06db5786a10892b3949188573b815636 ijmolsci_a2022v23n9p4958.pdf d79aa3addd1a87c4b0c70b97ec6882e2ccfbf530 ijmolsci_a2022v23n9p4958.pdf 13b32c6c235ff3cd9152343386f9aa6aaf6cd7d5875828cbebd491addbf3d52a ijmolsci_a2022v23n9p4958.pdf Title: An In Silico Methodology That Facilitates Decision Making in the Engineering of Nanoscale Protein Materials Subject: Under the need for new functional and biocompatible materials for biomedical applications, protein engineering allows the design of assemblable polypeptides, which, as convenient building blocks of supramolecular complexes, can be produced in recombinant cells by simple and scalable methodologies. However, the stability of such materials is often overlooked or disregarded, becoming a potential bottleneck in the development and viability of novel products. In this context, we propose a design strategy based on in silico tools to detect instability areas in protein materials and to facilitate the decision making in the rational mutagenesis aimed to increase their stability and solubility. As a case study, we demonstrate the potential of this methodology to improve the stability of a humanized scaffold protein (a domain of the human nidogen), with the ability to oligomerize into regular nanoparticles usable to deliver payload drugs to tumor cells. Several nidogen mutants suggested by the method showed important and measurable improvements in their structural stability while retaining the functionalities and production yields of the original protein. Then, we propose the procedure developed here as a cost-effective routine tool in the design and optimization of multimeric protein materials prior to any experimental testing. Keywords: nanomaterials; protein stability; nanomedicine; mutagenesis Author: Eloi Parladé, Eric Voltà-Durán, Olivia Cano-Garrido, Julieta M. Sánchez, Ugutz Unzueta, Hèctor López-Laguna, Naroa Serna, Montserrat Cano, Manuel Rodríguez-Mariscal, Esther Vazquez and Antonio Villaverde Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu May 5 04:20:39 2022 CEST ModDate: Thu May 5 04:25:40 2022 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 12 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3077427 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- OHKNFJ+VnURWPalladioL Type 1 Custom yes yes no 10 0 AYUEVD+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 XCTOZO+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 GVKDYG+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 ETLENW+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 46 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 49 0 AXWEOY+CMSY10 Type 1 Builtin yes yes no 74 0 HVSBKL+PazoMath Type 1 Builtin yes yes no 78 0 FKPFBV+PazoMath-Italic Type 1 Builtin yes yes no 82 0 JHYTSG+CMR10 Type 1 Builtin yes yes no 86 0 NGOKVJ+EURM10 Type 1 Builtin yes yes no 90 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2022-05-07 02:28:08 CEST RepresentationInformation: ijmolsci_a2022v23n9p4958.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-05-06 10:37:58 CEST Size: 3077427 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 302 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Children: Item: Title: Rational Refining Procedure Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Protein Production and Characterization Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Morphometric Characterization and Zeta Potential Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Determination of Intrinsic Fluorescence and Unfolding Temperature Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Monomethyl Auristatin E Conjugation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Protein Modeling and Statistical Analyses Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Results and Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Children: Item: Title: In Silico Procedure Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Protein Expression and Purification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Stability Study, Size Distribution, and Nanoparticle Formation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Patents Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Info: Title: An In Silico Methodology That Facilitates Decision Making in the Engineering of Nanoscale Protein Materials Author: Eloi Parladé, Eric Voltà-Durán, Olivia Cano-Garrido, Julieta M. Sánchez, Ugutz Unzueta, Hèctor López-Laguna, Naroa Serna, Montserrat Cano, Manuel Rodríguez-Mariscal, Esther Vazquez and Antonio Villaverde Subject: Under the need for new functional and biocompatible materials for biomedical applications, protein engineering allows the design of assemblable polypeptides, which, as convenient building blocks of supramolecular complexes, can be produced in recombinant cells by simple and scalable methodologies. However, the stability of such materials is often overlooked or disregarded, becoming a potential bottleneck in the development and viability of novel products. In this context, we propose a design strategy based on in silico tools to detect instability areas in protein materials and to facilitate the decision making in the rational mutagenesis aimed to increase their stability and solubility. As a case study, we demonstrate the potential of this methodology to improve the stability of a humanized scaffold protein (a domain of the human nidogen), with the ability to oligomerize into regular nanoparticles usable to deliver payload drugs to tumor cells. Several nidogen mutants suggested by the method showed important and measurable improvements in their structural stability while retaining the functionalities and production yields of the original protein. Then, we propose the procedure developed here as a cost-effective routine tool in the design and optimization of multimeric protein materials prior to any experimental testing. 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