Studying ion channel conformation dynamics by encoding coumarin as unnatural amino acid.

Monitoring the conformational changes of proteins is critical to understand their function. Ion channels are membrane-bound minute machines controlling the passage of ions across biological membranes. The precise labeling of ion channels with fluorescent probes allows studying their dynamics and facilitates their characterization by high-resolution optical techniques. Here we describe a protocol for the use of a small fluorescent reporter, incorporated by expansion of the genetic code in the host cell. An important advantage of using small probes is that they are less likely to perturb protein structure, function, and trafficking. In our hands, Tyr-coumarin proved to be useful to measure the conformational changes occurring in the narrow space of the permeation pathway in single capsaicin receptors. The method described here could be directly translated to the study of membrane receptors, non-electrogenic transporters, or membrane-bound enzymes.

Human CCAAT/enhancer-binding protein ß interacts with chromatin remodeling complexes of the imitation switch subfamily.

Transcription factor C/EBPß is involved in several cellular processes, such as proliferation, differentiation, and energy metabolism. This factor exerts its activity through recruitment of different proteins or protein complexes, including the ATP-dependent chromatin remodeling complex SWI/SNF. The C/EBPß protein is found as three major isoforms, C/EBPß1, -2, and -3. They are generated by translation at alternative AUG initiation codons of a unique mRNA, C/EBPß1 being the full-length isoform. It has been found that C/EBPß1 participates in terminal differentiation processes. Conversely, C/EBPß2 and -3 promote cell proliferation and are involved in malignant progression in a number of tissues. The mechanisms by which C/EBPß2 and -3 promote cell proliferation and tumor progression are not fully understood. In this work, we sought to identify proteins interacting with hC/EBPß using a proteomics approach. We found that all three isoforms interact with hSNF2H and hACF, components of ACF and CHRAC chromatin remodeling complexes, which belong to the imitation switch subfamily. Additional protein-protein interaction studies confirmed this finding and also showed that hC/EBPß directly interacts with hACF1. By overexpressing hC/EBPß, hSNF2H, and hACF1 in HepG2 cells and analyzing variations in expression of cyclin D1 and other C/EBPß target genes, we observed a functional interaction between C/EBPß and SNF2H/ACF1, characterized mainly by suppression of C/EBPß transactivation activity in the presence of SNF2H and ACF1. Consistent with these findings, induction of differentiation of HepG2 cells by 1% DMSO was accompanied by a reduction in the level of cyclin D1 expression and the appearance of hC/EBPß, hSNF2H, and hACF1 on the promoter region of this gene.