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

Exposure of a cryptic Hsp70 binding site determines the cytotoxicity of the ALS-associated SOD1-mutant A4V. 暴露于一个隐蔽的Hsp70结合位点决定了als相关sod1突变体A4V的细胞毒性。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa008

Filip Claes, Stanislav Rudyak, Angela S Laird, Nikolaos Louros, Jacinte Beerten, Maja Debulpaep, Emiel Michiels, Rob van der Kant, Joost Van Durme, Greet De Baets, Bert Houben, Meine Ramakers, Kristy Yuan, Serene S L Gwee, Sara Hernandez, Kerensa Broersen, Mikael Oliveberg, Barbara Moahamed, Janine Kirstein, Wim Robberecht, Frederic Rousseau, Joost Schymkowitz

{"title":"Exposure of a cryptic Hsp70 binding site determines the cytotoxicity of the ALS-associated SOD1-mutant A4V.","authors":"Filip Claes, Stanislav Rudyak, Angela S Laird, Nikolaos Louros, Jacinte Beerten, Maja Debulpaep, Emiel Michiels, Rob van der Kant, Joost Van Durme, Greet De Baets, Bert Houben, Meine Ramakers, Kristy Yuan, Serene S L Gwee, Sara Hernandez, Kerensa Broersen, Mikael Oliveberg, Barbara Moahamed, Janine Kirstein, Wim Robberecht, Frederic Rousseau, Joost Schymkowitz","doi":"10.1093/protein/gzaa008","DOIUrl":"https://doi.org/10.1093/protein/gzaa008","url":null,"abstract":"The accumulation of toxic protein aggregates is thought to play a key role in a range of degenerative pathologies, but it remains unclear why aggregation of polypeptides into non-native assemblies is toxic and why cellular clearance pathways offer ineffective protection. We here study the A4V mutant of SOD1, which forms toxic aggregates in motor neurons of patients with familial amyotrophic lateral sclerosis (ALS). A comparison of the location of aggregation prone regions (APRs) and Hsp70 binding sites in the denatured state of SOD1 reveals that ALS-associated mutations promote exposure of the APRs more than the strongest Hsc/Hsp70 binding site that we could detect. Mutations designed to increase the exposure of this Hsp70 interaction site in the denatured state promote aggregation but also display an increased interaction with Hsp70 chaperones. Depending on the cell type, in vitro this resulted in cellular inclusion body formation or increased clearance, accompanied with a suppression of cytotoxicity. The latter was also observed in a zebrafish model in vivo. Our results suggest that the uncontrolled accumulation of toxic SOD1A4V aggregates results from insufficient detection by the cellular surveillance network.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 10","pages":"443-457"},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzaa008","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37927125","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: 6

Design, creation and in vitro testing of a reduced immunogenicity humanized anti-CD25 monoclonal antibody that retains functional activity. 保留功能活性的低免疫原性人源抗cd25单克隆抗体的设计、制备和体外测试

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa017

Marcia Stickler, Anita Reddy, Joanna M Xiong, Melanie H Wong, Yoshiko Akamatsu, Paul R Hinton, Fiona A Harding

{"title":"Design, creation and in vitro testing of a reduced immunogenicity humanized anti-CD25 monoclonal antibody that retains functional activity.","authors":"Marcia Stickler, Anita Reddy, Joanna M Xiong, Melanie H Wong, Yoshiko Akamatsu, Paul R Hinton, Fiona A Harding","doi":"10.1093/protein/gzaa017","DOIUrl":"https://doi.org/10.1093/protein/gzaa017","url":null,"abstract":"Humanized and fully human sequence-derived therapeutic antibodies retain the capacity to induce anti-drug antibodies. Daclizumab (humanized version of the murine anti-Tac antibody; E.HAT) was selected for a proof of concept application of engineering approaches to reduce potential immunogenicity due to its demonstrated immunogenicity in the clinic. Reduced immunogenicity variants of E.HAT were created by identifying and modifying a CD4+ T cell epitope region in the VH region. Variant epitope region peptides were selected for their reduced capacity to induce CD4+ T cell proliferative responses in vitro. Variant antibody molecules were created, and CD25 affinity and potency were similar to the unmodified parent antibody. Fab fragments from the variant antibodies induced a lower frequency and magnitude of responses in human peripheral blood mononuclear cells proliferation tests. By the empirical selection of two amino acid mutations, fully functional humanized E.HAT antibodies with reduced potential to induce immune responses in vitro were created.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 12","pages":"543-554"},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/protein/gzaa017","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38213005","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

