Protein Engineering Design & Selection最新文献_第8页

Shared unfolding pathways of unrelated immunoglobulin-like β-sandwich proteins. 不相关免疫球蛋白样β-三明治蛋白的共享展开途径。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzz040

Rudesh D Toofanny, Sara Calhoun, Amanda L Jonsson, Valerie Daggett

{"title":"Shared unfolding pathways of unrelated immunoglobulin-like β-sandwich proteins.","authors":"Rudesh D Toofanny, Sara Calhoun, Amanda L Jonsson, Valerie Daggett","doi":"10.1093/protein/gzz040","DOIUrl":"https://doi.org/10.1093/protein/gzz040","url":null,"abstract":"The Dynameomics project contains native state and unfolding simulations of 807 protein domains, where each domain is representative of a different metafold; these metafolds encompass ~97% of protein fold space. There is a long-standing question in structural biology as to whether proteins in the same fold family share the same folding/unfolding characteristics. Using molecular dynamics simulations from the Dynameomics project, we conducted a detailed study of protein unfolding/folding pathways for 5 protein domains from the immunoglobulin (Ig)-like β-sandwich metafold (the highest ranked metafold in our database). The domains have sequence similarities ranging from 4 to 15% and are all from different SCOP superfamilies, yet they share the same overall Ig-like topology. Despite having very different amino acid sequences, the dominant unfolding pathway is very similar for the 5 proteins, and the secondary structures that are peripheral to the aligned, shared core domain add variability to the unfolding pathway. Aligned residues in the core domain display consensus structure in the transition state primarily through conservation of hydrophobic positions. Commonalities in the obligate folding nucleus indicate that insights into the major events in the folding/unfolding of other domains from this metafold may be obtainable from unfolding simulations of a few representative proteins.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 7","pages":"331-345"},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzz040","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37483331","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: 1

Molecular dynamics simulations suggest stabilizing mutations in a de novo designed α/β protein. 分子动力学模拟表明，新设计的 α/β 蛋白中存在稳定突变。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa005

Matthew Gill, Michelle E McCully

{"title":"Molecular dynamics simulations suggest stabilizing mutations in a de novo designed α/β protein.","authors":"Matthew Gill, Michelle E McCully","doi":"10.1093/protein/gzaa005","DOIUrl":"10.1093/protein/gzaa005","url":null,"abstract":"Designing functional proteins that can withstand extreme heat is beneficial for industrial and protein therapeutic applications. Thus, elucidating the atomic-level determinants of thermostability is a major interest for rational protein design. To that end, we compared the structure and dynamics of a set of previously designed, thermostable proteins based on the activation domain of human procarboxypeptidase A2 (AYEwt). The mutations in these designed proteins were intended to increase hydrophobic core packing and inter-secondary-structure interactions. To evaluate whether these design strategies were successfully deployed, we performed all-atom, explicit-solvent molecular dynamics (MD) simulations of AYEwt and three designed variants at both 25 and 100°C. Our MD simulations agreed with the relative experimental stabilities of the designs based on their secondary structure content, Cα root-mean-square deviation/fluctuation, and buried-residue solvent accessible surface area. Using a contact analysis, we found that the designs stabilize inter-secondary structure interactions and buried hydrophobic surface area, as intended. Based on our analysis, we designed three additional variants to test the role of helix stabilization, core packing, and a Phe → Met mutation on thermostability. We performed the additional MD simulations and analysis on these variants, and these data supported our predictions.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 7","pages":"317-329"},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/89/31/gzaa005.PMC7052480.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37667323","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

Functional analysis of chimeric TrCel6A enzymes with different carbohydrate binding modules. 不同碳水化合物结合模块嵌合TrCel6A酶的功能分析。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa003

Stefan Jarl Christensen, Silke Flindt Badino, Ana Mafalda Cavaleiro, Kim Borch, Peter Westh

