4th EucheMs chemistry congress

Poster Session 1
s887
chem. Listy 106, s587–s1425 (2012)
Poster session 1 - life sciences
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dnA dAMAGe reCoGnition By xPA/rAd14 in
nuCLeotide exCiSion rePAir
K. GASteiGer 1 , S. KoCh 1 , u. LiSChKe 1 ,
S. SChneider 2 , t. CAreLL 1
1 Ludwig-Maximilians-Universität, Chemistry, München,
Germany
2 Technische Universität München, Biochemistry, München,
Germany
DNA is constantly subjected to damaging agents such as
UV- or γ-irradiation, as well as oxidation and hydrolysis or
chemical carcinogenes. To avoid degeneration of the genetic
information, nature developed a variety of DNA repair pathways
to repair the resulting lesions. One of the main genome
maintenance systems in eukaryotes is the Nucleotide Excision
Repair (NER). NER is a multistep process that includes damage
recognition, assembly of repair factors at the damaged site, dual
incision on either side of the lesion containing oligonucleotide,
which results in an excision of the lesion, followed by DNA
synthesis to fill the gap. A defective NER response in humans
leads to a high predisposition to skin cancer (Xermoderma
Pigmentosum). [1] XPC/Rad 4 is known to detect disrupted duplex
structures [2] , whereas XPA/Rad14 is thought to be required for the
binding of bulky DNA adducts generated by chemical
carcinogens, like aromatic amines. We want to elucidate the exact
mechanism of the recognition of bulky adducts by NER
recognition factors. Therefore we investigate the binding affinity
of the human XPA and its yeast homolog Rad14 to DNA
containing bulky lesions by EMSA. The crystal structure of Rad14
in complex with an acetylaminofluorene-dG containing
oligonucleotide will give us insight into the mechanism of damage
recognition of bulky adducts in NER.
references:
1. M. T. Hess, D. Gunz, H. Naegeli, Nucleic Acids Research
1996, 24 (5), 824-828.
2. J. H. Min, N. P. Pavletich, Nature 2007, 449, 570-575.
Keywords: NER; XPA/Rad14;
4 th EucheMs chemistry congress
P - 0 0 5 6
MonitorinG ChAnneL Protein–MediAted
trAnSLoCAtion of ArGinine–riCh PePtideS By
A SuPrAMoLeCuLAr SenSinG enSeMBLe
G. GhALe 1 , h. weinGArt 1 , M. winterhALter 1 ,
w. M. nAu 1
1 Jacobs University Bremen, School of Engineering and Science,
Bremen, Germany
The influx of xenobiotics such as antibiotics, peptides, and
other biologically relevant molecules through channel proteins,
or in general, through lipid bilayers, is crucial for medicinal,
biotechnological, and analytical applications and also for
understanding the translocation processes. [1–4] Consequently,
assays to detect the flux of molecules through these channels with
high sensitivity, versatility, and scalability for high-throughput
screening is in high demand in the pharmaceutical industry, and
biochemical research laboratories. [5–7] However, the techniques for
accurate monitoring of this process are limited, in particular those
allowing continuous real-time measurement of concentration
changes.
Drawing inspiration from supramolecular tandem assays, [8–11]
we introduce herein a simple yet effective fluorescence based
strategy to study the diffusion of molecules through the membrane
or channel proteins, namely supramolecular tandem membrane
assay. The underlying principle is to encapsulate a macrocyclic
host and a fluorescent probe inside the liposomes. This leads to a
compartmentalization of the sensing ensemble inside the
proteoliposomes, such that one can follow the permeation of
analytes in real-time via fluorescence, because the host/dye
complex will respond only to analytes which enter into the inside
of the vesicles, where they displace the dye from the macrocycle
and trigger the fluorescence response.
Owing to their impermeablity through the lipid membrane,
a anionic macrocyclic receptor p-sulfonatocalix[4]arene (CX4)
and a highly fluorescent dye lucigenin (LCG) were selected as a
host/dye reporter pair to investigate the translocation of
protamine, an arginine-rich antimicrobial peptide through a
bacterial outer membrane channel protein F (OmpF). [10, 12, 13] The
translocation of protamine into the liposomes was monitored as a
time-resolved increase in fluorescence intensity due to the release
of the dye LCG from the CX4 cavity. Furthermore, using the
tandem membrane assay method, we were able to screen for other
translocating analytes, the activity of mutagenized OmpF, and the
effect of channel modulators on the translocation process.
references:
1. Han, J.; Nilius, B.; Earm, Y. E.; Noble, D. Progress in
Biophysics & Molecular Biology 2010, 103, 1.
2. Housden, N. G.; Wojdyla, J. A.; Korczynska, J.;
Grishkovskaya, I.; Kirkpatrick, N.; Brzozowski, A. M.;
Kleanthous, C. Proc. Natl. Acad. Sci. U. S. A. 2010, 107,
21412.
3. Imming, P.; Sinning, C.; Meyer, A. Nat. Rev. Drug
Discovery 2006, 5, 821.
4. Nestorovich, E. M.; Danelon, C.; Winterhalter, M.;
Bezrukov, S. M. Proc. Natl. Acad. Sci. U. S. A. 2002, 99,
9789.
5. Dunlop, J.; Bowlby, M.; Peri, R.; Vasilyev, D.; Arias, R.
Nat. Rev. Drug Discovery 2008, 7, 358.
6. Mahendran, K. R.; Hajjar, E.; Mach, T.; Lovelle, M.;
Kumar, A.; Sousa, I.; Spiga, E.; Weingart, H.; Gameiro, P.;
Winterhalter, M.; Ceccarelli, M. J. Phys. Chem. B 2010,
114, 5170.
AUGUst 26–30, 2012, PrAGUE, cZEcH rEPUbLIc