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

15A/2003G322

Studies of ribosomal protein S1 from Thermus thermophilus at different ionicconditions in solution by X-ray small-angle scattering
Alexander Timchenko1,3, Olga Selivanova1, Vyacheslav Shiryaev1, Kazumoto Kimura2, Hiroshi Kihara3*
1
Institute of Protein Research, Pushchino, Russia, 142292;
2
Division of Medical Informatics, Dokkyo University, Mibu, Tochigi 321-0200, Japan3
Department of Physics, Kansai Medical University, Uyamahigashi, Hirakata 573-1136 Japan
Introduction
Ribosomal protein S1 is the largest protein of eubacterial
ribosomes with a molecular mass of 61 kDa and a length of557 amino acid residues. It is an RNA-binding protein
involved in retention of mRNA during initiation oftranslation and, maybe, during elongation. The most
specific feature of the primary structure of Escherichia
coli protein S1 is the presence of six homologous aminoacid repeats of about 70 amino acids long separated by
spacers of 10-15 residues. Physical studies of isolated
protein S1 from E.coli showed that the protein in solution
manifested a highly extended, non-compact conformationwith the longest dimension (23nm) of the whole ribosome.
At the same time our recent sedimentation andmicrocalorimetry data [1] have shown the compact
structure of S1 Th. th.. at low ionic strength. To study thedetailed structural behaviour of this protein in solution at
different ionic conditions we used SAXS techniques.
Experimental
Ribosomal protein S1 has been recently identified in
Thermus thermophilus [1] and its gene has been expressedin E.coli overproducing strain BL21(DE3)pET21d-tthS1
and S1 has been successfully purified in the sufficientamounts for physical measurements. The protein wasstored at 4°C in the precipitated form in (NH4)2SO4.
Samples for measurements were prepared by solving theprecipitate either in 25 mM HEPES-NaOH buffer, pH 7.5(buffer A) or in 25 mM HEPES-NaOH (or NaOD) buffer,pH 7.5, with 100 mM NaCl (buffer B) and subsequent
dialysis against the same buffers at room temperature
overnight. The used protein concentrations were in therange of 1.5-10.0 mg/ml. Synchrotron X-ray measurements
were done on the small-angle camera BL-15A (PhotonFactory, Tsukuba) using CCD-detector. The range ofscattering vectors Q= 0.08-2 nm-1.
Results
Scattering patterns for S1 in both buffers plotted in theGuinier coordinates showed the nonlinear behaviour with
the initial ordinate indicating the association of protein
molecules. Thus, we can only estimate the value of radiusof gyration (Rg). These values are in the range 7-11 nm tobe considerably higher the expected one (about 3nm) for

globular protein of 61kD. For S1 in the buffer A theessential concentration dependence of scattering pattern isobserved, and the associates correspond to trimers andhigher order oligomers. For S1 in the buffer B suchdependence is weak, and the associates correspondpreferentially to dimmers and trimers. The more structural
information we can obtain from the scattering patterns
plotted in the Kratky coordinates (see Fig.1). One can see
the bell-shaped plot for S1 in the buffer B corresponding tocompact particles. The value Rg estimated from theposition of maximum on the Kratky plot was 2.9 nm whichis expected for globular protein of 61kD. Thus, S1particles at the moderate ionic strength associates in therandom order building dimers, trimers with the globular
conformation of monomer inside them. Judging the Fig.1,
S1 particles at the low ionic strength form the globularlike high-order associates with the globular conformationof monomer inside them. The future model calculations
will give the more detailed composition of oligomers.
This work was supported by the RFBR projects no. 04-0497319.

Fig.1 Kratky plot of SAXS data for S1 in buffer B (fullcircles) and in buffer A (empty circles).
References
1) 1. Selivanova O.M. et al. , J.Biol.Chem. ,278(38):36311-4, 2003.
* E-mail:kihara@makino.kmu.ac.jp

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