{"title":"Functional analysis of chimeric TrCel6A enzymes with different carbohydrate binding modules.","authors":"Stefan Jarl Christensen, Silke Flindt Badino, Ana Mafalda Cavaleiro, Kim Borch, Peter Westh","doi":"10.1093/protein/gzaa003","DOIUrl":"https://doi.org/10.1093/protein/gzaa003","url":null,"abstract":"The glycoside hydrolase (GH) family 6 is an important group of enzymes that constitute an essential part of industrial enzyme cocktails used to convert lignocellulose into fermentable sugars. In nature, enzymes from this family often have a carbohydrate binding module (CBM) from the CBM family 1. These modules are known to promote adsorption to the cellulose surface and influence enzymatic activity. Here, we have investigated the functional diversity of CBMs found within the GH6 family. This was done by constructing five chimeric enzymes based on the model enzyme, TrCel6A, from the soft-rot fungus Trichoderma reesei. The natural CBM of this enzyme was exchanged with CBMs from other GH6 enzymes originating from different cellulose degrading fungi. The chimeric enzymes were expressed in the same host and investigated in adsorption and quasi-steady-state kinetic experiments. Our results quantified functional differences of these phylogenetically distant binding modules. Thus, the partitioning coefficient for substrate binding varied 4-fold, while the maximal turnover (kcat) showed a 2-fold difference. The wild-type enzyme showed the highest cellulose affinity on all tested substrates and the highest catalytic turnover. The CBM from Serendipita indica strongly promoted the enzyme's ability to form productive complexes with sites on the substrate surface but showed lower turnover of the complex. We conclude that the CBM plays an important role for the functional differences between GH6 wild-type enzymes.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 9","pages":"401-409"},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzaa003","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37679100","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: 7

Erratum to: Affinity versus specificity in coupled binding and folding reactions. 偶联结合和折叠反应的亲和与特异性的勘误。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzz038

S. Gianni, P. Jemth

引用次数: 0

Thermostability improvement of Aspergillus awamori glucoamylase via directed evolution of its gene located on episomal expression vector in Pichia pastoris cells. 毕赤酵母细胞胞外表达载体基因定向进化改良川曲霉葡萄糖淀粉酶的热稳定性。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzz048

Alexander Schmidt, Alexey Shvetsov, Elena Soboleva, Yury Kil, Vladimir Sergeev, Marina Surzhik

{"title":"Thermostability improvement of Aspergillus awamori glucoamylase via directed evolution of its gene located on episomal expression vector in Pichia pastoris cells.","authors":"Alexander Schmidt, Alexey Shvetsov, Elena Soboleva, Yury Kil, Vladimir Sergeev, Marina Surzhik","doi":"10.1093/protein/gzz048","DOIUrl":"https://doi.org/10.1093/protein/gzz048","url":null,"abstract":"Novel thermostable variants of glucoamylase (GA) from filamentous fungus Aspergillus awamori X100 were constructed using the directed evolution approach based on random mutagenesis by error-prone PCR of the catalytic domain region of glucoamylase gene located on a new episomal expression vector pPEHα in Pichia pastoris cells. Out of 3000 yeast transformants screened, six new thermostable GA variants with amino acid substitutions Val301Asp, Thr390Ala, Thr390Ala/Ser436Pro, Leu7Met/His391Tyr, Asn9His/Ile82Phe and Ser8Arg/Gln338Leu were identified and studied. To estimate the effect of each substitution in the double mutants, we have constructed the relevant single mutants of GA by site-directed mutagenesis and analyzed their thermal properties. Results of the analysis showed that only Ile82Phe and Ser8Arg substitutions by themselves increased enzyme thermostability. While the substitutions Leu7Met, Asn9His and Gln338Leu decreased the thermal stability of GA, the synergistic effect of double mutant variants Leu7Met/His391Tyr, Asn9His/Ile82Phe and Ser8Arg/Gln338Leu resulted in significant thermostability improvement as compared to the wild type GA. Thr390Ala and Thr390Ala/Ser436Pro mutant variants revealed the highest thermostability with free activation energy changes ΔΔG of 2.99 and 3.1 kJ/mol at 80°C, respectively.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 6","pages":"251-259"},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzz048","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37503413","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: 11

Toward the design of efficient transglycosidases: the case of the GH1 of Thermus thermophilus. 高效转糖苷酶的设计:以嗜热热菌GH1为例。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzz032

