Protein Engineering, Design and Selection最新文献_第4页

Chimeric approach for narrowing a membrane-inserting region within human perforin 缩小人穿孔膜插入区域的嵌合方法

Protein Engineering, Design and Selection

Pub Date : 2017-02-01 DOI: 10.1093/protein/gzw069

Amy E. Neely, Kimberly A Mandigo, R. Robinson, T. Ness, M. H. Weiland

Perforin is a pore-forming, immune protein that functions to deliver an apoptotic cocktail of proteins into a target pathogen. Recent studies of the bacterial cholesterol-dependent cytolysins (CDCs) have provided a model for perforin's pore-forming mechanism. Both perforin and CDC family members share a conserved &bgr;-sheet flanked by two clusters of &agr;-helices. Within the CDCs, these helices refold into two transmembrane &bgr;-hairpins, TMH1 and TMH2. Based upon structural conservation and electron microscopy imaging, the analogous helices within perforin are predicted to also be membrane inserting; however, these regions are approximately twice the length of the CDC TMHs. To test the membrane-insertion potential of one of these regions, chimeras were created using a well-characterized CDC, perfringolysin-O (PFO), as the backbone of these constructs. PFO's TMH2 region was replaced with perforin's corresponding helical region. Although hemolytic activity was observed, the chimera was poorly soluble. A second chimera contained the same region truncated to match the length of the PFO TMH2 region. The truncated chimera demonstrated improved solubility, significant hemolytic activity and the ability to form pores characteristic of those created by PFO. These results provide the first evidence that perforin's helices function as TMHs and more importantly narrows the residues responsible for membrane insertion.

穿孔素是一种形成孔的免疫蛋白，其功能是将凋亡蛋白的混合物传递到目标病原体中。近年来对细菌胆固醇依赖性细胞溶素(CDCs)的研究为穿孔素的成孔机制提供了模型。perforin和CDC家族成员共享一个保守的&bgr;-薄片，两侧是两个&agr;-螺旋簇。在cdc中，这些螺旋重新折叠成两个跨膜发夹，TMH1和TMH2。基于结构守恒和电子显微镜成像，预测穿孔内的类似螺旋也为膜插入;然而，这些区域的长度大约是疾控中心tmh的两倍。为了测试其中一个区域的膜插入潜力，嵌合体是用一种表征良好的CDC，即perfringolysin-O (PFO)作为这些结构的主干来创建的。将PFO的TMH2区替换为穿孔素相应的螺旋区。虽然观察到溶血活性，但嵌合体难溶。第二个嵌合体包含相同的区域，截断以匹配PFO TMH2区域的长度。截断的嵌合体表现出更好的溶解度，显著的溶血活性和形成PFO所产生的孔的能力。这些结果提供了穿孔素螺旋作为TMHs功能的第一个证据，更重要的是缩小了负责膜插入的残基。

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引用次数: 0

Dual chemistry catalyzed by human acireductone dioxygenase 人酸还原酮双加氧酶催化的双化学反应

Protein Engineering, Design and Selection

Pub Date : 2017-01-05 DOI: 10.1093/protein/gzw078

A. Deshpande, T. Pochapsky, G. Petsko, D. Ringe

Acireductone dioxygenase (ARD) from the methionine salvage pathway of Klebsiella oxytoca is the only known naturally occurring metalloenzyme that catalyzes different reactions in vivo based solely on the identity of the divalent transition metal ion (Fe2+ or Ni2+) bound in the active site. The iron-containing isozyme catalyzes the cleavage of substrate 1,2-dihydroxy-3-keto-5-(thiomethyl)pent-1-ene (acireductone) by O2 to formate and the ketoacid precursor of methionine, whereas the nickel-containing isozyme uses the same substrates to catalyze an off-pathway shunt to form methylthiopropionate, carbon monoxide and formate. This dual chemistry was recently demonstrated in vitro by ARD from Mus musculus (MmARD), providing the first example of a mammalian ARD exhibiting metal-dependent catalysis. We now show that human ARD (HsARD) is also capable of metal-dependent dual chemistry. Recombinant HsARD was expressed and purified to obtain a homogeneous enzyme with a single transition metal ion bound. As with MmARD, the Fe2+-bound HsARD shows the highest activity and catalyzes on-pathway chemistry, whereas Ni2+, Co2+ or Mn2+ forms catalyze off-pathway chemistry. The thermal stability of the HsARD isozymes is a function of the metal ion identity, with Ni2+-bound HsARD being the most stable followed by Co2+ and Fe2+, and Mn2+-bound HsARD being the least stable. As with the bacterial ARD, solution NMR data suggest that HsARD isozymes can have significant structural differences depending upon the metal ion bound.

