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.