Probing the allosteric NBD-TMD crosstalk in the ABC transporter MsbA by solid-state NMR.

The ABC transporter MsbA plays a critical role in Gram-negative bacteria in the regulation of the outer membrane by translocating core-LPS across the inner membrane. Additionally, a broad substrate specificity for lipophilic drugs has been shown. The allosteric interplay between substrate binding in the transmembrane domains and ATP binding and turnover in the nucleotide-binding domains must be mediated via the NBD/TMD interface. Previous studies suggested the involvement of two intracellular loops called coupling helix 1 and 2 (CH1, CH2). Here, we demonstrate by solid-state NMR spectroscopy that substantial chemical shift changes within both CH1 and CH2 occur upon substrate binding, in the ATP hydrolysis transition state, and upon inhibitor binding. CH2 is domain-swapped within the MsbA structure, and it is noteworthy that substrate binding induces a larger response in CH2 compared to CH1. Our data demonstrate that CH1 and CH2 undergo structural changes as part of the TMD-NBD cross-talk.

Rhodamine-Sensitized Two-Photon Activation of a Red Light-Absorbing BODIPY Photocage.

Two-photon (2P) activatable probes are of high value in biological and medical chemistry since near infrared (NIR) light can penetrate deeply even in blood-perfused tissue and due to the intrinsic three-dimensional activation properties. Designing two-photon chromophores is challenging. However, the two-photon absorption qualities of a photocage can be improved with an intramolecular sensitizer, which transfers the absorbed light onto the cage. We herein present the synthesis and photophysical characterization of a 2P-sensitive uncaging dyad based on rhodamine 101 as donor fluorophore and a redshifted BODIPY as acceptor photocage. Liberation of p-nitroaniline (PNA) upon one-photon photolysis was confirmed by HPLC analysis. The photoreaction was found to be accompanied by a considerable change of the fluorescence properties of the chromophores. The possibility of a fluorescent read-out enabled the detection of two-photon induced uncaging by confocal fluorescence microscopy.