Chemical Synthesis of a Full-Length G-Protein-Coupled Receptor β-Adrenergic Receptor with Defined Modification Patterns at the C-Terminus.

The β-adrenergic receptor (βAR) is a G-protein-coupled receptor (GPCR) that responds to the hormone adrenaline and is an important drug target in the context of respiratory diseases, including asthma. βAR function can be regulated by post-translational modifications such as phosphorylation and ubiquitination at the C-terminus, but access to the full-length βAR with well-defined and homogeneous modification patterns critical for biochemical and biophysical studies remains challenging. Here, we report a practical synthesis of differentially modified, full-length βAR based on a combined native chemical ligation (NCL) and sortase ligation strategy.

Analysis of βAR-G and βAR-G complex formation by NMR spectroscopy.

The β-adrenergic receptor (βAR) is a prototypical G protein-coupled receptor (GPCR) that preferentially couples to the stimulatory G protein G and stimulates cAMP formation. Functional studies have shown that the βAR also couples to inhibitory G protein G, activation of which inhibits cAMP formation [R. P.

Thermodynamic secrets of multidrug resistance: A new take on transport mechanisms of secondary active antiporters.

Multidrug resistance (MDR) presents a growing challenge to global public health. Drug extrusion transporters play a critical part in MDR; thus, their mechanisms of substrate recognition are being studied in great detail. In this work, we review common structural features of key transporters involved in MDR.

Crystal structures of MdfA complexed with acetylcholine and inhibitor reserpine.

The DHA12 family of transporters contains a number of prokaryotic and eukaryote membrane proteins. Some of these proteins share conserved sites intrinsic to substrate recognition, structural stabilization and conformational changes. For this study, we chose the MdfA transporter as a model DHA12 protein to study some general characteristics of the vesicular neurotransmitter transporters (VNTs), which all belong to the DHA12 family.

Substrate-bound structure of the E. coli multidrug resistance transporter MdfA.

Multidrug resistance is a serious threat to public health. Proton motive force-driven antiporters from the major facilitator superfamily (MFS) constitute a major group of multidrug-resistance transporters. Currently, no reports on crystal structures of MFS antiporters in complex with their substrates exist.

Structure of the YajR transporter suggests a transport mechanism based on the conserved motif A.

The major facilitator superfamily (MFS) is the largest family of secondary active transporters and is present in all life kingdoms. Detailed structural basis of the substrate transport and energy-coupling mechanisms of these proteins remain to be elucidated. YajR is a putative proton-driven MFS transporter found in many Gram-negative bacteria.

An efficient strategy for high throughput screening of recombinant integral membrane protein expression and stability.

Membrane proteins account for about 30% of the genomes sequenced to date and play important roles in a variety of cellular functions. However, determining the three-dimensional structures of membrane proteins continues to pose a major challenge for structural biologists due to difficulties in recombinant expression and purification. We describe here a high throughput pipeline for Escherichia coli based membrane protein expression and purification.