Decrypting the structure, function, and molecular interactions of complex molecular machines in their cellular context and at atomic resolution is of prime importance for understanding fundamental physiological processes. Nuclear magnetic resonance is a wellestablished imaging method that can visualize cellular entities at the micrometer scale and can be used to obtain 3D atomic structures under in vitro conditions. Here, we introduce a solid-state NMR approach that provides atomic level insights into cell-associated molecular components. By combining dedicated protein production and labeling schemes with tailored solid-state NMR pulse methods, we obtained structural information of a recombinant integral membrane protein and the major endogenous molecular components in a bacterial environment. Our approach permits studying entire cellular compartments as well as cell-associated proteins at the same time and at atomic resolution. cellular envelope | Escherichia coli | lipoprotein | PagL | magic angle spinning P hysiological processes rely on the concerted action of molecular entities in and across different cellular compartments. Whereas advancements in molecular imaging have provided unprecedented insights into the macromolecular organization in the subnanometer range (1), studying atomic structure and motion in situ has been challenging for structural biology. NMR has provided insight into cellular processes (2-4) and can determine entire 3D molecular structures inside living cells (5) provided that molecular entities tumble rapidly in a cellular setting. In principle, solid-state NMR (ssNMR) spectroscopy offers a complementary spectroscopic tool to monitor molecular structure and dynamics at atomic resolution in a complex setting (see ref. 6 for a recent review). Indeed, ssNMR has already been used to study individual molecular components in the context of natural bilayers (7,8), bacterial cell walls (9), and cellular organelles (10).Here, we introduce a general approach to investigate structure and dynamics of an arbitrary molecular target and its potential molecular partners in a cellular setting. Our studies focuses on the Gram-negative bacterial cell that is characterized by a molecularly complex but architecturally unique envelope, consisting of two lipid bilayers, the inner and outer membrane (IM, OM), separated by the periplasm containing the peptidoglycan (PG) layer (Fig. 1A). The IM is a phospholipid bilayer and harbors α-helical proteins, whereas the OM is asymmetrical and consists of phospholipids, lipopolysaccharides (LPS), lipoproteins, and β-barrelfold integral membrane proteins. LPS forms the outermost layer of the OM and protects the cell against harmful compounds from the environment. PG is a large macromolecule that gives the cell its shape and rigidity.Using uniformly 13 C, 15 N-labeled cellular preparations of Escherichia coli, we characterized the structure and dynamics of a recombinant integral membrane protein (PagL) and other major endogenous molecular components of the cell envelope in...
A peek inside: Dynamic nuclear polarization (DNP) enhances the spectroscopic sensitivity of solid‐state NMR measurements of uniformly (13C,15N)‐labeled preparations of Escherichia coli cells by more than an order of magnitude (see picture; MW=microwaves, ε=enhancement factor). The major molecular components in the cells can be characterized in this way.
Background: Outer membrane protein assembly is an incompletely understood process in Gram-negative bacteria. Results: A Neisseria meningitidis mutant lacking the lipoprotein GNA2091 is affected in growth and accumulates unassembled outer membrane proteins. Conclusion:We identified a novel component involved in outer membrane biogenesis. Significance: Our findings contribute to the understanding of a fundamental process occurring in Gram-negative bacteria.
The genome of the Gram-negative bacterium Pseudomonas putida harbours a complete set of xcp genes for a type II protein secretion system (T2SS). This study shows that expression of these genes is induced under inorganic phosphate (Pi ) limitation and that the system enables the utilization of various organic phosphate sources. A phosphatase of the PhoX family, previously designated UxpB, was identified, which was produced under low Pi conditions and transported across the cell envelope in an Xcp-dependent manner demonstrating that the xcp genes encode an active T2SS. The signal sequence of UxpB contains a twin-arginine translocation (Tat) motif as well as a lipobox, and both processing by leader peptidase II and Tat dependency were experimentally confirmed. Two different tat gene clusters were detected in the P. putida genome, of which one, named tat-1, is located adjacent to the uxpB and xcp genes. Both Tat systems appeared to be capable of transporting the UxpB protein. However, expression of the tat-1 genes was strongly induced by low Pi levels, indicating a function of this system in survival during Pi starvation.
