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On the Path to Optimal Alchemistry 通往最佳炼金术之路
IF 3 4区 生物学 Q2 Chemistry Pub Date : 2023-08-31 DOI: 10.1007/s10930-023-10137-1
Magnus Lundborg, Jack Lidmar, Berk Hess

Alchemical free energy calculations have become a standard and widely used tool, in particular for calculating and comparing binding affinities of drugs. Although methods to compute such free energies have improved significantly over the last decades, the choice of path between the end states of interest is usually still the same as two decades ago. We will show that there is a fundamentally arbitrary, implicit choice of parametrization of this path. To address this, the notion of the length of a path or a metric is required. A metric recently introduced in the context of the accelerated weight histogram method also proves to be very useful here. We demonstrate that this metric can not only improve the efficiency of sampling along a given path, but that it can also be used to improve the actual choice of path. For a set of relevant use cases, the combination of these improvements can increase the efficiency of alchemical free energy calculations by up to a factor 16.

炼金术自由能的计算已成为一种标准和广泛使用的工具,特别是用于计算和比较药物的结合亲和力。虽然计算自由能的方法在过去的几十年里有了很大的改进,但在最终感兴趣的状态之间的路径选择通常仍然和20年前一样。我们将证明,这条路径的参数化基本上是任意的,隐式的选择。为了解决这个问题,需要使用路径长度或度量的概念。最近在加速权重直方图方法中引入的一个度量在这里也被证明是非常有用的。我们证明了该度量不仅可以提高沿给定路径的采样效率,而且还可以用于改进实际路径的选择。对于一组相关的用例,这些改进的组合可以将炼金术自由能计算的效率提高16倍。
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引用次数: 0
Staphylococcus aureus Bacteriophage 52 Endolysin Exhibits Anti-Biofilm and Broad Antibacterial Activity Against Gram-Positive Bacteria 金黄色葡萄球菌噬菌体52内溶素对革兰氏阳性菌具有抗生物膜和广泛的抗菌活性
IF 3 4区 生物学 Q2 Chemistry Pub Date : 2023-08-27 DOI: 10.1007/s10930-023-10145-1
Mujib Abdulkadir Abdurahman, İnci Durukan, Tuba Dinçer, Serap Pektaş, Ersin Karataş, Ali Osman Kiliç

Bacteriophage endolysins have been shown to hold great promise as new antibacterial agents for animal and human health in food preservation. In the present study, endolysin from Staphylococcus aureus subsp. aureus ATCC 27692-B1 bacteriophage 52 (LysSA52) was cloned, expressed, and characterized for its antimicrobial properties. Following DNA extraction from bacteriophage 52, a 1446-bp DNA fragment containing the endolysin gene (lysSA52) was obtained by PCR amplification and cloned into pET SUMO expression vector. The positive clone was validated by sequencing and open-reading frame analysis. The LysSA52 sequence shared high homology with staphylococcal phage endolysins of the SA12, SA13, and DSW2 phages and others. The cloned lysSA52 gene encoding 481 amino acids endolysin was expressed in Escherichia coli BL21 with a calculated molecular mass of 66 kDa (LysSA52). This recombinant endolysin LysSA52 exhibited lytic activity against 8 of 10 Gram-positive bacteria via agar spot-on lawn antimicrobial assay, including methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Streptococcus pneumonia, Streptococcus pyogenes, Enterococcus faecium, Enterococcus faecalis, and Bacillus atrophaeus. In addition, the 0.50 mg/mL, LysSA52 endolysins reduced about 60% of the biofilms of S. aureus and S. epidermidis established on a microtiter plate in 12 h treatment. The data from this study indicate that LysSA52 endolysin could be used as an antibacterial protein to prevent and treat infections caused by staphylococci and several other Gram-positive pathogenic bacteria irrespective of their antibiotic resistance.

