Highly selective cross-linking Highly selective cross-linking techniques techniques Dmitry Kolpashchikov Dmitry Kolpashchikov Department of Medicine Department of Medicine Columbia University Columbia University 650 West 168 Street, BB#806 650 West 168 Street, BB#806 New York, NY 10032 New York, NY 10032 Tel: 212-342-5610 Tel: 212-342-5610 Fax: 212-305-3475 Fax: 212-305-3475 E.mail: E.mail: [email protected][email protected]Kolpashchikov D.M. Superselective labelling of proteins: approaches and techniques. J. Biomol. Struct.Dyn. 2003 55-64. Novosibirsk Institute of Bioorganic Chemistry, Novosibirsk, Novosibirsk Institute of Bioorganic Chemistry, Novosibirsk, Russia; Russia; National Institute of Genetics, Mishima, Japan; National Institute of Genetics, Mishima, Japan; National Institute of Environmental Health Sciences, North National Institute of Environmental Health Sciences, North Carolina, USA; Carolina, USA; Columbia University, New York, USA. Columbia University, New York, USA.
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• • Affinity Cross-linking is less efficient then X-ray Affinity Cross-linking is less efficient then X-ray crystallography and NMR for structural studies of crystallography and NMR for structural studies of purified biopolymers;purified biopolymers;
• • The method might be useful for the studying of The method might be useful for the studying of multicomponent systems, such as complex multicomponent systems, such as complex reconstitutedreconstitutedsystems, cellular extracts and living cells;systems, cellular extracts and living cells;
• • Being highly selective cross-linking techniques could Being highly selective cross-linking techniques could be used for drug design or as an analytical tools.be used for drug design or as an analytical tools.
Labelling using binary photoaffinity Labelling using binary photoaffinity reagentreagent
Novosibirsk Institute of Bioorganic Chemistry, Novosibirsk, RussiaNovosibirsk Institute of Bioorganic Chemistry, Novosibirsk, Russia
Catalytically competent labelling
M. A.Grachev, T. I. Kolocheva, E. A. Lukhtanov and A. A. Mustaev, Eur. J. Biochem. 163, 113-121 (1987).
Mustaev A, Zaychikov E, Grachev M, Kozlov M, Severinov K, Epshtein V, Korzheva N, Bereshchenko O, Markovtsov V, Lukhtanov E, Tsarev I, Maximova T, Kashlev M, Bass I, Nikiforov V, Goldfarb A. Strategies and methods of cross-linking of RNA polymerase active center.Methods Enzymol. 2003;371:191-206.
binding sites for two ligands
→
→
reagent
Influenza virus RNA polymerase
ATP analogue (I,II or III) [ -32P]GTP
(Lys, His)
(Lys, after NaBH4 treatment)
(Lys, after NaBH4 treatment)
vRNAPB1
PB2
PA
RNP
NP
R-Ap*G
*
*
*
Kolpashchikov DM, Honda A., Ishihama A. Structure-function relationship of the influenza virus RNA polymerase: primer-binding site on the PB1 subunit. Biochemistry. 2004, 43, 5882-7
Binary photoaffinity reagent
→→
→
Binary photo-affinity reagent for labeling of DNA polymerases
Kolpashchikov D.M., Rechkunova N.I., Dobrikov M.I., Khodyreva S.N., Lebedeva N.A., Lavrik O.I (1999) Sensitized photomodification of mammalian DNA polymerase beta. A new approach for highly selective affinity labeling of polymerases. FEBS Lett., V. 448, P. 141-144.
Lavrik OI, Kolpashchikov DM, Prasad R, Sobol RW, Wilson SH. Binary system for selective photoaffinity labeling of base excision repair DNA polymerases.Nucleic Acids Res. 2002; 30, e73
Binary photoaffinity reagent can improve cross-linking efficiency
Lebedeva N.A., Kolpashchikov D.M., Rechkunova N.I., Khodyreva S.N., Lavrik O.I. (2001) A binary system of photoreagents for high-efficiency labeling of DNA polymerases. Biochem. Biophys. Res. Commun. V. 287, P. 530-535.
Babendure JR, Adams SR, Tsien RY. Aptamers switch on fluorescence of triphenylmethane dyes J Am Chem Soc. 2003,125,14716-7.
MG
MG
MGMG
MG
Fluorescence
0 20 40 60 80
1
2
3
4
5
5’- GA GAG AGT TGG GTG CGA GAT
5’-GGA AG AGT TGG GTG CGA GAT
5’-GGA GAG A T TGG GTG CGA GAT
5’-GGA GAG AGT TGG GTG CGA GA
No DNA analyte0.7
65.8
14.0
1.6
11.3
Dezhurov S.V., Dr. Lebedeva N.A., Prof. Lavrik O.I. Dezhurov S.V., Dr. Lebedeva N.A., Prof. Lavrik O.I. (Novosibirsk Institute of Bioorganic Chemistry);(Novosibirsk Institute of Bioorganic Chemistry);
Dr. Honda A., Prof. Ishihama A., (National Institute of Dr. Honda A., Prof. Ishihama A., (National Institute of Genetics, Mishima, Japan);Genetics, Mishima, Japan);
Dr. Prasad R., Dr. Sobol R.W., Prof. Wilson S.H. (National Dr. Prasad R., Dr. Sobol R.W., Prof. Wilson S.H. (National Institute of Environmental Health Sciences, North Carolina, Institute of Environmental Health Sciences, North Carolina, USA);USA);
Semova S., Dr. Stojanovic M.N., (Columbia University, New Semova S., Dr. Stojanovic M.N., (Columbia University, New York).York).