B. David, P. Arnaud, C. Tellier, Y. Sanejouand

引用次数: 7

Dissecting the statistical properties of the linear extrapolation method of determining protein stability. 剖析了测定蛋白质稳定性的线性外推法的统计特性。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa010

Kresten Lindorff-Larsen

引用次数: 5

Attempts to develop an enzyme converting DHIV to KIV. 试图开发一种将hiv转化为KIV的酶。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzz042

Kenji Oki, Frederick S Lee, Stephen L Mayo

{"title":"Attempts to develop an enzyme converting DHIV to KIV.","authors":"Kenji Oki, Frederick S Lee, Stephen L Mayo","doi":"10.1093/protein/gzz042","DOIUrl":"https://doi.org/10.1093/protein/gzz042","url":null,"abstract":"Dihydroxy-acid dehydratase (DHAD) catalyzes the dehydration of R-2,3-dihydroxyisovalerate (DHIV) to 2-ketoisovalerate (KIV) using an Fe-S cluster as a cofactor, which is sensitive to oxidation and expensive to synthesize. In contrast, sugar acid dehydratases catalyze the same chemical reactions using a magnesium ion. Here, we attempted to substitute the high-cost DHAD with a cost-efficient engineered sugar acid dehydratase using computational protein design (CPD). First, we tried without success to modify the binding pocket of a sugar acid dehydratase to accommodate the smaller, more hydrophobic DHIV. Then, we used a chemically activated substrate analog to react with sugar acid dehydratases or other enolase superfamily enzymes. Mandelate racemase from Pseudomonas putida (PpManR) and the putative sugar acid dehydratase from Salmonella typhimurium (StPutD) showed beta-elimination activity towards chlorolactate (CLD). CPD combined with medium-throughput selection improved the PpManR kcat/KM for CLD by four-fold. However, these enzyme variants did not show dehydration activity towards DHIV. Lastly, assuming phosphorylation could also be a good activation mechanism, we found that mevalonate-3-kinase (M3K) from Picrophilus torridus (PtM3K) exhibited adenosine triphosphate (ATP) hydrolysis activity when mixed with DHIV, indicating phosphorylation activity towards DHIV. Engineering PpManR or StPutD to accept 3-phospho-DHIV as a substrate was performed, but no variants with the desired activity were obtained.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 6","pages":"261-270"},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzz042","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37486616","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: 2

Statistical noise from recombinant plasmids can be abated via complementation of a ribosomal protein gene deletion. 重组质粒的统计噪声可以通过核糖体蛋白基因缺失的补充来减弱。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa007

Ichiro Matsumura, Donian Chyong

{"title":"Statistical noise from recombinant plasmids can be abated via complementation of a ribosomal protein gene deletion.","authors":"Ichiro Matsumura, Donian Chyong","doi":"10.1093/protein/gzaa007","DOIUrl":"https://doi.org/10.1093/protein/gzaa007","url":null,"abstract":"The phenotypes conferred by recombinant plasmids upon host cells often exhibit variability between replicate populations. This statistical noise is mostly a consequence of adaptive evolution in response to fitness burdens imposed by the plasmids themselves. We developed a novel strategy, 'ribosome pegging', to exclude common unwanted mutations that benefit host cells at the expense of heterologous gene expression. Plasmids that constitutively co-expressed the fluorescent reporter tagRFP and ribosomal protein L23 (rplW) were used to transform Escherichia coli cells that lacked the essential chromosomal rplW gene. Cells within the population that expressed too little L23, or too much, were evidently inviable. Ribosome pegging obviates the need for antibiotics, thus facilitating the deployment of recombinant bacteria in uncontrolled environments. We show that ribosome-pegged E. coli carrying a plasmid that constitutively expresses L23 and an artificially evolved enzyme protects fruit flies from otherwise toxic doses of the insecticide malathion.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 10","pages":"433-441"},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzaa007","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37867398","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: 0

Another week, another breakthrough: immunotherapy. 又一个星期，又一个突破:免疫疗法。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzz045

James S Huston

引用次数: 0