Protein Engineering Design & Selection最新文献_第9页

Generation of a 100-billion cyclic peptide phage display library having a high skeletal diversity. 产生具有高骨骼多样性的1000亿个环状肽噬菌体展示文库。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2021-02-15 DOI: 10.1093/protein/gzab018

Vanessa Carle, Xu-Dong Kong, Alice Comberlato, Chelsea Edwards, Cristina Díaz-Perlas, Christian Heinis

引用次数: 4

Molecular flexibility in computational protein design: an algorithmic perspective. 计算蛋白设计中的分子灵活性:一个算法的视角。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2021-02-15 DOI: 10.1093/protein/gzab011

Younes Bouchiba, Juan Cortés, Thomas Schiex, Sophie Barbe

{"title":"Molecular flexibility in computational protein design: an algorithmic perspective.","authors":"Younes Bouchiba, Juan Cortés, Thomas Schiex, Sophie Barbe","doi":"10.1093/protein/gzab011","DOIUrl":"https://doi.org/10.1093/protein/gzab011","url":null,"abstract":"Computational protein design (CPD) is a powerful technique for engineering new proteins, with both great fundamental implications and diverse practical interests. However, the approximations usually made for computational efficiency, using a single fixed backbone and a discrete set of side chain rotamers, tend to produce rigid and hyper-stable folds that may lack functionality. These approximations contrast with the demonstrated importance of molecular flexibility and motions in a wide range of protein functions. The integration of backbone flexibility and multiple conformational states in CPD, in order to relieve the inaccuracies resulting from these simplifications and to improve design reliability, are attracting increased attention. However, the greatly increased search space that needs to be explored in these extensions defines extremely challenging computational problems. In this review, we outline the principles of CPD and discuss recent effort in algorithmic developments for incorporating molecular flexibility in the design process.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2021-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzab011","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38958995","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 4

Substitution of distal and active site residues reduces product inhibition of E1 from Acidothermus Cellulolyticus. 远端和活性位点残基的取代降低了酸热菌降解纤维素的产物E1的抑制作用。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2021-02-15 DOI: 10.1093/protein/gzab031

Samantha R Summers, Sarah Alamdari, Casey J Kraft, Roman Brunecky, Jim Pfaendtner, Joel L Kaar

{"title":"Substitution of distal and active site residues reduces product inhibition of E1 from Acidothermus Cellulolyticus.","authors":"Samantha R Summers, Sarah Alamdari, Casey J Kraft, Roman Brunecky, Jim Pfaendtner, Joel L Kaar","doi":"10.1093/protein/gzab031","DOIUrl":"https://doi.org/10.1093/protein/gzab031","url":null,"abstract":"Cellulases are largely afflicted by inhibition from their reaction products, especially at high-substrate loading, which represents a major challenge for biomass processing. This challenge was overcome for endoglucanase 1 (E1) from Acidothermus cellulolyticus by identifying a large conformational change involving distal residues upon binding cellobiose. Having introduced alanine substitutions at each of these residues, we identified several mutations that reduced cellobiose inhibition of E1, including W212A, W213A, Q247A, W249A and F250A. One of the mutations (W212A) resulted in a 47-fold decrease in binding affinity of cellobiose as well as a 5-fold increase in the kcat. The mutation further increased E1 activity on Avicel and dilute-acid treated corn stover and enhanced its productivity at high-substrate loadings. These findings were corroborated by funnel metadynamics, which showed that the W212A substitution led to reduced affinity for cellobiose in the +1 and +2 binding sites due to rearrangement of key cellobiose-binding residues.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2021-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39624356","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

Protease-stable DARPins as promising oral therapeutics. 蛋白酶稳定的DARPins作为有前途的口服治疗药物。

IF 2.6 4区生物学

Protein Engineering Design & Selection Pub Date : 2021-02-15 DOI: 10.1093/protein/gzab028

