Photon Factory Activity Report 2005 #23 Part B (2006)
Biological Science

15A/2003G322

Comparative analysis of stability of human NF-κB p50 subunit and its mutantforms C62W and R59E against urea studied by small-angle x-ray scattering
Alexander TIMCHENKO1, Maria TIMCHENKO2, Viktoria SHYP1, Kazumoto KIMURA3, Hiroshi
KIHARA4*
1
Institute of Protein Research, Pushchino, Russia, 142290;
2
Institute of Theoretical and Experimental Biophysics, Pushchino, Russia, 142290;
3
Department of Biochemistry Dokkyo University, Mibu, Tochigi 321-0293, Japan
4
Department of Physics, Kansai Medical Univ., Uyamahigashi, Hirakata Osaka 573-1136, Japanproteins as seen from Figures. At 5 M of urea Kratky plotshows plateau indicating the coil conformation due tounfolding of proteins. At 3 M of urea the position of peakshifts to smaller scattering vector values reflecting theassociation of proteins. The similar behavior of scattering
patterns on urea concentration for all studied proteins means
the proximity of stability of mutant forms of p50 subunit tothat of wild type. It permits to consider the mutant forms asperspective ones for NF-κB inhibition in a cell.
0.010
0M 0M
0.008

IQ

20.006
4M
4M
0.004
3M
3M
0.002

0.000
0.00

5M
5M

0.02

0.04

0.06

0.08

0.10

0.12oQ(A-1)

Fig.1 Kratky plot for wild type of p50 subunit at theindicating urea concentrations.
0.008

0.006

3M
4M

20M

IQ

Introduction
The human transcription factor NF-κB is an inducible
DNA-binding protein which promotes the expression of alot of genes the majority of which participate in the hostimmune response [1]. Recently it was shown that thehyperactivation of factor NF-κB plays a crucial role intumorigenesis resulting in the hyperexpression of oncogenes.
Thus the problem of NF-κB inhibition is intensively studied
now [2]. One of the possible way to inhibit the NF-κB is theusage of mutant forms of NF-κB incapable for DNA
binding but keeping the stability and dimerization properties
close to those of wild type of protein. Here we present thecomparative analysis of stability of wild type p50 subunit ofNF-κB and its mutant forms C62W and R59E withconsiderably reduced DNA-binding activity at different urea
concentrations.
Experimental
Wild type and mutant forms C62W and R59E of human p50
subunit of NF-κB (replacement of Cys62 for Trp and Arg59
for Glu, respectively) were isolated from the E.coli
BL21(DE3) carried the plasmids encoding corresponding
genes. For the construction of genes encoding mutated p50
molecules a plasmid pEt-14b encoding wild type of p50
protein (kindly provided by A.Israel) was used as template.
The buffer conditions are: 7.5mM HEPES (pH8.0), 34mM
NaCl, 1mM MgCl2, 0.5mM DTT, 0.05mM EDTA. Proteinconcentrations were in the range 1.5-2.0 mg/ml. Proteins
were incubated with the 3, 4 and 5M of urea. Synchrotron
X-ray measurements were done on the small-angle camera
BL-15A (Photon Factory, Tsukuba) using CCD-detector.
The range of scattering vectors Q= 0.008-0.15 nm-1.
Results
To follow the protein conformation it is better to plot
scattering patterns in Kratky coordinates (I*Q2 versus Q).
One can see in Fig.1 and Fig.2 that Kratky plots exhibit bell
shape for all three studied proteins without urea (data forC62W are omitted to avoid crowding), indicating a compact
structure of proteins. The position of maximum corresponds
to a dimer of proteins. Addition of urea up to 4 M
concentration does not change the compact structure of0.004
5M
0.002

0.000
0.00

0.02

0.04

0.06
o

0.08

0.10

0.12

-1

Q(A )

Fig.2
Kratky plot for R59E mutant form of p50 subunit at theindicating urea concentrations.
References
1) Perkins, TIBS, 25, 434, 2000
2) Huang et al. Oncogene, 20, 4188, 2001

* kihara@makino.kmu.ac.jp

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