噬菌体内溶素作为一种新型抗菌剂,在食品保鲜方面具有广阔的应用前景。在本研究中,来自金黄色葡萄球菌亚群的内溶素。对金黄色葡萄球菌ATCC 27692-B1噬菌体52 (LysSA52)进行了克隆、表达和抑菌鉴定。从噬菌体52中提取DNA,通过PCR扩增得到含有内溶素基因(lysSA52)的1446 bp DNA片段,并将其克隆到pET SUMO表达载体中。阳性克隆经测序和开放阅读框分析证实。LysSA52序列与葡萄球菌噬菌体SA12、SA13和DSW2等噬菌体的内溶酶具有高度同源性。克隆的lysSA52基因编码481个氨基酸的内溶素,在大肠杆菌BL21中表达,计算分子量为66 kDa (lysSA52)。重组内溶素LysSA52对10种革兰氏阳性细菌中的8种表现出裂解活性,包括耐甲氧西林金黄色葡萄球菌、表皮葡萄球菌、溶血葡萄球菌、肺炎链球菌、化脓性链球菌、粪肠球菌、粪肠球菌和萎缩芽孢杆菌。此外,0.50 mg/mL的LysSA52内溶素处理12 h后,在微滴板上建立的金黄色葡萄球菌和表皮葡萄球菌生物膜减少了约60%。本研究的数据表明,无论葡萄球菌和其他几种革兰氏阳性致病菌的耐药性如何,LysSA52内溶素都可以作为一种抗菌蛋白来预防和治疗其引起的感染。
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引用次数: 0
Molecular Modeling and Optimization of Type II E.coli l-Asparginase Activity by in silico Design and in vitro Site-directed Mutagenesis 通过计算机设计和体外定点突变优化II型大肠杆菌l-天冬氨酸酶活性的分子模型。
IF 3 4区 生物学 Q2 Chemistry Pub Date : 2023-08-27 DOI: 10.1007/s10930-023-10149-x
Mahdieh Mahboobi, Ali-Hatef Salmanian, Hamid Sedighian, Bijan Bambai

Introduction

L-asparaginase (also known as L-ASNase) is a crucial therapeutic enzyme that is widely used in treatment of ALL (acute lymphoblastic leukemia) as a chemotherapeutic drug. Besides, this enzyme is used in the food industry as a food processing reagent to reduce the content of acrylamide in addition to the clinical industry. The improvement of activity and kinetic parameters of the L-ASNase enzyme may lead to higher efficiency resulting in practical achievement. In order to achieve this goal, we chosen glycine residue in position 88 as a potential mutation with advantageous outcomes.

Method

In this study, firstly to find the appropriate mutation on glycine 88, various in silico analyses, such as MD simulation and molecular docking, were carried out. Then, the rational design was adopted as the best strategy for molecular modifications of the enzyme to improve its enzymatic properties.

Result

Our in silico findings show that the four mutations G88Q, G88L, G88K, and G88A may be able to increase L-ASNase’s asparaginase activity. The catalytic efficiency of each enzyme (kcat/Km) is the most important feature for comparing the catalytic activity of the mutants with the wild type form. The laboratory experiments showed that the kcat/Km for the G88Q mutant is 36.32% higher than the Escherichia coli K12 ASNase II (wild type), which suggests that L-ASNase activity is improved at lower concentration of L-ASN. Kinetic characterization of the mutants L-ASNase activity confirmed the high turnover rate (kcat) with ASN as substrate relative to the wild type enzyme.

Conclusion

In silico analyses and laboratory experiments demonstrated that the G88Q mutation rather than other mutation (G88L, G88K, and G88A) could improve the kinetics of L-ASNase.

