High‐Resolution Protein 3D Structure Determination in Living Eukaryotic Cells. (25th April 2019)

• Record Type: Journal Article
• Authors: Tanaka, Takashi; Ikeya, Teppei; Kamoshida, Hajime; Suemoto, Yusuke; Mishima, Masaki; Shirakawa, Masahiro; Güntert, Peter; Ito, Yutaka
• Is Part Of: Angewandte Chemie. Volume 131:Number 22(2019)
• Page Start: 7362
• Page End: 7366
• Publication Date: 2019-04-25
• Subjects: Eukaryotische Zellen -- Makromolekulares Crowding -- NMR-Strukturbestimmung -- NOESY -- Proteine; Chemistry -- Periodicals; 540
• DOI: 10.1002/ange.201900840
• Languages: English
• ISSNs: 0044-8249

Abstract

Abstract: Proteins in living cells interact specifically or nonspecifically with an enormous number of biomolecules. To understand the behavior of proteins under intracellular crowding conditions, it is indispensable to observe their three‐dimensional (3D) structures at the atomic level in a physiologically natural environment. We demonstrate the first de novo protein structure determinations in eukaryotes with the sf9 cell/baculovirus system using NMR data from living cells exclusively. The method was applied to five proteins, rat calmodulin, human HRas, human ubiquitin, T. thermophilus HB8 TTHA1718, and Streptococcus protein G B1 domain. In all cases, we could obtain structural information from well‐resolved in‐cell 3D nuclear Overhauser effect spectroscopy (NOESY) data, suggesting that our method can be a standard tool for protein structure determinations in living eukaryotic cells. For three proteins, we achieved well‐converged 3D structures. Among these, the in‐cell structure of protein G B1 domain was most accurately determined, demonstrating that a helix‐loop region is tilted away from a β‐sheet compared to the conformation in diluted solution. Abstract : Protein‐Shake : 3D‐Proteinstrukturen wurden auf Basis von Informationen aus lebenden eukaryotischen Zellen bestimmt. Mit intrazellulärer NMR‐Spektroskopie wurden gut aufgelöste 3D‐Spektren und Strukturinformationen für fünf Modellproteine erhalten. Ein deutlicher Strukturunterschied wurde gegenüber verdünnter Lösung beobachtet, wahrscheinlich infolge dichter Molekülpackung oder anderer intrazellulärer Effekte.