产氧克雷伯菌蛋氨酸回收途径中的酸性还原酮双加氧酶(ARD)是目前已知的唯一一种天然存在的金属酶，它能仅根据结合在活性位点的二价过渡金属离子(Fe2+或Ni2+)的身份在体内催化不同的反应。含铁同工酶通过O2催化底物1,2-二羟基-3-酮-5-(硫甲基)戊-1-烯(酸还原酮)裂解生成甲酸和蛋氨酸的酮酸前体，而含镍同工酶使用相同的底物催化非通路分流生成甲基硫丙酸、一氧化碳和甲酸。这种双重化学反应最近在体外由小家鼠的ARD (MmARD)证实，提供了哺乳动物ARD表现出金属依赖性催化的第一个例子。我们现在表明，人类ARD (HsARD)也具有依赖金属的双重化学能力。表达并纯化重组HsARD，获得具有单一过渡金属离子结合的均相酶。与MmARD一样，Fe2+结合的HsARD表现出最高的活性，并能催化通路上的化学反应，而Ni2+、Co2+或Mn2+则能催化通路外的化学反应。HsARD同工酶的热稳定性与金属离子特性有关，其中Ni2+结合的HsARD最稳定，其次是Co2+和Fe2+， Mn2+结合的HsARD最不稳定。与细菌ARD一样，溶液核磁共振数据表明，HsARD同工酶可以根据金属离子结合而具有显着的结构差异。

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引用次数: 10

On the effect of alkaline pH and cofactor availability in the conformational and oligomeric state of Escherichia coli glutamate decarboxylase 碱性pH和辅助因子可用性对大肠杆菌谷氨酸脱羧酶构象和寡聚状态的影响

Protein Engineering, Design and Selection

Pub Date : 2017-01-05 DOI: 10.1093/protein/gzw076

Fabio Giovannercole, C. Mérigoux, C. Zamparelli, D. Verzili, G. Grassini, Malcolm Buckle, P. Vachette, D. Biase

Escherichia coli glutamate decarboxylase (EcGad) is a homohexameric pyridoxal 5'-phosphate (PLP)-dependent enzyme. It is the structural component of the major acid resistance system that protects E. coli from strong acid stress (pH < 3), typically encountered in the mammalian gastrointestinal tract. In fact EcGad consumes one proton/catalytic cycle while yielding γ-aminobutyrate and carbon dioxide from the decarboxylation of l-glutamate. Two isoforms of Gad occur in E. coli (GadA and GadB) that are 99% identical in sequence. GadB is the most intensively investigated. Prompted by the observation that some transcriptomic and proteomic studies show EcGad to be expressed in conditions far from acidic, we investigated the structural organization of EcGadB in solution in the pH range 7.5-8.6. Small angle X-ray scattering, combined with size exclusion chromatography, and analytical ultracentrifugation analysis show that the compact and entangled EcGadB hexameric structure undergoes dissociation into dimers as pH alkalinizes. When PLP is not present, the dimeric species is the most abundant in solution, though evidence for the occurrence of a likely tetrameric species was also obtained. Trp fluorescence emission spectra as well as limited proteolysis studies suggest that PLP plays a key role in the acquisition of a folding necessary for the canonical catalytic activity.

大肠杆菌谷氨酸脱羧酶(EcGad)是一种同源六聚体吡哆醛5'-磷酸(PLP)依赖性酶。它是主要抗酸系统的结构组成部分，保护大肠杆菌免受强酸应激(pH < 3)，通常在哺乳动物胃肠道中遇到。事实上，EcGad消耗一个质子/催化循环，同时从l-谷氨酸脱羧产生γ-氨基丁酸盐和二氧化碳。大肠杆菌中存在两种Gad亚型(GadA和GadB)，它们的序列99%相同。GadB是研究最深入的。由于一些转录组学和蛋白质组学研究表明EcGadB在远离酸性的条件下表达，我们研究了EcGadB在pH范围为7.5-8.6的溶液中的结构组织。小角度x射线散射，结合粒径排除层析和分析性超离心分析表明，紧凑和纠缠的EcGadB六聚体结构随着pH碱化而解离成二聚体。当PLP不存在时，溶液中的二聚体物种是最丰富的，尽管也获得了可能发生四聚体物种的证据。色氨酸荧光发射光谱以及有限的蛋白质水解研究表明，PLP在获得典型催化活性所需的折叠中起着关键作用。