简介:L-天冬酰胺酶(也称为L-ASNase)是一种重要的治疗酶,作为一种化疗药物,广泛用于治疗急性淋巴细胞白血病。此外,这种酶在食品工业中被用作食品加工试剂,以降低丙烯酰胺的含量。L-ASNase酶的活性和动力学参数的提高可能导致更高的效率,从而产生实际的成就。为了实现这一目标,我们选择88位的甘氨酸残基作为具有有利结果的潜在突变。方法:本研究首先对甘氨酸88进行了分子动力学模拟和分子对接等多种计算机分析,以寻找合适的突变位点。然后,采用合理的设计作为对酶进行分子修饰的最佳策略,以改善其酶性质。结果:我们的计算机研究结果表明,G88Q、G88L、G88K和G88A四个突变可能能够提高L-ASNase的天冬酰胺酶活性。每种酶的催化效率(kcat/Km)是比较突变体与野生型催化活性的最重要特征。实验室实验表明,G88Q突变体的kcat/Km比大肠杆菌K12 ASNase II(野生型)高36.32%,这表明低浓度的L-ASN可以提高L-ASNase的活性。突变体L-ASNase活性的动力学表征证实了与野生型酶相比,以ASN为底物的高周转率(kcat)。结论:计算机分析和实验室实验表明,G88Q突变而不是其他突变(G88L、G88K和G88A)可以改善L-ASNase的动力学。
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引用次数: 1
The Effect of Ultrasonication on the Fibrillar/ Oligomeric Structures of Aβ1−42 at Different Concentrations 超声对不同浓度Aβ1−42纤维/寡聚体结构的影响
IF 3 4区 生物学 Q2 Chemistry Pub Date : 2023-08-27 DOI: 10.1007/s10930-023-10138-0
Nassim Faridi, Maryam Sanjari-Pour, Ping Wang, S. Zahra Bathaie

The number of disease states linked the aberrant regular protein conformations to oligomers and amyloid fibrils. Amyloid beta 1–42 (Aβ1−42) peptide is very hydrophobic and quickly forms the β-rich structure and fibrillar protein aggregates in some solutions and buffer conditions. Ultrasonication pulses can disrupt amyloid fibrils to smaller fragments and produce Aβ1−42 peptides of different sizes and oligomers. Herein, we investigated the effects of buffer and ultrasonication on Aβ1−42 structure at low and high concentrations. After ultrasonication, the Western blot results showed that Aβ1−42 fibrils were disaggregated into different sizes. The transmission electron microscopy results indicated Aβ1−42 at low concentration (25 µM) in Ham’s/F12 phenol red-free culture medium formed short-size fragments and oligomers. In comparison, Aβ1−42 at higher concentration (100 µM) formed fibrils that break down into smaller fragments after ultrasonication. However, after regrowth, it formed mature fibrils again. Cell viability assay indicated that Aβ1−42 oligomers formed at a low concentration (25 µM) were more toxic to PC12 cells than other forms. In conclusion, by applying ultrasonication pulses and controlling peptide concentration and buffer condition, we can rich Aβ1−42 aggregates with a particular size and molecular structure.

疾病状态的数量将异常的规则蛋白质构象与低聚物和淀粉样原纤维联系起来。淀粉样蛋白β 1-42 (a - β1−42)肽具有很强的疏水性,在某些溶液和缓冲条件下迅速形成富β结构和纤维蛋白聚集体。超声脉冲可以将淀粉样蛋白原纤维破坏成更小的片段,并产生不同大小的a - β1 - 42肽和低聚物。在此,我们研究了缓冲液和超声波对低浓度和高浓度Aβ1−42结构的影响。超声处理后,Western blot结果显示a - β1−42原纤维被分解成不同大小。透射电镜结果显示,低浓度(25µM)的Aβ1−42在Ham’s/F12无酚红培养基中形成短片段和低聚物。相比之下,a - β1−42在较高浓度(100µM)下形成原纤维,超声作用后成更小的碎片。然而,再生后,它又形成了成熟的原纤维。细胞活力测定表明,低浓度(25µM)形成的a - β1−42寡聚物对PC12细胞的毒性大于其他形式。综上所述,通过施加超声脉冲,控制肽浓度和缓冲条件,可以丰富具有特定大小和分子结构的a β1−42聚集体。
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引用次数: 0
Expression, Purification, and Crystallization of the Vγ9Vδ2 T-cell Receptor Recognizing Protein/Peptide Antigens Vγ9Vδ2 T细胞受体识别蛋白/肽抗原的表达、纯化和结晶。
IF 3 4区 生物学 Q2 Chemistry Pub Date : 2023-08-25 DOI: 10.1007/s10930-023-10151-3
Chaofei Cheng, Zhendong Zhao, Guangzhi Liu

