Combined 1H-Detected solid-state NMR spectroscopy and electron cryotomography to study membrane proteins across resolutions in native environments

Membrane proteins remain challenging targets for structural biology, despite much effort, as their native environment is heterogeneous and complex. Most methods rely on detergents to extract membrane proteins from their native environment, but this removal can significantly alter the structure and function of these proteins. Here, we overcome these challenges with a hybrid method to study membrane proteins in their native membranes, combining high-resolution solid-state nuclear magnetic resonance spectroscopy and electron cryotomography using the same sample. Our method allows the structure and function of membrane proteins to be studied in their native environments, across different spatial and temporal resolutions, and the combination is more powerful than each technique individually. We use the method to demonstrate that the bacterial membrane protein YidC adopts a different conformation in native membranes and that substrate binding to YidC in these native membranes differs from purified and reconstituted system

SecA mediated protein translocation in Escherichia coli

The movement of proteins across or integration of proteins into the cytoplasmic membrane of Escherichia coli is mediated by the multimeric membrane protein complex translocase. The core of the translocase consists of a motor protein, the ATPase SecA, and a protein-conducting channel, formed by the integral membrane proteins SecY and SecE. The SecYE complex is highly conserved, with homologs in the cytoplasmic membrane of Archae, the chloroplast thylakoid membrane, and the eukaryotic endoplasmatic reticulum (ER). SecA is unique for the bacterial post-translational translocation pathway. It is absent both in Archaea and in the eukaryotic ER, but a homolog exists in plant chloroplasts. SecA is an soluble protein that distributes between cytoplasmic and membrane associated states. The interaction with the cytoplasmic membrane occurs via low affinity interactions with anionic phospholipids and by a high affinity interaction with the SecYEG complex. At the membrane SecA forms a receptor for preproteins and drives their stepwise movement through the SecYEG channel [93, 173]. ... Zie: Summary.

SecA mediated protein translocation in escherichia coli

+103hlm.;25c

Arginine 357 of SecY is needed for SecA-dependent initiation of preprotein translocation

The Escherichia coli SecYEG complex forms a transmembrane channel for both protein export and membrane protein insertion. Secretory proteins and large periplasmic domains of membrane proteins require for translocation in addition the SecA ATPase. The conserved arginine 357 of SecY is essential for a yet unidentified step in the SecA catalytic cycle. To further dissect its role, we have analysed the requirement for R357 in membrane protein insertion. Although R357 substitutions abolish post-translational translocation, they allow the translocation of periplasmic domains targeted co-translationally by an N-terminal transmembrane segment. We propose that R357 is essential for the initiation of SecA-dependent translocation only.

Kinetic Analysis of the Translocation of Fluorescent Precursor Proteins into Escherichia coli Membrane Vesicles

Protein secretion in Escherichia coli is mediated by translocase, a multi-subunit membrane protein complex with SecA as ATP-driven motor protein and the SecYEG complex as translocation pore. A fluorescent assay was developed to facilitate kinetic studies of protein translocation. Single cysteine mutants of proOmpA were site-specific labeled with fluorescent dyes, and the SecA and ATP-dependent translocation into inner membrane vesicles and SecYEG proteoliposomes was monitored by means of protease accessibility and in gel fluorescent imaging. The translocation of fluorescently labeled proOmpA was largely independent on the position and the size of the fluorescent label (up to a size of 13–16 Å). A fluorophore at the +4 position blocked translocation, but inhibition was completely relieved in the PrlA4 mutant. The kinetics of translocation of the fluorescently labeled proOmpA could be directly monitored by means of fluorescence quenching. Inner membrane vesicles containing wild-type SecYEG were found to translocate proOmpA with a turnover of 4.5 molecules proOmpA/SecYEG complex/min and an apparent Km of 180 nM, whereas the PrlA4 mutant showed an almost 10-fold increase in turnover rate and a 3-fold increase of the apparent Km for proOmpA translocation.

Competitive Binding of the SecA ATPase and Ribosomes to the SecYEG Translocon

During co-translational membrane insertion of membrane proteins with large periplasmic domains, the bacterial SecYEG complex needs to interact both with the ribosome and the SecA ATPase. Although the binding sites for SecA and the ribosome overlap, it has been suggested that these ligands can interact simultaneously with SecYEG. We used surface plasmon resonance and fluorescence correlation spectroscopy to examine the interaction of SecA and ribosomes with the SecYEG complex present in membrane vesicles and the purified SecYEG complex present in a detergent-solubilized state or reconstituted into nanodiscs. Ribosome binding to the SecYEG complex is strongly stimulated when the ribosomes are charged with nascent chains of the monotopic membrane protein FtsQ. This binding is competed by an excess of SecA, indicating that binding of SecA and ribosomes to SecYEG is mutually exclusive.