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引用次数: 4

Generation of camelid VHH bispecific constructs via in-cell intein-mediated protein trans-splicing 通过细胞内蛋白介导的反式剪接产生骆驼类VHH双特异性构建体

Protein Engineering, Design and Selection

Pub Date : 2017-01-01 DOI: 10.1093/protein/gzw057

Yukihiko Shibuya, N. Haga, R. Asano, H. Nakazawa, T. Hattori, D. Takeda, Aruto Sugiyama, R. Kurotani, I. Kumagai, M. Umetsu, K. Makabe

Production of various combinations of bispecific variable domain of heavy chain of heavy chain-only antibody (VHH) constructs to evaluate their therapeutic potential usually requires several gene-engineering steps. Here, we present an alternative method of creating bispecific VHH constructs in vivo through protein trans-splicing (PTS) reaction; this method may reduce the number of gene manipulation steps required. As a proof-of-concept, we constructed a bispecific antibody (bsAb) containing an anti-epidermal growth factor receptor VHH and anti-green fluorescent protein VHH, and we evaluated and confirmed its bispecificity. We also tested antibody labeling by fluorescent protein tagging using the PTS reaction. Compared with the conventional gene construction method, bsAb construction via PTS is a promising alternative approach for generating multiple bsAb combinations.

生产重链或仅重链抗体(VHH)结构体的各种双特异性可变结构域组合以评估其治疗潜力通常需要几个基因工程步骤。在这里，我们提出了一种通过蛋白质反式剪接(PTS)反应在体内创建双特异性VHH构建物的替代方法;这种方法可以减少所需的基因操作步骤的数量。作为概念验证，我们构建了含有抗表皮生长因子受体VHH和抗绿色荧光蛋白VHH的双特异性抗体(bsAb)，并对其双特异性进行了评估和确认。我们还使用PTS反应测试了荧光蛋白标记的抗体标记。与传统的基因构建方法相比，通过PTS构建bsAb是一种很有前途的生成多个bsAb组合的替代方法。

引用次数: 8

Chaetomium thermophilum formate dehydrogenase has high activity in the reduction of hydrogen carbonate (HCO3 −) to formate 热毛毛菌甲酸脱氢酶在还原碳酸氢(HCO3−)生成甲酸中具有高活性

Protein Engineering, Design and Selection

Pub Date : 2017-01-01 DOI: 10.1093/protein/gzw062

Aşkın Sevinç Aslan, J. Valjakka, Jouni Ruupunen, D. Yıldırım, N. Turner, O. Turunen, Barış Binay

While formate dehydrogenases (FDHs) have been used for cofactor recycling in chemoenzymatic synthesis, the ability of FDH to reduce CO2 could also be utilized in the conversion of CO2 to useful products via formate (HCOO−). In this study, we investigated the reduction of CO2 in the form of hydrogen carbonate (HCO3 −) to formate by FDHs from Candida methylica (CmFDH) and Chaetomium thermophilum (CtFDH) in a NADH-dependent reaction. The catalytic performance with HCO3 − as a substrate was evaluated by measuring the kinetic rates and conducting productivity assays. CtFDH showed a higher efficiency in converting HCO3 − to formate than CmFDH, whereas CmFDH was better in the oxidation of formate. The pH optimum of the reduction was at pH 7–8. However, the high concentrations of HCO3 − reduced the reaction rate. CtFDH was modeled in the presence of HCO3 − showing that it fits to the active site. The active site setting for hydride transfer in CO2 reduction was modeled. The hydride donated by NADH would form a favorable contact to the carbon atom of HCO3 −, resulting in a surplus of electrons within the molecule. This would cause the complex formed by hydrogen carbonate and the hydride to break into formate and hydroxide ions.