γδ T cells, especially Vγ9Vδ2 T cells, play an important role in mycobacterial infection. We have identified some Vγ9Vδ2 T cells that recognize protein/peptide antigens derived from mycobacteria, which may induce protective immune responses to mycobacterial infection. To clarify the structural basis of the molecular recognition mechanism, we tried many methods to express the Vγ9Vδ2 T-cell receptor (TCR). The Vγ9Vδ2 TCR was not expressed well in a prokaryotic expression system or a baculovirus expression system, even after extensive optimization. In a mammalian cell expression system, the Vγ9Vδ2 TCR was expressed in the form of a soluble heterodimer, which was suitable for crystal screening. Reduced-temperature cultivation (cold shock) increased the yield of the recombinant TCR. The recombinant purified TCR was used for crystal trials, and crystals that could be used for X-ray diffraction were obtained. Although we have not yet determined the crystal structure of the Vγ9Vδ2 TCR, we have established a procedure for Vγ9Vδ2 TCR expression and purification, which is useful for basic research and potentially for clinical application.

γδT细胞,特别是Vγ9Vδ2 T细胞,在分枝杆菌感染中起着重要作用。我们已经鉴定了一些Vγ9Vδ2 T细胞,它们识别来自分枝杆菌的蛋白质/肽抗原,可能诱导对分枝杆菌感染的保护性免疫反应。为了阐明分子识别机制的结构基础,我们尝试了多种方法来表达Vγ9Vδ2 T细胞受体(TCR)。即使经过广泛的优化,Vγ9Vδ2 TCR在原核表达系统或杆状病毒表达系统中也不能很好地表达。在哺乳动物细胞表达系统中,Vγ9Vδ2 TCR以可溶性异二聚体的形式表达,适合于晶体筛选。低温培养(冷激)提高了重组TCR的产量。将重组纯化的TCR用于晶体试验,并获得可用于X射线衍射的晶体。虽然我们还没有确定Vγ9Vδ2 TCR的晶体结构,但我们已经建立了Vγ9Vδ2 TCR的表达和纯化程序,这对基础研究和临床应用都很有用。
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引用次数: 0
gmXtal: Cooking Crystals with GROMACS gmXtal:用 GROMACS 烹饪晶体。
IF 1.9 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2023-08-25 DOI: 10.1007/s10930-023-10141-5
Pavel Buslaev, Gerrit Groenhof

Molecular dynamics (MD) simulations are routinely performed of biomolecules in solution, because this is their native environment. However, the structures used in such simulations are often obtained with X-ray crystallography, which provides the atomic coordinates of the biomolecule in a crystal environment. With the advent of free electron lasers and time-resolved techniques, X-ray crystallography can now also access metastable states that are intermediates in a biochemical process. Such experiments provide additional data, which can be used, for example, to optimize MD force fields. Doing so requires that the simulation of the biomolecule is also performed in the crystal environment. However, in contrast to simulations of biomolecules in solution, setting up a crystal is challenging. In particular, because not all solvent molecules are resolved in X-ray crystallography, adding a suitable number of solvent molecules, such that the properties of the crystallographic unit cell are preserved in the simulation, can be difficult and typically is a trial-and-error based procedure requiring manual interventions. Such interventions preclude high throughput applications. To overcome this bottleneck, we introduce gmXtal, a tool for setting up crystal simulations for MD simulations with GROMACS. With the information from the protein data bank (rcsb.org) gmXtal automatically (i) builds the crystallographic unit cell; (ii) sets the protonation of titratable residues; (iii) builds missing residues that were not resolved experimentally; and (iv) adds an appropriate number of solvent molecules to the system. gmXtal is available as a standalone tool https://gitlab.com/pbuslaev/gmxtal.

