Highly selective crosslinking techniques

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Highly selective cross-linking techniques
Novosibirsk Institute of Bioorganic Chemistry,
Novosibirsk, Russia National Institute of
Genetics, Mishima, Japan National Institute of
Environmental Health Sciences, North Carolina,
USA Columbia University, New York, USA.

• Dmitry Kolpashchikov
• Department of Medicine
• Columbia University
• 650 West 168 Street, BB806
• New York, NY 10032
• Tel [phone]
• Fax [phone]
• E.mail dk2111_at_columbia.edu

Kolpashchikov D.M. Superselective labelling of
proteins approaches and techniques. J. Biomol.
Struct.Dyn. 2003 55-64.
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Classical affinity cross-linking
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Affinity Cross-linking is less efficient then
X-ray crystallography and NMR for structural
studies of purified biopolymers The method
might be useful for the studying of
multicomponent systems, such as complex
reconstitutedsystems, cellular extracts and
living cells Being highly selective
cross-linking techniques could be used for drug
design or as an analytical tools.
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Mechanism-based enzyme inhibitors Rando R.R.
Methods Enzymol. 19774628-41. Mechanism-based
irreversible enzyme inhibitorsTryptophan
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-azideY. Raviv, Y. Salomon,
C. Gitler and T. Bercovici, PNAS U.S.A. 84,
6103-6107 (1987).
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• Catalytically competent labelling
• Labelling using binary photoaffinity reagent

Novosibirsk Institute of Bioorganic Chemistry,
Novosibirsk, Russia
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Catalytically competent labelling
binding sites for two ligands
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reagent
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Influenza virus RNA polymerase
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RNP
PA
PB2
ATP analogue (I,II or III)
a -32PGTP
vRNA
PB1
NP
R-ApG
(Lys, His)
(Lys, after NaBH4 treatment)
(Lys, after NaBH4 treatment)
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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
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Binary photoaffinity reagent
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Binary photo-affinity reagent for labeling of DNA
polymerases
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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.
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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
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Binary photoaffinity reagent can improve
cross-linking efficiency
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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.
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Babendure JR, Adams SR, Tsien RY. Aptamers switch
on fluorescence of triphenylmethane dyes J Am
Chem Soc. 2003,125,14716-7.
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MG
Fluorescence
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• Dezhurov S.V., Dr. Lebedeva N.A., Prof. Lavrik
  O.I. (Novosibirsk Institute of Bioorganic
  Chemistry)
• Dr. Honda A., Prof. Ishihama A., (National
  Institute of Genetics, Mishima, Japan)
• Dr. Prasad R., Dr. Sobol R.W., Prof. Wilson S.H.
  (National Institute of Environmental Health
  Sciences, North Carolina, USA)
• Semova S., Dr. Stojanovic M.N., (Columbia
  University, New York).