甲酸脱氢酶(FDHs)在化学酶合成中用于辅助因子回收，FDH减少CO2的能力也可以用于通过甲酸(HCOO−)将CO2转化为有用的产物。在这项研究中，我们研究了在nadh依赖的反应中，来自甲基念珠菌(CmFDH)和嗜热毛毛菌(CtFDH)的fdh将碳酸氢形式的CO2 (HCO3−)还原为甲酸。以HCO3−为底物，通过测定动力学速率和产率来评价其催化性能。CtFDH将HCO3−转化为甲酸的效率高于CmFDH，而CmFDH对甲酸的氧化效果更好。还原的最佳pH值为pH 7 ~ 8。然而，高浓度的HCO3−降低了反应速率。CtFDH在HCO3−存在下建模，表明它适合活性位点。模拟了CO2还原过程中氢化物转移的活性位点设置。NADH提供的氢化物会与碳原子HCO3−形成有利的接触，导致分子内的电子过剩。这将导致碳酸氢和氢化物形成的络合物分解成甲酸盐和氢氧根离子。

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引用次数: 29

Allosteric control of antibody-prion recognition through oxidation of a disulfide bond between the CH and CL chains. 通过氧化 CH 链和 CL 链之间的二硫键，对抗体-磷脂识别进行异构控制。

Protein Engineering, Design and Selection

Pub Date : 2017-01-01 Epub Date: 2016-11-29 DOI: 10.1093/protein/gzw065

Jun Zhao, Ruth Nussinov, Buyong Ma

Molecular details of the recognition of disordered antigens by their cognate antibodies have not been studied as extensively as folded protein antigens and much is still unknown. To follow the conformational changes in the antibody and cross-talk between its subunits and with antigens, we performed molecular dynamics (MD) simulations of the complex of Fab and prion-associated peptide in the apo and bound forms. We observed that the inter-chain disulfide bond in constant domains restrains the conformational changes of Fab, especially the loops in the CH1 domain, resulting in inhibition of the cross-talk between Fab subdomains that thereby may prevent prion peptide binding. We further identified several negative and positive correlations of motions between the peptide and Fab constant domains, which suggested structural cross-talks between the constant domains and the antigen. The cross-talk was influenced by the inter-chain disulfide bond, which reduced the number of paths between them. Importantly, network analysis of the complex and its bound water molecules observed that those water molecules form an integral part of the Fab/peptide complex network and potential allosteric pathways. On-going work focuses on developing strategies aimed to incorporate these new network communications-including the associated water molecules-toward the grand challenge of antibody design.

人们对无序抗原的同源抗体识别无序抗原的分子细节还没有像折叠蛋白抗原那样进行广泛的研究，而且还有许多未知。为了跟踪抗体的构象变化以及抗体亚基之间和抗体与抗原之间的相互作用，我们对Fab和朊病毒相关肽的复合物进行了分子动力学（MD）模拟。我们观察到，恒定结构域中的链间二硫键限制了Fab的构象变化，尤其是CH1结构域中的环，从而抑制了Fab亚结构域之间的交叉作用，进而阻止了朊病毒肽的结合。我们进一步确定了肽与 Fab 常域之间的几种负相关和正相关运动，这表明常域与抗原之间存在结构上的交叉对话。这种交叉作用受到链间二硫键的影响，二硫键减少了它们之间的路径数量。重要的是，对复合物及其结合的水分子进行的网络分析发现，这些水分子构成了 Fab/肽复合物网络和潜在异构途径的组成部分。目前的工作重点是制定策略，将这些新的网络通信（包括相关的水分子）纳入抗体设计的巨大挑战中。

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引用次数: 0

Stabilization of luciferase from Renilla reniformis using random mutations 随机突变稳定Renilla reniformis荧光素酶

Protein Engineering, Design and Selection

Pub Date : 2017-01-01 DOI: 10.1093/protein/gzw056

M. Shigehisa, Norie Amaba, S. Arai, Chisato Higashi, Ryo Kawanabe, Ayano Matsunaga, F. A. Laksmi, M. Tokunaga, M. Ishibashi

We expressed luciferase (RLuc) from Renilla reniformis in Escherichia coli. RLuc was purified using a Ni-NTA column and subsequently characterized. It was unstable in acidic solutions and at 30°C. To increase the stability of RLuc, the Rluc gene was randomly mutated using error-prone polymerase chain reaction. E. coli harboring the mutated gene was screened by detecting luminescence on a plate containing the substrate coelenterazine at 34°C. Three mutants, i.e. N264SS287P, N178D and F116LI137V, were obtained. The solubilities and specific activities of these mutants were higher than those of the wild type. Furthermore, the N264SS287P mutant maintained stability at a temperature approximately 5°C higher than that of the wild type, while denaturation of the F116LI137V mutant started at a temperature that was 5°C lower than the wild type, and ended at a temperature that was 7°C higher. We examined the obtained mutations using thermal shift assays and a computer program Coot in this study.