Die Festkçrper-NMR(ssNMR)-Spektroskopie bietet zunehmende Mçglichkeiten, um komplexe Biomoleküle auf atomarer Ebene zu studieren (siehe z. B. Lit. [1]). Ein wichtiges Anwendungsgebiet betrifft Membranproteine, die nach Rekonstitution in synthetischen Lipiddoppelschichten ssNMRspektroskopisch untersucht werden kçnnen. Während es solche Präparationen erlauben, funktionelle Aspekte des Zielproteins zu untersuchen, kann der Einfluss der nativen zellulären Umgebung auf Struktur und Funktion des Proteins nicht studiert werden. Wir haben vor kurzem einen allgemeinen Ansatz eingeführt, um komplexe molekulare Strukturen, einschließlich integraler Membranproteine in nativer zellulärer Umgebung durch ssNMR-Spektroskopie unter Probenrotation im magischen Winkel [2] (MAS) zu untersuchen. [3] Wir konnten zeigen, dass mithilfe spezieller Probenpräparationsmethoden die Aufnahme hochaufgelçster ssNMR-Spektren von einheitlich 13 C, 15 N-markierten Proben vollständiger Escherichia-coli-Zellen (ZE) sowie deren Zellhüllen (ZH) mçglich ist. Deren Morphologie bleibt unter experimentellen Standardbedingungen der ssNMR-Spektroskopie erhalten, und die 13 C-und 15 N-Kreuzpolarisationssignale (CP-MAS) sind zeitlich invariant. Allerdings wird die spektroskopische Empfindlichkeit mit zunehmender Molekülkomplexität, vor allem im Fall von ZE-Proben, kritisch.In den letzten Jahren hat sich die dynamische Kernpolarisation (DNP) zu einem Routinewerkzeug zur Empfindlichkeitserhçhung in der multidimensionalen ssNMR-Spektroskopie entwickelt. [4] Signalverstärkungen um einen Faktor von bis zu 148 wurden an mikro/nanokristallinen Biomolekülen, wie z. B. einem amyloidogenen Peptid [5] und einem deuterierten Protein [6] beobachtet. Signalerhçhungen zwischen 18-und 46-fach wurden für membranständige Polypeptide, [7] Purpurmembranen [8] und Bakteriophagen [8b] beschrieben.Hier beschreiben wir die Anwendung von ssNMR-Experimenten an 13 C, 15 N-markierten E.-coli-Zellen, die PagL, ein Protein der äußeren Zellmembran, [9] überproduziert enthalten. In Abbildung 1 vergleichen wir 13 C-and 15 N-CP-MAS-Spektren von einheitlich 13 C, 15 N-markierten kompletten Zellen mit Zellhüllen, die von PagL-überproduzierenden E.coli-Zellen isoliert wurden. Die Daten wurden in An-und Abwesenheit der Mikrowellenstrahlung aufgenommen. Bei hçherer Temperatur (271 K) offenbarten die ssNMR-Daten der Zellhülle atomare Details von PagL und endogenen membranständigen Makromolekülen wie dem Hauptlipoprotein Lpp sowie von nicht-proteinhaltigen Komponenten wie Lipopolysacchariden (LPS), Peptidoglycan (PG) und Phospholipiden. [3] Bei tiefer Temperatur und DNP-Bedingungen beobachteten wir für beide Präparationen eine si-Abbildung 1. Vergleich von 13 C-(links) und 15 N-CP-MAS-Spektren (rechts) an (U-13 C, 15 N)-markierten ZHs (a) und ZEs (b) mit ("an", oben) und ohne ("aus", unten) DNP, mit einer Mikrowellenbestrahlungszeit von 10 s. Signifikante spektrale Unterschiede zwischen den Proben sind mit Pfeilen gekennzeichnet. Sternchen stehen für MAS-Seitenbanden, und Zuordnungen der molekulare...
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