Photon Factory Activity Report 2014 #32 (2015) B
BL-6A/2013G732

SAXS study of N177S mutant of Bence-Jones protein BIF
Alexander Timchenko1, Maria Timchenko2, Masaji Shinjo4, Hiroshi Kihara4-6*
1

Institute of Protein Research, Pushchino , Russia, 142290;
2
Institute of Theoreticaland Experimental Biophysics, Pushchino, Russia, 142290;
3
Department of Physics, Kansai Medical University, Hirakata, Osaka 573-1010, Japan4
SR Center, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu 525-8577, Japan
5
Nagoya University SR Ressearch Center, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan6Himeji-Hinomoto College, 890 Koro, Kodera-cho, Himeji 679-2151, Japan

Introduction
Multiple myeloma (MM) is one of the incurable diseases.
One of the severe complication of MM is a renal
amyloidosis caused by Bence-Jones proteins fibril
formation. Earlier it was thought that the key role inamyloid deposits formation belongs to the variable domain(VL) of light chains. Several years ago Bence-Jones
protein BIF was found a in the urine of one patient which
amyloidogenity was due to the single change of Ser177 toAsn in a constant domain (CL) of light chains [1]. It was
the first case of participation of CL of light chain inamyloidogenity. Here we present SAXS pattern of N177S
mutant of BIF proposed to be not amyloidogenic.

oligomeric one. The addition of urea (buffer 2) causes
breakdown of oligomers. The addition of DTT (buffer 3)
greatly diminishes the oligomerization. We investigated theshape of oligomers plotting dependence logI-logQ (Fig. 1).
One can see that in both cases we have linear dependencewith incline 1.1 (correlation coefficient r=0.941) for buffer
3 and incline 2.0 (r=0.946) for buffer 1. It means that inbuffer 1 shape of oligomer is flat-like one and rod-like one
in buffer 3. These data are in agreement with our atomic
force microscopy observations where we registeredmultimeric plane structure in buffer 1, and dendritic
multimers in buffer 3 and small particles in buffer 2.
-1.6

Results
Unfortunately, due to highly amyloidogenic properties ofBIF it is practically impossible to carry out SAXS
experiments with it. Therefore we studied N177S in details.
It was obtained for N177S in buffer 1 Rg=(62.8±0.6) Å,
Mw=(1100±10) kDa; in buffer 2 Rg=(39.9±1.0) Å,
Mw=(32±2) kDa;
in buffer 3 Rg=(55.6±1.8) Å,
Mw=(112±5) kDa. It means that in buffer 1 protein is-2.0-2.4
log I

Experimental
We obtained recombinant protein BIF (Mw=25 kDa) and
its analogue (N177S) with the substitution of Asn177 toSer and studied the process of fibril formation in threebuffer systems which were chosen to reflect environments
within the nephron. Buffer 1: 50 mM Na-P, pH 7.2, 0.1 M
NaCl. Buffer 2: 50 mM Na-P, pH 6.5, 0.4 M NaCl, 0.4 M
urea. Buffer 3: buffer 1+ 20 mM DTT. The protein
concentration was 0.37 mg/ml. Synchrotron X-ray
measurements were done on a small-angle camera BL-6A(Photon Factory, Tsukuba) using PILATUS 100K detector.
The range of scattering vectors Q=0.01-0.25 Å-1. For better
accuracy we evaluated radius of gyration (Rg) andmolecular mass (Mw) of protein from the distance
distribution function P(r) calculated by GNOM program[1] in the range of Q=0.015-0.15 Å-1.

-2.8-3.2-3.6-2.0-1.9-1.8-1.7-1.6

-1.5

-1.4

-1.3

-1.2log Q

Fig.1 The dependence log I versus log Q for N177S inbuffer 1 (filled circles) and in buffer 3 (open circles).
References
1) Solomon A. et al., PNAS., 95, 9547, 1998.
2) Svergun D., J.Appl.Cryst., 25, 495, 1992.
* E-mail:kiharah1234@gmail.com