Photon Factory Activity Report 2004 #22 Part B (2005)
Biological Science

15A/2003G321

κB and its complexes with DNA duplexes at
Oligomeric state of human p50 NF-κ
reducing conditions studied by SAXSAlexander Timchenko1,4, Maria Timchenko2, Marina Ivanovskaya2, Kazumoto Kimura3, Hiroshi
Kihara4*
1
Institute of Protein Research, Pushchino, Russia, 142292
2
Institute of Physico-Chemical Biology of Moscow State University, Russia, 117333
3
Division of Medical Informatics, Dokkyo University, Mibu, Tochigi 321-0200, Japan4
Department of Physics, Kansai Medical University, Uyamahigashi, Hirakata 573-1136, JapanIntroduction
The human transcription factor NF-țB plays the importantrole in the maintenance of immune homeostasis. Besides it
regulates the expression of the genes responsible foroccurrence of some oncologic and infectious diseases,
including AIDS [1]. At various pathologies the determining
moment is hyper-activation of factor NF-țB and theformation of a specific complex of this protein with its
recognition sequence in DNA. It is known, that the DNA
binding of NF-țB in a cell is redox-sensitive. To present
time many problems concerning its function are alreadysolved [2]. The problem of the regulation of NF-κB activity
to prevent pathology is intensively studied now. To checkthe efficiency of protein-DNA interaction usually p50
subunit of NF-κB, responsible for DNA binding, is used.
Here we present the study of solution structure ofrecombinant p50 subunit NF-κB conjugated with 19 Nterminal amino acids, including 6 His (p50-His6) and its
complexes with synthetic DNA duplexes.

from the analysis of P(R) function presented in Fig.1. One
can see that the addition of reducing agent (DTT) sharply
decreases the amplitude of P(R) function witnessing thedisruption of oligomers preferentially to dimers. This factsheds light on the known importance of reducing agents.forprotein-DNA binding. Addition of specific DNA-duplexescauses the further disruption of oligomers with theappearence of monomers (peak near 5nm). At the same time
the nonspecific DNA-duplex practically does not influenceon the P(R) fuction in agreement with the independent dataon the high dissociation constant of such complex. The
above results may contribute the valuable information about
NF-κB-DNA interactions. Further model calculations maypresent stoichiometry of associates in details. This work wassupported in part by the RFBR projects no.04-04-97255.

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Results
Here we used p50-His6 to diminish maximally theinfluence of additional part in protein on its function.
Guinier plot of all samples had a curvature at the very small
scattering angles reflecting some association of protein.
Kratky plot exhibited bell shape indicating compact
structure of protein. More information can be extracted

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Users' Report

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p(r)

Experimental
Human p50 subunit of NF-kB were isolated from theBL21(DE3) strain of E.coli carried the pEt-14B plasmid
kindly provided by A.Israel (France). The buffer conditions
are: 7.5mM HEPES (pH8.0), 34mM NaCl, 1mM MgCl2,0.5mM DTT, 0.05mM EDTA. Protein concentration was
3.7 mg/ml. 20-mer synthetic specific and nonspecific DNA
duplexes were added to protein solution in protein/DNAmolar ratio 1:1 and 4:1, respectively. Synchrotron X-ray
measurements were done on the small-angle camera BL15A (Photon Factory, Tsukuba) using CCD-detector. Therange of scattering vectors Q= 0.008-0.2 nm-1. P(R) distance
distribution function was calculated by program GNOM [3].

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4050r(nm)Fig.1 P(R) function for free p50-His6 without DTT (Ɣ),
with DTT (o) and its complex with specific DNAduplex (molar ratio 2:1) (Ÿ) and the nonspecific one(molar ratio 4:1) (+).
References
1) Perkins, TRENDS in Cell Biology, 14, 64, 2004.
2) Ghosh, Nature, 373, 303, 1995
3) Semenyuk, J. Appl. Crystallog., 24, 537, 1991.
*E-mail:kihara@makino.kmu.ac.jp