دورية أكاديمية
أعرض في EDS

High-throughput structure determination of an intrinsically disordered protein using cell-free protein crystallization.

التفاصيل البيبلوغرافية
العنوان: High-throughput structure determination of an intrinsically disordered protein using cell-free protein crystallization.
المؤلفون: Kojima M; School of Life Science and Technology, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan., Abe S; School of Life Science and Technology, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan., Furuta T; School of Life Science and Technology, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan., Hirata K; Synchrotron Radiation Life Science Instrumentation Unit, RIKEN/SPring-8 Center, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan., Yao X; School of Life Science and Technology, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan., Kobayashi A; School of Life Science and Technology, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan., Kobayashi R; School of Life Science and Technology, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan., Ueno T; School of Life Science and Technology, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan.; Research Center for Autonomous Systems Materialogy (ASMat), Institute of Innovative Research, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan.
المصدر: Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2024 Jun 18; Vol. 121 (25), pp. e2322452121. Date of Electronic Publication: 2024 Jun 11.
نوع المنشور: Journal Article
اللغة: English
بيانات الدورية: Publisher: National Academy of Sciences Country of Publication: United States NLM ID: 7505876 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1091-6490 (Electronic) Linking ISSN: 00278424 NLM ISO Abbreviation: Proc Natl Acad Sci U S A Subsets: MEDLINE
أسماء مطبوعة: Original Publication: Washington, DC : National Academy of Sciences
مواضيع طبية MeSH: Intrinsically Disordered Proteins*/chemistry , Crystallization* , Proto-Oncogene Proteins c-myc*/chemistry , Proto-Oncogene Proteins c-myc*/genetics , Proto-Oncogene Proteins c-myc*/metabolism , Cell-Free System*, Crystallography, X-Ray/methods ; Humans ; Protein Conformation ; Models, Molecular ; Protein Binding
مستخلص: Intrinsically disordered proteins (IDPs) play a crucial role in various biological phenomena, dynamically changing their conformations in response to external environmental cues. To gain a deeper understanding of these proteins, it is essential to identify the determinants that fix their structures at the atomic level. Here, we developed a pipeline for rapid crystal structure analysis of IDP using a cell-free protein crystallization (CFPC) method. Through this approach, we successfully demonstrated the determination of the structure of an IDP to uncover the key determinants that stabilize its conformation. Specifically, we focused on the 11-residue fragment of c-Myc, which forms an α-helix through dimerization with a binding partner protein. This fragment was strategically recombined with an in-cell crystallizing protein and was expressed in a cell-free system. The resulting crystal structures of the c-Myc fragment were successfully determined at a resolution of 1.92 Å and we confirmed that they are identical to the structures of the complex with the native binding partner protein. This indicates that the environment of the scaffold crystal can fix the structure of c-Myc. Significantly, these crystals were obtained directly from a small reaction mixture (30 µL) incubated for only 72 h. Analysis of eight crystal structures derived from 22 mutants revealed two hydrophobic residues as the key determinants responsible for stabilizing the α-helical structure. These findings underscore the power of our CFPC screening method as a valuable tool for determining the structures of challenging target proteins and elucidating the essential molecular interactions that govern their stability.
Competing Interests: Competing interests statement:The authors declare no competing interest.
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معلومات مُعتمدة: JP19H02830 JP20K21244 MEXT | Japan Society for the Promotion of Science (JSPS); JPMJTR20U1 MEXT | JST | Adaptable and Seamless Technology Transfer Program through Target-Driven R and D (A-STEP); JP18K05140 MEXT | Japan Society for the Promotion of Science (JSPS)
فهرسة مساهمة: Keywords: X-ray crystallography; cell-free protein crystallization; intrinsically disordered protein; nano crsytal
المشرفين على المادة: 0 (Intrinsically Disordered Proteins)
0 (Proto-Oncogene Proteins c-myc)
تواريخ الأحداث: Date Created: 20240611 Date Completed: 20240611 Latest Revision: 20240626
رمز التحديث: 20240626
مُعرف محوري في PubMed: PMC11194560
DOI: 10.1073/pnas.2322452121
PMID: 38861600
قاعدة البيانات: MEDLINE
الوصف
تدمد:1091-6490
DOI:10.1073/pnas.2322452121