Graphical Abstract

分子动力学(MD)模拟通常是对溶液中的生物分子进行模拟,因为这是生物分子的原生环境。然而,此类模拟中使用的结构通常是通过 X 射线晶体学获得的,X 射线晶体学提供了晶体环境中生物大分子的原子坐标。随着自由电子激光器和时间分辨技术的出现,X 射线晶体学现在还能获取作为生化过程中间产物的蜕变态。这些实验提供了额外的数据,可用于优化 MD 力场等。这样做需要在晶体环境中对生物分子进行模拟。然而,与溶液中的生物分子模拟相比,晶体的建立具有挑战性。特别是,由于并非所有的溶剂分子都能在 X 射线晶体学中解析,因此要添加适当数量的溶剂分子,从而在模拟中保留晶体学单元格的特性,可能会非常困难,而且通常是一个需要人工干预的试错过程。这种干预排除了高通量应用。为了克服这一瓶颈,我们推出了 gmXtal,这是一种利用 GROMACS 进行 MD 模拟的晶体模拟设置工具。利用蛋白质数据库(rcsb.org)中的信息,gmXtal 可自动:(i) 建立晶体学单元格;(ii) 设置可滴定残基的质子化;(iii) 建立实验中未解析的缺失残基;(iv) 为系统添加适当数量的溶剂分子。
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引用次数: 0
Computer-Aided Multi-Epitope Based Vaccine Design Against Monkeypox Virus Surface Protein A30L: An Immunoinformatics Approach 猴痘病毒表面蛋白A30L的计算机辅助多表位疫苗设计:一种免疫信息学方法。
IF 3 4区 生物学 Q2 Chemistry Pub Date : 2023-08-24 DOI: 10.1007/s10930-023-10150-4
S. V. Ramprasadh, Santhosh Rajakumar, S. Srinivasan, D. Susha, Sameer Sharma, Rajan Chourasiya

Monkeypox, a viral zoonotic disease resembling smallpox, has emerged as a significant national epidemic primarily in Africa. Nevertheless, the recent global dissemination of this pathogen has engendered apprehension regarding its capacity to metamorphose into a sweeping pandemic. To effectively combat this menace, a multi-epitope vaccine has been meticulously engineered with the specific aim of targeting the cell envelope protein of Monkeypox virus (MPXV), thereby stimulating a potent immunological response while mitigating untoward effects. This new vaccine uses T-cell and B-cell epitopes from a highly antigenic, non-allergenic, non-toxic, conserved, and non-homologous A30L protein to provide protection against the virus. In order to ascertain the vaccine design with the utmost efficacy, protein–protein docking methodologies were employed to anticipate the intricate interactions with Toll-like receptors (TLR) 2, 3, 4, 6, and 8. This meticulous approach led the researchers to discern an optimal vaccine architecture, bolstered by affirmative prognostications derived from both molecular dynamics (MD) simulations and immune simulations. The current research findings indicate that the peptides ATHAAFEYSK, FFIVVATAAV, and MNSLSIFFV exhibited antigenic properties and were determined to be non-allergenic and non-toxic. Through the utilization of codon optimization and in-silico cloning techniques, our investigation revealed that the prospective vaccine exhibited a remarkable expression level within Escherichia coli. Moreover, upon conducting immune simulations, we observed the induction of a robust immune response characterized by elevated levels of both B-cell and T-cell mediated immunity. Moreover, as the initial prediction with in-silico techniques has yielded promising results these epitope-based vaccines can be recommended to in vitro and in silico studies to validate their immunogenic properties.

猴痘是一种类似天花的病毒性人畜共患疾病,已成为主要在非洲流行的一种重要的全国性流行病。尽管如此,这种病原体最近在全球的传播引起了人们对其转变为全面流行病的能力的担忧。为了有效对抗这一威胁,我们精心设计了一种多表位疫苗,其特定目的是靶向猴痘病毒(MPXV)的细胞包膜蛋白,从而刺激强大的免疫反应,同时减轻不良反应。这种新疫苗使用来自高度抗原性、非致敏性、无毒、保守和非同源A30L蛋白的T细胞和B细胞表位来提供对病毒的保护。为了确定具有最大效力的疫苗设计,采用蛋白质-蛋白质对接方法来预测与Toll样受体(TLR)2、3、4、6和8的复杂相互作用。这种细致的方法使研究人员在分子动力学(MD)模拟和免疫模拟的肯定预测的支持下,发现了最佳的疫苗结构。目前的研究结果表明,肽ATHAAFEYSK、FFIVATAAV和MNSLSIFFV表现出抗原特性,并被确定为非致敏性和无毒性。通过利用密码子优化和计算机克隆技术,我们的研究表明,该疫苗在大肠杆菌中表现出显著的表达水平。此外,在进行免疫模拟时,我们观察到了以B细胞和T细胞介导的免疫水平升高为特征的强大免疫反应的诱导。此外,由于计算机技术的初步预测已经产生了有希望的结果,这些基于表位的疫苗可以推荐用于体外和计算机研究,以验证其免疫原性。
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引用次数: 1
Synthesis and Characterisation of Chickpea Peptides-Zinc Chelates Having ACE2 Inhibitory Activity 具有ACE2抑制活性的鹰嘴豆肽锌螯合物的合成与表征
IF 3 4区 生物学 Q2 Chemistry Pub Date : 2023-08-23 DOI: 10.1007/s10930-023-10133-5
Nurkhodja Mukhamedov, Akmal Asrorov, Ansor Yashinov, Muzaffar Kayumov, Ahmidin Wali, Sharafitdin Mirzaakhmedov, Haji Akber Aisa, Abulimiti Yili

