Conformational Flexibility of the Protein Insertase BamA in the Native Asymmetric Bilayer Elucidated by ESR Spectroscopy

Abstract

The b-barrel assembly machinery(BAM) consisting of the central b-barrel BamA and four other lipoproteins mediates the folding of the majority of the outer membrane proteins.B amA is placed in an asymmetric bilayer and its lateral gate is suggested to be the functional hotspot. Here we used in situ pulsed electron-electron double resonance spectroscopytoc haracterize BamA in the native outer membrane. In the detergent micelles,the data is consistent with mainly an inward-open conformation of BamA. The native membrane considerably enhanced the conformational heterogeneity.T he lateral gate and the extracellular loop 3exist in an equilibrium between different conformations.T he outer membrane provides af avorable environment for occupying multiple conformational states independent of the lipoproteins.Our results reveal ahighly dynamic behavior of the lateral gate and other key structural elements and provide direct evidence for the conformational modulation of amembrane protein in situ.

Gd3+–Trityl–Nitroxide Triple Labeling and Distance Measurements in the Heterooligomeric Cobalamin Transport Complex in the Native Lipid Bilayers

“…Despite the vast amount of the structural, biochemical, and biophysical data available, what triggers the CNCbl release from BtuB is still a matter of debate. 33−38 Following the protocols previously established, 8,39,40 here, we labeled BtuB T426C (using MTS-OX063 or MTSL) directly in the native OM. Briefly, after overexpression of BtuB in E. coli, cells were lysed and the total membrane fraction was separated.…”

“…The BtuCD–F complex finally transports cobalamin into the cytoplasm at the expense of ATP binding and hydrolysis. Despite the vast amount of the structural, biochemical, and biophysical data available, what triggers the CNCbl release from BtuB is still a matter of debate. − Following the protocols previously established, ,, here, we labeled BtuB T426C (using MTS-OX063 or MTSL) directly in the native OM. Briefly, after overexpression of BtuB in E.…”

“…Observing the intermolecular interactions and their dynamics within a functional protein network calls for new approaches having high sensitivity and selectivity. − Pulsed electron–electron double resonance spectroscopy (PELDOR or DEER) is the most popular tool to measure long-range distances in proteins, even within their native surroundings. − The methane thiosulfonate nitroxide (NO) spin label (MTSL) combined with cysteine substitution is the most popular approach for labeling proteins . Other spin labels based on Gd 3+ , Mn 2+ , Cu 2+ , and trityl radicals are increasingly used, in particular for in situ studies. − Typical PELDOR experiments employing an identical spin pair limit the output to a single distance.…”

EPR Spectroscopy Provides New Insights into Complex Biological Reaction Mechanisms

“… 37 EPR experiments were used to target conformational and dynamical changes of the MscL ion channel. 38 Moreover, following in situ conformational changes of membrane transporters in a lipid environment 39 and in intact cells 40 opened many new routes for understanding mechanisms of ligand and ion transfer across the membrane. Electron spin–echo envelope modulation (ESEEM) spectroscopy has also been used to provide information on the gating mechanism of transporters and channels.…”

Lateral gating mechanism and plasticity of the β-barrel assembly machinery complex in micelles and Escherichia coli

“…Structures captured distinct states from this broad conformational space. Interestingly, when observed in the native OMs, BamA showed a heterogeneous conformation spanning multiple states ( 34 ). A systematic observation of BamAB, BamACDE, and BamABCDE complexes under identical conditions is necessary to exclude the differential effects of the environments and to elucidate the gating mechanism.…”