我们在大肠杆菌中表达了Renilla reniformis的荧光素酶(RLuc)。用Ni-NTA柱纯化RLuc并对其进行表征。它在酸性溶液和30°C时不稳定。为了增加RLuc的稳定性，采用易出错的聚合酶链反应随机突变RLuc基因。通过在含有底物coelenterazine的平板上检测34℃下的发光来筛选携带突变基因的大肠杆菌。获得了N264SS287P、N178D和F116LI137V三个突变体。这些突变体的溶解度和比活性均高于野生型。此外，N264SS287P突变体在比野生型高约5℃的温度下保持稳定，而F116LI137V突变体的变性开始温度比野生型低5℃，结束温度比野生型高7℃。在本研究中，我们使用热移法和计算机程序来检查获得的突变。

引用次数: 12

Rational design of glycoengineered interferon-&bgr; analogs with improved aggregation state: experimental validation 糖工程干扰素的合理设计具有改进聚合状态的类似物:实验验证

Protein Engineering, Design and Selection

Pub Date : 2017-01-01 DOI: 10.1093/protein/gzw058

M. Samoudi, Z. Minuchehr, S. Harcum, F. Tabandeh, N. Omid Yeganeh, M. Khodabandeh

Recombinant human interferon-&bgr; (rhIFN-&bgr;) used clinically has lower efficacy than expected due to protein instabilities such as aggregation. Increasing molecular stability, glycoengineering has been used to improve clinical efficacy for a number of therapeutics; however, often labor-intensive trail-and-error approaches are used to identify additional glycosylation sites. In this study two rhIFN-&bgr; analogs with one additional glycosylation site, L6T and S75N, identified by a rational in silico approach, were characterized. These rhIFN-&bgr; analogs were synthesized in parallel with a Chinese hamster ovary (CHO) codon-optimized natural human IFN-&bgr; (Opt-IFN-&bgr;) and expressed in CHO cells using the same expression system. The molecular weights for both analogs were observed to be higher than Opt-IFN-&bgr;, consistent with hyper-glycosylation. The in vitro biological assay showed the hyper-glycosylated analogs and the Opt-IFN-&bgr; had similar activity. The aggregation studies demonstrated that both analogs had lower tendencies to aggregate compared to the Opt-IFN-&bgr;. These experimental studies validate the in silico strategy to predict suitable glycosylation sites that would be glycosylated, while maintaining biological function. Moreover, this work describes hyper-glycosylated rhIFN-&bgr; analogs with improved solubility (i.e. lower aggregation). These findings, together with the rational in silico design, will allow us to increase protein glycosylation with the goal to enhance therapeutic efficacy.

重组人干扰素;(rrhin -&bgr;)由于蛋白质聚集等不稳定性，临床使用的疗效低于预期。增加分子稳定性，糖工程已被用于改善临床疗效的治疗方法;然而，通常使用劳动密集的追踪和错误方法来识别额外的糖基化位点。在本研究中，两个rrhin -&bgr;通过理性硅法鉴定了具有一个额外糖基化位点L6T和S75N的类似物。这些rhIFN -&bgr;与中国仓鼠卵巢(CHO)密码子优化的天然人IFN-&bgr;(Opt-IFN-&bgr;)，并使用相同的表达系统在CHO细胞中表达。观察到两种类似物的分子量均高于Opt-IFN-&bgr;，与高糖基化一致。体外生物实验显示高糖基化类似物和Opt-IFN-&bgr;有类似的活动。聚集研究表明，与Opt-IFN-&bgr;相比，这两种类似物的聚集倾向较低。这些实验研究验证了在保持生物功能的同时预测合适的糖基化位点的计算机策略。此外，这项工作描述了高糖基化的rrhin -&bgr;具有改善溶解度(即较低聚集)的类似物。这些发现，加上合理的硅设计，将使我们能够增加蛋白质糖基化，以提高治疗效果。

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引用次数: 6

Design and expression of a chimeric vaccine candidate for avian necrotic enteritis 禽坏死性肠炎候选嵌合疫苗的设计与表达