Tryptic hydrolysates of protein fractions obtained by the Osborne method from chickpea (Cicer arietinum L.) seeds interacted with zinc ions and the results of chelation were monitored by the Energy Dispersive X-Ray (EDX) technique. The glutelin hydrolysate (GluHyd) reacted with zinc ions and depicted a relatively higher zinc content. For this reason, the zinc complex of the glutelin hydrolysate (GluHyd-Zn) was studied deeper, and 11 peptides were identified in its more zinc-containing second fraction obtained after gel filtration. The peptide HKERVQLHIIPTAVGK showed a relatively higher chelating capacity (57.86 ± 2.14%). According to the result of the ICP-OS analysis, 1 mg peptide could chelate 381.61 ± 133.39 µg zinc, and the molar ratio of peptide-zinc was about 1:4. Spectral methods proved that side chain and C-termini carboxyl groups of the peptide mostly were involved in chelation and N atoms of amino side chains, imidazole group of histidine, and N-termini at some extents were occupied by the metal ions. Modeling of zinc-peptide interaction was done using Molecular Operating Environment (MOE) software. The results of the docking correlate with the experimental data.

ACE2 inhibitory effect of HKERVQLHIIPTAVGK-Zn complex (IC50 = 1.5 mg/mL) was better than that of HKERVQLHIIPTAVGK (IC50 = 2.2 mg/mL).

用能量色散x射线(EDX)技术对鹰嘴豆(Cicer arietinum L.)种子奥斯本法获得的胰蛋白酶水解产物与锌离子的相互作用及螯合结果进行了监测。谷蛋白水解物(GluHyd)与锌离子反应,锌含量相对较高。因此,我们对谷蛋白水解产物的锌络合物(GluHyd-Zn)进行了更深入的研究,并在凝胶过滤后获得的含锌量更高的第二部分中鉴定了11个肽段。肽HKERVQLHIIPTAVGK具有较高的螯合能力(57.86±2.14%)。ICP-OS分析结果显示,1 mg肽能螯合381.61±133.39µg锌,肽与锌的摩尔比约为1:4。光谱方法证明,肽的侧链和c端羧基主要参与螯合,氨基侧链的N原子、组氨酸的咪唑基团和N端部分被金属离子占据。利用分子操作环境(MOE)软件对锌-肽相互作用进行建模。对接结果与实验数据相吻合。HKERVQLHIIPTAVGK- zn复合物对ACE2的抑制作用(IC50 = 1.5 mg/mL)优于HKERVQLHIIPTAVGK复合物(IC50 = 2.2 mg/mL)。
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引用次数: 0
Domain Shuffling and Site-Saturation Mutagenesis for the Enhanced Inhibitory Potential of Amaranthaceae α-Amylase Inhibitors 区域洗牌和位点饱和诱变增强苋科α-淀粉酶抑制剂的抑制潜力
IF 3 4区 生物学 Q2 Chemistry Pub Date : 2023-08-19 DOI: 10.1007/s10930-023-10148-y
Ashwini S. Rane, Vineetkumar S. Nair, Rakesh S. Joshi, Ashok P. Giri

