2
One way to stabilize membrane proteins after
purification and to make them amenable for
subsequent biophysical analysis is solubilization
with detergent additives [4].
Identifying the right detergent and buffer conditions
however is a challenge by itself, since the low
concentrations and yields of membrane proteins
usually pose a bottleneck for buffer screening
approaches by methods such as DSC or CD-
spectroscopy. Moreover, commonly used
fluorometric approaches with reporter dyes – such
as DSF with SyproOrange – are not compatible
with detergent-containing formulations.
Here we present a label-free, fluorometric high-
throughput approach to monitor thermal stability of
a transmembrane protein.
Using the Prometheus NT.48, we performed a
detergent screen on the tellurite resistance protein
A from Haemophilus influenzae (HiTehA). The
integral membrane protein TehA together with
TehB, a soluble cytoplasmic protein, confers
resistance to tellurite in bacteria by an unknown
mechanism [5]. Later studies showed that the
integral membrane protein TehA possibly
transports lipophilic quaternary ammonium
compounds [6]. Sequence analyses predicted an
architecture containing 10 transmembrane helices
(Figure 1A) [6]. Crystal structure analysis of HiTehA
confirmed this architecture, and revealed a novel
transmembrane fold (Figure 1B): five helix-tandems
are arranged in a quasi-five-fold symmetry,
resulting in five inner and five outer helices
traversing the cell membrane, surrounding the
central pore [7].
Table 1: List of detergents used in this study
