Highly selective cross-linking Highly selective cross-linking techniquestechniques

Dmitry KolpashchikovDmitry KolpashchikovDepartment of Medicine Department of Medicine

Columbia University Columbia University 650 West 168 Street, BB#806650 West 168 Street, BB#806

New York, NY 10032New York, NY 10032Tel: 212-342-5610Tel: 212-342-5610Fax: 212-305-3475Fax: 212-305-3475

E.mail: E.mail: dk2111@columbia.edudk2111@columbia.edu

Kolpashchikov D.M. Superselective labelling of proteins: approaches and techniques. J. Biomol. Struct.Dyn. 2003 55-64.

Novosibirsk Institute of Bioorganic Chemistry, Novosibirsk, Russia;Novosibirsk Institute of Bioorganic Chemistry, Novosibirsk, Russia;

National Institute of Genetics, Mishima, Japan;National Institute of Genetics, Mishima, Japan;

National Institute of Environmental Health Sciences, North Carolina, USA;National Institute of Environmental Health Sciences, North Carolina, USA;

Columbia University, New York, USA.Columbia University, New York, USA.

Classical affinity cross-linking

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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.

Mechanism-based enzyme inhibitorsMechanism-based enzyme inhibitors

Rando R.R. Methods Enzymol. 1977;46:28-41. Mechanism-based irreversible enzyme inhibitors;;

Tryptophan residue-mediated energy transfer photolabellingTryptophan residue-mediated energy transfer photolabelling

M. P. Goeldner and C. G. Hirth, Proc. Natl. Acad. Sci. U.S.A. 77, 6439-6442 (1980);

Photosensitized labelling by 5-iodonaphthalene-1-azidePhotosensitized labelling by 5-iodonaphthalene-1-azide

Y. Raviv, Y. Salomon, C. Gitler and T. Bercovici, PNAS U.S.A. 84, 6103-6107 (1987).

Catalytically competent labelling Catalytically competent labelling

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

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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

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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).