Rudo A Simeon, Yu Zeng, Vikas Chonira, Andrea Martinez Aguirre, Mauricio Lasagna, Marko Baloh, Joseph A Sorg, Cecilia Tommos, Zhilei Chen

{"title":"Protease-stable DARPins as promising oral therapeutics.","authors":"Rudo A Simeon, Yu Zeng, Vikas Chonira, Andrea Martinez Aguirre, Mauricio Lasagna, Marko Baloh, Joseph A Sorg, Cecilia Tommos, Zhilei Chen","doi":"10.1093/protein/gzab028","DOIUrl":"10.1093/protein/gzab028","url":null,"abstract":"Clostridioides difficile is an enteric bacterium whose exotoxins, TcdA and TcdB, inactivate small GTPases within the host cells, leading to bloody diarrhea. In prior work, our group engineered a panel of potent TcdB-neutralizing designed ankyrin repeat proteins (DARPin) as oral therapeutics against C. difficile infection. However, all these DARPins are highly susceptible to digestion by gut-resident proteases, i.e. trypsin and chymotrypsin. Close evaluation of the protein sequence revealed a large abundance of positively charged and aromatic residues in the DARPin scaffold. In this study, we significantly improved the protease stability of one of the DARPins, 1.4E, via protein engineering. Unlike 1.4E, whose anti-TcdB EC50 increased >83-fold after 1-hour incubation with trypsin (1 mg/ml) or chymotrypsin (0.5 mg/ml), the best progenies-T10-2 and T10b-exhibit similar anti-TcdB potency as their parent in PBS regardless of protease treatment. The superior protease stability of T10-2 and T10b is attributed to the removal of nearly all positively charged and aromatic residues except those directly engaged in target binding. Furthermore, T10-2 was found to retain significant toxin-neutralization ability in ex vivo cecum fluid and can be easily detected in mouse fecal samples upon oral administration. Both T10-2 and T10b enjoy a high thermo- and chemo-stability and can be expressed very efficiently in Escherichia coli (>100 mg/l in shaker flasks). We believe that, in additional to their potential as oral therapeutics against C. difficile infection, T10-2 and T10b can also serve as a new generation DARPin scaffold with superior protease stability.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2021-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8861517/pdf/gzab028.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10431620","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Increasing loop flexibility affords low-temperature adaptation of a moderate thermophilic malate dehydrogenase from Geobacillus stearothermophilus. 增加环的灵活性提供了中等嗜热苹果酸脱氢酶从嗜热硬脂地杆菌低温适应。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2021-02-15 DOI: 10.1093/protein/gzab026

Yuya Shimozawa, Tomoki Himiyama, Tsutomu Nakamura, Yoshiaki Nishiya

{"title":"Increasing loop flexibility affords low-temperature adaptation of a moderate thermophilic malate dehydrogenase from Geobacillus stearothermophilus.","authors":"Yuya Shimozawa, Tomoki Himiyama, Tsutomu Nakamura, Yoshiaki Nishiya","doi":"10.1093/protein/gzab026","DOIUrl":"https://doi.org/10.1093/protein/gzab026","url":null,"abstract":"Malate dehydrogenase (MDH) catalyzes the reversible reduction of nicotinamide adenine dinucleotide from oxaloacetate to L-malate. MDH from moderate thermophilic Geobacillus stearothermophilus (gs-MDH) has high thermal stability and substrate specificity and is used as a diagnostic reagent. In this study, gs-MDH was engineered to increase its catalytic activity at low temperatures. Based on sequential and structural comparison with lactate dehydrogenase from G. stearothermophilus, we selected G218 as a mutation site to increase the loop flexibility pivotal for MDH catalysis. The G218 mutants showed significantly higher specific activities than the wild type at low temperatures and maintained thermal stability. The crystal structure of the G218Y mutant, which had the highest catalytic efficiency among all the G218 mutants, suggested that the flexibility of the mobile loop was successfully increased by the bulky side chain. Therefore, this study demonstrated the low-temperature adaptation of MDH by facilitating conformational changes during catalysis.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2021-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39681041","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

