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Volume 10, Issue 8 (Suppl)

J Proteomics Bioinform, an open access journal

ISSN: 0974-276X

Structural Biology 2017

September 18-20, 2017

International Conference on

Structural Biology

September 18-20, 2017 Zurich, Switzerland

The dynamics of a protein during its insertion into a membrane

Andreas Kuhn, Dirk Spann

and

Maximilian Haase

University of Hohenheim, Germany

ost membrane proteins are inserted co-translationally by the Sec-translocase or the YidC/Oxa1/Alb3 insertases. The

folding of these proteins occurs within the membrane during the interaction with the insertases. We have purified

and reconstituted YidC, the membrane insertase of

Escherichia coli

. The protein spans the membrane 6 times, and the

recently solved structure shows a hydrophilic cavity and a greasy slide between the transmembrane segments TM3 and TM5.

Hydrophobic residues of TM3 and TM5 interact with the substrate, with a prospective transmembrane segment of an inserting

membrane protein as documented by disulfide crossinking experiments. The membrane insertion process can be studied with

the reconstituted vesicle system. The purified substrate proteins are solubilized in 10% isopranol or kept unfolded with urea

or GuHCl. When the substrate proteins are added to the proteoliposomes by dilution 1:100, they rapidly bind to YidC and

become membrane inserted within 2 msec. FRET-based kinetic measurements show that the substrate proteins approach YidC

to a close distance during the insertion event. Time-resolved fluorenscence anisotropy shows that the periplasmic domain

of YidC moves when a substrate protein was added. This suggests that both the insertase and the substrate protein undergo

conformational motions.

Biography

Andreas Kuhn has his expertise in protein folding of membrane proteins. Studies include reconstituted systems with bacterial translocases and insertase, as well

in vivo

studies with

Escherichia coli

. For biophysical experiments, the membrane proteins are purified and their folding is monitored spectroscopically in real time

after their addition to liposomes. Andreas Kuhn obtained his PhD from the Universities Basel and Freiburg im Breisgau 1982. After a Postdoc at UCLA with Bill

Wickner he continued at the Biozentrum Basel from 1986 to 1989 and accepted a professorship at the University Karlsruhe. Since 1996 he is at the University of

Hohenheim in Stuttgart.

Andreas.Kuhn@uni-hohenheim.de

Andreas Kuhn et al., J Proteomics Bioinform 2017, 10:8(Suppl)

DOI: 10.4172/0974-276X-C1-0100

Figure1:

YidC-mediated insertion of the Pf3 coat protein