Amaranthaceae α-amylase inhibitors (AAIs) are knottin-type proteins with selective inhibitory potential against coleopteran α-amylases. Their small size and remarkable stability make them exciting molecules for protein engineering to achieve superior selectivity and efficacy. In this report, we have designed a set of AAI pro- and mature peptides chimeras. Based on in silico analysis, stable AAI chimeras having a stronger affinity with target amylases were selected for characterization. In vitro studies validated that chimera of the propeptide from Chenopodium quinoa α-AI and mature peptide from Beta vulgaris α-AI possess 3, 7.6, and 4.26 fold higher inhibition potential than parental counterparts. Importantly, recombinant AAI chimera retained specificity towards target coleopteran α-amylases. In addition, to improve the inhibitory potential of AAI, we performed in silico site-saturation mutagenesis. Computational analysis followed by experimental data showed that substituting Asparagine at the 6th position with Methionine had a remarkable increase in the specific inhibition potential of Amaranthus hypochondriacus α-AI. These results provide structural–functional insights into the vitality of AAI propeptide and a potential hotspot for mutagenesis to enhance the AAI activity. Our investigation will be a toolkit for AAI’s optimization and functional differentiation for future biotechnological applications.

苋科α-淀粉酶抑制剂(AAIs)是一种对鞘翅目α-淀粉酶具有选择性抑制潜力的结蛋白型蛋白。它们的小尺寸和显著的稳定性使它们成为蛋白质工程中令人兴奋的分子,以实现优越的选择性和功效。在本报告中,我们设计了一套AAI前肽和成熟肽嵌合体。基于硅分析,选择与目标淀粉酶亲和力较强的稳定AAI嵌合体进行表征。体外实验证实,藜麦前肽α-AI与甜菜成熟肽α-AI嵌合体的抑制潜能分别比亲本高3倍、7.6倍和4.26倍。重要的是,重组AAI嵌合体保留了对目标鞘翅目α-淀粉酶的特异性。此外,为了提高AAI的抑制潜能,我们进行了硅位点饱和诱变。计算分析和实验数据表明,用蛋氨酸取代第6位的天冬酰胺可显著提高苋α-AI的特异性抑制电位。这些结果为了解AAI前肽的活性提供了结构-功能方面的见解,并为增强AAI活性提供了潜在的诱变热点。我们的研究将成为未来生物技术应用中AAI优化和功能分化的工具包。
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引用次数: 0
Improved Antimicrobial Activity of Bovine Lactoferrin Peptide (LFcinB) Based on Rational Design 基于合理设计提高牛乳铁蛋白肽(LFcinB)的抗菌活性。
IF 3 4区 生物学 Q2 Chemistry Pub Date : 2023-08-11 DOI: 10.1007/s10930-023-10142-4
Xiaokun Hong, Xueqian Liu, Bingmei Su, Juan Lin

Bovine lactoferrin peptide (LFcinB), as an antimicrobial peptide, is expected to be an alternative of antibiotics owing to its broad-spectrum antimicrobial activity and specific mechanism. However, the weak antimicrobial activity, high hemolysis, and poor stability of LFcinB limited its applications in the field of biomedicine, food and agriculture. In order to improve the antimicrobial activity of LFcinB, five mutants were designed rationally, of which mutant LF4 (M10W/P16R/A24L) showed highest antimicrobial activity. The bioinformatics analysis indicated that the improved antimicrobial activity of LF4 was related to its increased cations, higher amphiphilicity and the extension of the β-sheet in the structure. These studies will highlight the important role of bioinformatic tools in designing ideal biopeptides and lay a foundation for further development of antimicrobial peptides.

牛乳铁蛋白肽(LFcinB)作为一种抗菌肽,由于其广谱抗菌活性和特异性机制,有望成为抗生素的替代品。然而,LFcinB的抗菌活性弱、溶血性高、稳定性差,限制了其在生物医学、食品和农业领域的应用。为了提高LFcinB的抗菌活性,合理设计了5个突变体,其中突变体LF4(M10W/P16R/A24L)的抗菌活性最高。生物信息学分析表明,LF4抗菌活性的提高与其阳离子的增加、更高的两亲性和结构中β-片的延伸有关。这些研究将突出生物信息学工具在设计理想的生物肽中的重要作用,并为抗菌肽的进一步开发奠定基础。
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引用次数: 0
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The Protein Journal
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