Accurate and efficient structure-based computational mutagenesis for modeling fluorescence levels of Aequorea victoria green fluorescent protein mutants. 准确、高效的基于结构的计算诱变法模拟维多利亚绿荧光蛋白突变体的荧光水平。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2020-09-14 DOI: 10.1093/protein/gzaa022

Majid Masso

{"title":"Accurate and efficient structure-based computational mutagenesis for modeling fluorescence levels of Aequorea victoria green fluorescent protein mutants.","authors":"Majid Masso","doi":"10.1093/protein/gzaa022","DOIUrl":"https://doi.org/10.1093/protein/gzaa022","url":null,"abstract":"A computational mutagenesis technique was used to characterize the structural effects associated with over 46 000 single and multiple amino acid variants of Aequorea victoria green fluorescent protein (GFP), whose functional effects (fluorescence levels) were recently measured by experimental researchers. For each GFP mutant, the approach generated a single score reflecting the overall change in sequence-structure compatibility relative to native GFP, as well as a vector of environmental perturbation (EP) scores characterizing the impact at all GFP residue positions. A significant GFP structure-function relationship (P < 0.0001) was elucidated by comparing the sequence-structure compatibility scores with the functional data. Next, the computed vectors for GFP mutants were used to train predictive models of fluorescence by implementing random forest (RF) classification and tree regression machine learning algorithms. Classification performance reached 0.93 for sensitivity, 0.91 for precision and 0.90 for balanced accuracy, and regression models led to Pearson's correlation as high as r = 0.83 between experimental and predicted GFP mutant fluorescence. An RF model trained on a subset of over 1000 experimental single residue GFP mutants with measured fluorescence was used for predicting the 3300 remaining unstudied single residue mutants, with results complementing known GFP biochemical and biophysical properties. In addition, models trained on the subset of experimental GFP mutants harboring multiple residue replacements successfully predicted fluorescence of the single residue GFP mutants. The models developed for this study were accurate and efficient, and their predictions outperformed those of several related state-of-the-art methods.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2020-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzaa022","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38478437","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

Improved catalytic activity and stability of cellobiohydrolase (Cel6A) from the Aspergillus fumigatus by rational design. 通过合理设计提高烟曲霉纤维生物水解酶(Cel6A)的催化活性和稳定性。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2020-09-14 DOI: 10.1093/protein/gzaa020

Subba Reddy Dodda, Nibedita Sarkar, Piyush Jain, Kaustav Aikat, Sudit S Mukhopadhyay

{"title":"Improved catalytic activity and stability of cellobiohydrolase (Cel6A) from the Aspergillus fumigatus by rational design.","authors":"Subba Reddy Dodda, Nibedita Sarkar, Piyush Jain, Kaustav Aikat, Sudit S Mukhopadhyay","doi":"10.1093/protein/gzaa020","DOIUrl":"https://doi.org/10.1093/protein/gzaa020","url":null,"abstract":"Cheap production of glucose is the current challenge for the production of cheap bioethanol. Ideal protein engineering approaches are required for improving the efficiency of the members of the cellulase, the enzyme complex involved in the saccharification process of cellulose. An attempt was made to improve the efficiency of the cellobiohydrolase (Cel6A), the important member of the cellulase isolated from Aspergillus fumigatus (AfCel6A). Structure-based variants of AfCel6A were designed. Amino acids surrounding the catalytic site and conserved residues in the cellulose-binding domain were targeted (N449V, N168G, Y50W and W24YW32Y). I mutant 3 server was used to identify the potential variants based on the free energy values (∆∆G). In silico structural analyses and molecular dynamics simulations evaluated the potentiality of the variants for increasing thermostability and catalytic activity of Cel6A. Further enzyme studies with purified protein identified the N449V is highly thermo stable (60°C) and pH tolerant (pH 5-7). Kinetic studies with Avicel determined that substrate affinity of N449V (Km =0.90 ± 0.02) is higher than the wild type (1.17 ± 0.04) and the catalytic efficiency (Kcat/Km) of N449V is ~2-fold higher than wild type. All these results suggested that our strategy for the development of recombinant enzyme is a right approach for protein engineering.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2020-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzaa020","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38478434","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 5

Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid. 基于arac的生物传感器对三乙酸内酯和茴香酸的工程灵敏度和特异性。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2020-09-14 DOI: 10.1093/protein/gzaa027

Zhiqing Wang, Aarti Doshi, Ratul Chowdhury, Yixi Wang, Costas D Maranas, Patrick C Cirino

{"title":"Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid.","authors":"Zhiqing Wang, Aarti Doshi, Ratul Chowdhury, Yixi Wang, Costas D Maranas, Patrick C Cirino","doi":"10.1093/protein/gzaa027","DOIUrl":"https://doi.org/10.1093/protein/gzaa027","url":null,"abstract":"We previously described the design of triacetic acid lactone (TAL) biosensor 'AraC-TAL1', based on the AraC regulatory protein. Although useful as a tool to screen for enhanced TAL biosynthesis, this variant shows elevated background (leaky) expression, poor sensitivity and relaxed inducer specificity, including responsiveness to orsellinic acid (OA). More sensitive biosensors specific to either TAL or OA can aid in the study and engineering of polyketide synthases that produce these and similar compounds. In this work, we employed a TetA-based dual-selection to isolate new TAL-responsive AraC variants showing reduced background expression and improved TAL sensitivity. To improve TAL specificity, OA was included as a 'decoy' ligand during negative selection, resulting in the isolation of a TAL biosensor that is inhibited by OA. Finally, to engineer OA-specific AraC variants, the iterative protein redesign and optimization computational framework was employed, followed by 2 rounds of directed evolution, resulting in a biosensor with 24-fold improved OA/TAL specificity, relative to AraC-TAL1.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2020-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzaa027","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38626956","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 3

Protein Engineering, Design and Selection. 蛋白质工程，设计与选择。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2020-09-14 DOI: 10.1093/protein/gzaa024

R. Chica

引用次数: 16

Structure- and sequence-based design of synthetic single-domain antibody libraries. 基于结构和序列的合成单域抗体文库设计。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2020-09-14 DOI: 10.1093/protein/gzaa028

Alexander M Sevy, Ming-Tang Chen, Michelle Castor, Tyler Sylvia, Harini Krishnamurthy, Andrii Ishchenko, Chung-Ming Hsieh

{"title":"Structure- and sequence-based design of synthetic single-domain antibody libraries.","authors":"Alexander M Sevy, Ming-Tang Chen, Michelle Castor, Tyler Sylvia, Harini Krishnamurthy, Andrii Ishchenko, Chung-Ming Hsieh","doi":"10.1093/protein/gzaa028","DOIUrl":"https://doi.org/10.1093/protein/gzaa028","url":null,"abstract":"Single-domain antibody fragments known as VHH have emerged in the pharmaceutical industry as useful biotherapeutics. These molecules, which are naturally produced by camelids, share the characteristics of high affinity and specificity with traditional human immunoglobulins, while consisting of only a single heavy chain. Currently, the most common method for generating VHH is via animal immunization, which can be costly and time-consuming. Here we describe the development of a synthetic VHH library for in vitro selection of single domain binders. We combine structure-based design and next-generation sequencing analysis to build a library with characteristics that closely mimic the natural repertoire. To validate the performance of our synthetic library, we isolated VHH against three model antigens (soluble mouse PD-1 ectodomain, amyloid-β peptide, and MrgX1 GPCR) of different sizes and characteristics. We were able to isolate diverse binders targeting different epitopes with high affinity (as high as 5 nM) against all three targets. We then show that anti-mPD-1 binders have functional activity in a receptor blocking assay.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2020-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzaa028","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38737808","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 9