On the efficiency of NHS ester cross-linkers for stabilizing integral membrane protein complexes.

We have previously presented a straightforward approach based on high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) to study membrane proteins. In addition, the stoichiometry of integral membrane protein complexes could be determined by MALDI-MS, following chemical cross-linking via glutaraldehyde. However, glutaraldehyde polymerizes in solution and reacts nonspecifically with various functional groups of proteins, limiting its usefulness for structural studies of protein complexes.

Structure of AMP-PNP-bound BtuCD and mechanism of ATP-powered vitamin B12 transport by BtuCD-F.

The reaction mechanism of BtuCD-F-catalyzed vitamin B12 transport into Escherichia coli is currently unclear. Here we present the structure of the last missing state in the form of AMP-PNP-bound BtuCD, trapped by a disulfide cross-link. Our structural and biochemical data allow a consistent mechanism to be formulated, thus rationalizing the roles of substrate, ATP and substrate-binding protein.

High-mass matrix-assisted laser desorption ionization-mass spectrometry of integral membrane proteins and their complexes.

Analyzing purified membrane proteins and membrane protein complexes by mass spectrometry has been notoriously challenging and required highly specialized buffer conditions, sample preparation methods, and apparatus. Here we show that a standard matrix-assisted laser desorption/ionization (MALDI) protocol, if used in combination with a high-mass detector, allows straightforward mass spectrometric measurements of integral membrane proteins and their complexes, directly following purification in detergent solution. Molecular weights can be determined precisely (mass error ≤ 0.

Structure of AMP-PNP-bound vitamin B12 transporter BtuCD-F.

The ATP-binding cassette (ABC) transporter BtuCD mediates the uptake of vitamin B(12) across the inner membrane of Escherichia coli. Previous structures have shown the conformations of apo states, but the transport mechanism has remained unclear. Here we report the 3.

Asymmetric states of vitamin B₁₂ transporter BtuCD are not discriminated by its cognate substrate binding protein BtuF.

BtuCD is an ABC transporter catalyzing the uptake of vitamin B₁₂ across the Escherichia coli inner membrane. A previously reported X-ray structure of BtuCD in complex with the periplasmic vitamin B₁₂-binding protein BtuF revealed asymmetry of the transmembrane BtuC subunits. The functional relevance of this asymmetry has remained uncertain.