Photon Factory Activity Report 2016 #34 (2017)
BL-6A/2016G682

SAXS study of Y187N mutant of Bence-Jones protein BIF
Alexander Timchenko1, Maria Timchenko2, Yoshitaka Matsumura3, Hiroshi Kihara4,5*
1 Institute of Protein Research, Pushchino, Russia, 142290;
2 Institute of Theoretical and Experimental Biophysics, Pushchino, Russia, 142290;
3 Tokyo University of Pharmacy and Life Science, School of Life Science, Japan
4SR Center, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu 525-8577, Japan
5 Himeji-Hinomoto College, 890 Koro, Kodera-cho, Himeji 679-2151, Japan

2 Experimental
We obtained recombinant Y187N mutant of BIF (Mw=25kDa) and studied the process of fibril formation in twobuffer systems reflecting environments within the nephron.
Buffer 1: 50 mM Na-P, pH 7.2, 0.1 M NaCl. Buffer 2: 25
mM NaHCO3+NaOH pH 10.1. The protein concentration
was around 0.25 mg/ml. Synchrotron X-ray measurements
were done on a small-angle camera BL-6A (PhotonFactory, 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) from thedistance distribution function P(r) calculated by GNOM
program [1] in the range of Q=0.01-0.15 Å-1.
3 Results
SAXS experiments were carried out at small protein
concentration due to high amyloidogenic properties of BIF
and its mutants. Therefore one can see only overall
dimensions and shape of particles. The obtained radii ofgyration for Y187N in buffer 1 was Rg=(83.6±1.0) Å andin buffer 2 - Rg=(77.8±1.0) Å. It means that in both cases
protein was oligomeric. We investigated the shape ofoligomers plotting dependence logI-logQ (Fig. 1). One cansee that in both cases we have linear dependence withincline 1.66 (correlation coefficient r=0.928) for buffer 1
and incline 1.04 (r=0.943) for buffer 2. It means that inbuffer 2 the shape of oligomer is rod-like one and dendricelongated one in buffer 2.
These data are in agreement with our atomic force
microscopy observations where we registered rod-likestructure in buffer 2, and dendric elongated shape in buffer
1.
-1.8-2.0-2.2log I

1 Introduction
Multiple myeloma nephropathy occurs due to theformation of different aggregates by immunoglobulin lightchains (Bence-Jones protein) in kidneys. The mechanismof amyloid deposits formation is still unclear. Solomon etal. revealed in one of the patients amyloids consisted ofconstant domain (CL) of light chains. The analysis ofaminoacid sequence of such protein (BIF) showed thechange of Ser 177 to Asn. It was proposed that in manyneurodegenerative diseases the key role in amyloidogenesis
play proteins with Asn-rich regions. We decided to checkthis hypothesis and made the additional mutation Y187N.
Here we present SAXS pattern of Y187N mutant of BIF at
different ionic conditions simulating native pathway of thisprotein.

-2.4-2.6-2.8-3.0-2.2-2.1

-2.0

-1.9

-1.8

-1.7

-1.6

-1.5log Q

Fig.1 The dependence log I versus log Q for Y187N inbuffer 1 (filled circles) and in buffer 2 (open circles).
This work was supported by grant RFFI 16-04-01027.
References
1) Solomon A. et al., PNAS., 95, 9547, 1998.
2) Svergun D., J.Appl.Cryst., 25, 495, 1992.
* E-mail:kiharah1234@gmail.com