The solution structure of the C-terminal domain of NfeD reveals a novel membrane-anchored OB-fold (original) (raw)
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Biochemistry, 2008
FomA, the major outer membrane protein of Fusobacterium nucleatum, was expressed and purified in Escherichia coli and reconstituted from detergent in bilayer membranes of phosphatidylcholines with chain lengths from C(12:0) to C(17:0). The conformation and orientation of membrane-incorporated FomA were determined from polarized, attenuated total reflection, infrared (IR) spectroscopy, and lipid-protein interactions with FomA were characterized by using electron paramagnetic resonance (EPR) spectroscopy of spin-labeled lipids. Approximately 190 residues of membranous FomA are estimated to be in a -sheet configuration from IR band fitting, which is consistent with a 14-strand transmembrane -barrel structure. IR dichroism of FomA indicates that the -strands are tilted by ∼45°relative to the sheet/barrel axis and that the order parameter of the latter displays a discontinuity corresponding to hydrophobic matching with fluid C(13:0) lipid chains. The stoichiometry (N b ) 23 lipids/monomer) of lipid-protein interaction from EPR demonstrates that FomA is not trimeric in membranes of diC(14:0) phosphatidylcholine and is consistent with a monomeric -barrel of 14-16 strands. The pronounced selectivity of interaction found with anionic spin-labeled lipids places basic residues of the protein in the vicinity of the polar-apolar membrane interfaces, consistent with current topology models. Comparison with similar data from the 8-to 22-stranded E. coli outer membrane proteins, OmpA, OmpG, and FhuA, supports the above conclusions.
Membrane mediated phase separation of the bacterial nucleoid occlusion protein Noc
Scientific Reports
Liquid–liquid phase separation is a fundamental biophysical process to organize eukaryotic and prokaryotic cytosols. While many biomolecular condensates are formed in the vicinity of, or even on lipid membranes, little is known about the interaction of protein condensates and lipid bilayers. In this study, we characterize the recently unknown phase behavior of the bacterial nucleoid occlusion protein Noc. We find that, similarly to other ParB-like proteins, CTP binding tightly regulates Noc’s propensity to phase separate. As CTP-binding and hydrolysis also allows Noc to bind and spread on membranes, we furthermore establish Noc condensates as model system to investigate how lipid membranes can influence protein condensation and vice versa. Last, we show that Noc condensates can recruit FtsZ to the membrane, while this does not happen in the non-phase separated state. These findings suggest a new model of Noc mediated nucleoid occlusion, with membrane-mediated liquid–liquid phase sep...
Recent advances in the understanding of membrane protein assembly and structure
1999
For a variety of reasons–not the least biomedical importance–integral membrane proteins are now very much in focus in many areas of molecular biology, biochemistry, biophysics, and cell biology. Our understanding of the basic processes of membrane protein assembly, folding, and structure has grown significantly in recent times, both as a result of new methodological developments, more high-resolution structure data, and the possibility to analyze membrane proteins on a genome-wide scale.
Membrane Topology of the 60-kDa Oxa1p Homologue from Escherichia coli
Journal of Biological Chemistry, 1998
We have characterized the membrane topology of a 60-kDa inner membrane protein from Escherichia coli that is homologous to the recently identified Oxa1p protein in Saccharomyces cerevisiae mitochondria implicated in the assembly of mitochondrial inner membrane proteins. Hydrophobicity and alkaline phosphatase fusion analyses suggest a membrane topology with six transmembrane segments, including an N-terminal signal-anchor sequence not present in mitochondrial Oxa1p. In contrast to partial N-terminal fusion protein constructs, the full-length protein folds into a proteaseresistant conformation, suggesting that important folding determinants are present in the C-terminal part of the molecule.
The machinery of membrane protein assembly
2004
Introduction The prediction of the three-dimensional structure of a membrane protein (MP) from its sequence requires an understanding of two fundamental issues: mechanisms of the biological assembly of MPs and the principles behind the physical stability of MPs in their natural lipid bilayer milieu. The physical principles behind MP stability have recently been addressed in several reviews [1–3], as have the mechanisms of assembly [4–6].