG)3 and (AG)3YG(AG)2VGYG(AG)3YG(AG)3 treated with LiBr revealed a prevalent silk II conformation; moreover, the former contained a higher amount of random coil than the latter. This result was explained in relation to the different degrees of interruption of the (AG)n sequence. The Raman analysis of the AGSGAG-containing samples confirmed that the AGSGAG hexapeptide is a good model for the silk II crystalline domain. As the number of AGSGAG repeating units decreased, the random coil content increased. The study of the Y domain (I850/I830 intensity ratio) allowed us to hypothesize that in the packing characteristic of Silk I and Silk II conformations the Y residues experience different environments and hydrogen-bonding arrangements; the packing typical of silk I structure traps the tyrosyl side chains in environments more unfavorable to phenoxyl hydrogen-bonding interactions.     

Determination of the complete cDNA sequence, construction of expression systems, and elucidation of fibrinolytic activity for Tapes japonica lysozyme

The lysozyme of the marine bivalve, Tapes japonica (13.8 kDa), belongs to the invertebrate lysozyme family and displays both chitinase and isopeptidase activities. We determined the complete cDNA sequence and constructed effective expression systems for this enzyme using Escherichia coli (BL21) and Pichia pastoris. The native and recombinant proteins indicated lysozyme activity and isopeptidase activity, including the proteolysis of d-dimer, a plasminolytic product of stabilized polymeric fibrin. These results will be utilized for the structural and functional study of invertebrate lysozymes, and for the development of applications for thrombosis therapies.     

Solvent effects on the structure,dynamics and activity of lysozyme

Kuntz and Kauzmann have argued that dehydrating a protein results in conformational changes. In contrast, Rupleyet al. have developed a hydration model which involves no significant change in conformation; the onset of enzyme activity in hen egg-white lysozyme at hydration values of about 0.2 g water/g protein they ascribe rather to a solvation effect. Using a direct difference infra-red technique we can follow specific hydration events as water is added to a dry protein. Conformational studies of lysozyme using laser Raman spectroscopy indicate changes in conformation with hydration that are complete just before measurable activity is found. Parallel nuclear magnetic resonance measurements of exchangeability of the main chain amide hydrogens, as a function of hydration from near dryness, suggest a hydration-related increase in conformational flexibility which occurs before-and is probably necessary for-the Raman-detected conformational changes. Very recent inelastic neutron scattering measurements provides direct evidence of a flexibility change induced by hydration, which is apparently necessary before the enzyme can achieve adequate flexibility for it to begin to function.     

Effects of water activity and aqueous solvent ordering on thermal stability of lysozyme, α-chymotrypsinogen A, and alcohol dehydrogenase

Effects of water activity (a_W) and solvent ordering were separately analyzed on the thermal unfolding of lysozyme and α-chymotrypsinogen A, and also on the thermal deactivation of yeast alcohol dehydrogenase (YADH) in aqueous solutions with various additives. With the coexistence of additives, water activity was the determinant of the extent of the change in the thermal stability of proteins while solvent ordering was the determinant of the direction of the change. The parameter α, determined from the activity coefficient of water, representing the deviation of a_W from that of the ideal solution, was useful as a quantitative index of the solvent ordering showing good correlations with the unfolding temperature and enthalpy of lysozyme and α-chymotrypsinogen A and also with the thermal deactivation rate constant of YADH at a constant a_W. Solvent ordering seemed to affect the thermal stability of proteins mainly through its effect on the intramolecular hydrophobic interaction among amino acid residues in a protein molecule but the contribution of the electrostatic interaction including hydrogen bonding through the change in permittivity of solution was also suggested.     

家蚕拟抗微生物肽Gloverins基因（Bmglv）的原核表达及抗菌活性鉴定

家蚕Bombyx mori基因组全测序于2004年完成后,由家蚕基因组注释发现了35条拟抗微生物肽基因序列。本研究选取其中的5条Gloverins同系物（isoforms）基因(BmglvB,BmglvA2 ,BmglvA3,BmglvA5和BmglvA6 ),克隆至pET-21d载体,转化Escherichia coli Rosetta^TM（DE3）宿主菌进行表达;克隆表达的5个家蚕Gloverins同系物基因大部分以可溶形式表达,经Ni-NTA亲和层析纯化和Sephadex G-10脱盐处理后,采用平板孔穴抑菌法测定其抗菌活性。结果表明：注释发现的5条家蚕拟抗微生物肽Gloverins同系物基因,具有与在其他昆虫中已鉴定报道的Gloverin相似的抗革兰氏阴性细菌的功能,实验确认了它们就是家蚕Gloverins抗微生物肽基因。     

Enhanced antimicrobial activity of a peptide derived from human lysozyme by arylation of its tryptophan residues

Antimicrobial peptides are valuable agents to fight antibiotic resistance. These amphipatic species display positively charged and hydrophobic amino acids. Here, we enhance the local hydrophobicity of a model peptide derived from human lysozyme (107RKWVWWRNR115) by arylation of its tryptophan (Trp) residues, which renders a positive effect on Staphylococcus aureus and Staphylococcus epidermidis growth inhibition. This site‐selective modification was accessed by solid‐phase peptide synthesis using the non‐proteinogenic amino acid 2‐aryltryptophan, generated by direct C‐H activation from protected Trp. The modification brought about a relevant increase in growth inhibition: S. aureus was fully inhibited by arylation of Trp 112 and by only 10% by arylation of Trp 109 or 111, respect to the non‐arylated peptide. On the other hand, S. epidermidis was fully inhibited by the three arylated peptides and the parent peptide. The minimum inhibitory concentration was significantly reduced for S. aureus depending on the arylation site. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.