Nanobody stability engineering by employing the ΔTm shift; a comparison with apparent rate constants of heat-induced aggregation. 利用ΔTm位移的纳米体稳定性工程热诱导聚合的表观速率常数的比较。

IF 2.4 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzz017

Patrick Kunz, A. Ortale, N. Mücke, Katinka Zinner, J. Hoheisel

{"title":"Nanobody stability engineering by employing the ΔTm shift; a comparison with apparent rate constants of heat-induced aggregation.","authors":"Patrick Kunz, A. Ortale, N. Mücke, Katinka Zinner, J. Hoheisel","doi":"10.1093/protein/gzz017","DOIUrl":"https://doi.org/10.1093/protein/gzz017","url":null,"abstract":"The antigen-binding domains of camelid heavy-chain antibodies, also called nanobodies, gained strong attention because of their unique functional and biophysical properties. They gave rise to an entire spectrum of applications in biotechnology, research and medicine. Despite several reports about reversibly refolding nanobodies, protein aggregation plays a major role in nanobody thermoresistance, asking for strategies to engineer their refolding behavior. Here, we use measurements of nanobody aggregation kinetics to validate structural features in the nanobody fold that are suppressing heat-induced nanobody aggregation. Furthermore, the kinetic measurements yielded a detailed insight into the concept of the ΔTm shift, a metric for protein aggregation propensities obtained from differential scanning fluorimetry measurements. By relating the equilibrium measurements of the ΔTm shift to the kinetic measurements of heat-induced nanobody aggregation, a distinct relationship could be identified that allows a prediction of nanobody aggregation rates from a simple equilibrium measurement of ΔTm.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"61 1","pages":""},"PeriodicalIF":2.4,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86060422","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: 8

Conformational selection of allergen-antibody complexes-surface plasticity of paratopes and epitopes. 过敏原-抗体复合物的构象选择--副基团和表位的表面可塑性。

IF 2.6 4区生物学

Protein Engineering Design & Selection Pub Date : 2019-12-31 DOI: 10.1093/protein/gzaa014

Monica L Fernández-Quintero, Johannes R Loeffler, Franz Waibl, Anna S Kamenik, Florian Hofer, Klaus R Liedl

{"title":"Conformational selection of allergen-antibody complexes-surface plasticity of paratopes and epitopes.","authors":"Monica L Fernández-Quintero, Johannes R Loeffler, Franz Waibl, Anna S Kamenik, Florian Hofer, Klaus R Liedl","doi":"10.1093/protein/gzaa014","DOIUrl":"10.1093/protein/gzaa014","url":null,"abstract":"Antibodies have the ability to bind various types of antigens and to recognize different antibody-binding sites (epitopes) of the same antigen with different binding affinities. Due to the conserved structural framework of antibodies, their specificity to antigens is mainly determined by their antigen-binding site (paratope). Therefore, characterization of epitopes in combination with describing the involved conformational changes of the paratope upon binding is crucial in understanding and predicting antibody-antigen binding. Using molecular dynamics simulations complemented with strong experimental structural information, we investigated the underlying binding mechanism and the resulting local and global surface plasticity in the binding interfaces of distinct antibody-antigen complexes. In all studied allergen-antibody complexes, we clearly observe that experimentally suggested epitopes reveal less plasticity, while non-epitope regions show high surface plasticity. Surprisingly, the paratope shows higher conformational diversity reflected in substantially higher surface plasticity, compared to the epitope. This work allows a visualization and characterization of antibody-antigen interfaces and might have strong implications for antibody-antigen docking and in the area of epitope prediction.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":"32 11","pages":"513-523"},"PeriodicalIF":2.6,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7451023/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10707438","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

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