Application Notes

Detergent screen for solubilized membrane proteins – case study on the SLAC-protein HiTehA from haemophilus influenzae

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1 Thermal Unfolding of Membrane Proteins Application Note NT-PR-002 Detergent Screen for solubilized membrane proteins – Case study on the SLAC-protein HiTehA from Haemophilus influenzae Melanie Maschberger 1 , Stefanie Hüttl 2 , Thomas D. Mueller 2 and Dennis Breitsprecher 1 1 NanoTemper Technologies GmbH, Munich, Germany 2 Dept. Plant Physiology and Biophysics, Julius-von-Sachs Institute of the University Wuerzburg Abstract The biophysical characterization of integral membrane protein stability is often challenging due to several factors: First, the expression and purification of membrane proteins is often impeded by low expression levels and protein stability. As a result, yields are usually low and do not allow for a thorough analysis or a screening approach to determine optimal conditions. Second, the use of detergents – which are necessary to solubilize membrane proteins – often introduces artifacts and other secondary effects, and most importantly precludes the use of reporter dyes to monitor protein unfolding. Label free methods – such as DSC or CD spectroscopy – on the other hand require large quantities of proteins, and are limited in throughput. Here we use the 10 transmembrane-helix protein HiTehA, a protein of the slow anion channel family, to present label-free, native DSF as the method of choice to perform rapid and precise detergent screening projects for a solubilized membrane protein. Introduction Membrane proteins account for 20-30 % of the coding regions of all sequenced genomes and play crucial roles in many fundamental cell processes. For instance, ion channels, G-protein coupled receptors and carrier proteins are important in the regulation of a plethora of inter- and intramolecular processes. Defects in these proteins are often linked to a number of severe diseases thereby rendering them promising targets for novel drugs [1, 2]. However, obtaining sufficient quantities of a purified integral membrane protein for downstream experiments, such as structural or functional analysis in high-throughput screening approaches, can be challenging due to low yields and often poor stability [3]. Figure 1: Structure of HiTehA. A) Schematic representation of the domain organization of SLAC1-like transmembrane proteins. B) Crystal structure of HiTehA showing the quasi-five-fold symmetrical arrangement of transmembrane helices around the central pore.

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