Protein Engineering, Design and Selection

Pub Date : 2017-01-01 DOI: 10.1093/protein/gzw060

A. Rostami, F. Goshadrou, R. P. Langroudi, S. Z. Bathaie, A. Riazi, J. Amani, G. Ahmadian

Necrotic enteritis is an economically important disease of poultry mainly caused by Clostridium perfringens. The bacteria release multiple toxins of which NetB, alpha toxin and TpeL have been reported to play important roles in pathogenicity and/or severity of the disease. In this study, the sequence of clostridial toxins NetB, alpha toxin and TpeL were analyzed using bioinformatics tools to determine protein domains with high immunogenicity factor. Several chimeric trivalent proteins consisting of the immunogenic regions of the three toxins were designed and evaluated. The separate regions were fused together using rigid linkers. Based on a modeled tertiary structure, a proper combination was selected and expressed in a bacterial host (Escherichia coli) and successfully purified. The expression of the chimeric protein was further verified by western blotting. The ability of the immunized serum in recognizing each individual subunit of the chimeric protein was also examined. Circular dichroism was used to evaluate the predicted secondary structure of the chimeric protein. In vitro potency test demonstrated that the serum from a rabbit immunized with the chimeric protein is able to partially neutralize Alpha toxin, hence the construct can potentially be used as a vaccine against C. perfringens.

坏死性肠炎是由产气荚膜梭菌引起的一种重要的家禽疾病。据报道，细菌释放多种毒素，其中NetB、α毒素和TpeL在疾病的致病性和/或严重程度中发挥重要作用。本研究利用生物信息学工具分析了梭菌毒素NetB、α毒素和TpeL的序列，以确定具有高免疫原性因子的蛋白结构域。设计并评价了由三种毒素免疫原区组成的几种嵌合三价蛋白。分开的区域用刚性连接件粘合在一起。基于模拟的三级结构，选择合适的组合并在细菌宿主(大肠杆菌)中表达并成功纯化。western blotting进一步验证了嵌合蛋白的表达。免疫血清识别每个嵌合蛋白亚基的能力也被检测。利用圆二色性对预测的嵌合蛋白二级结构进行评价。体外效价试验表明，嵌合蛋白免疫的兔血清能够部分中和α毒素，因此该构建物可作为产气荚膜荚膜原细菌的疫苗。

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引用次数: 16

A robust cosolvent-compatible halohydrin dehalogenase by computational library design 基于计算库设计的稳健的助溶剂相容卤代醇脱卤酶

Protein Engineering, Design and Selection

Pub Date : 2016-12-19 DOI: 10.1093/protein/gzw068

Hesam Arabnejad, Marco Dal Lago, P. Jekel, Robert J. Floor, A. Thunnissen, A. C. Terwisscha van Scheltinga, Hein J. Wijma, D. Janssen

To improve the applicability of halohydrin dehalogenase as a catalyst for reactions in the presence of organic cosolvents, we explored a computational library design strategy (Framework for Rapid Enzyme Stabilization by Computational libraries) that involves discovery and in silico evaluation of stabilizing mutations. Energy calculations, disulfide bond predictions and molecular dynamics simulations identified 218 point mutations and 35 disulfide bonds with predicted stabilizing effects. Experiments confirmed 29 stabilizing point mutations, most of which were located in two distinct regions, whereas introduction of disulfide bonds was not effective. Combining the best mutations resulted in a 12-fold mutant (HheC-H12) with a 28°C higher apparent melting temperature and a remarkable increase in resistance to cosolvents. This variant also showed a higher optimum temperature for catalysis while activity at low temperature was preserved. Mutant H12 was used as a template for the introduction of mutations that enhance enantioselectivity or activity. Crystal structures showed that the structural changes in the H12 mutant mostly agreed with the computational predictions and that the enhanced stability was mainly due to mutations that redistributed surface charges and improved interactions between subunits, the latter including better interactions of water molecules at the subunit interfaces.

为了提高卤代醇脱卤酶在有机共溶剂存在下作为反应催化剂的适用性，我们探索了一种计算库设计策略(计算库快速酶稳定框架)，包括发现和计算机评估稳定突变。能量计算、二硫化物键预测和分子动力学模拟确定了218个点突变和35个二硫化物键具有预测的稳定作用。实验证实了29个稳定点突变，其中大部分位于两个不同的区域，而引入二硫键则无效。将最佳突变组合在一起，得到了12倍突变体(HheC-H12)，表观熔融温度提高28°C，对共溶剂的抗性显著提高。该突变体表现出较高的最适催化温度，同时保持了低温下的活性。突变体H12被用作模板，用于引入增强对映体选择性或活性的突变。晶体结构表明，H12突变体的结构变化与计算预测基本一致，稳定性的增强主要是由于突变重新分配了表面电荷和改善了亚基之间的相互作用，后者包括更好的水分子在亚基界面的相互作用。

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引用次数: 34