Photon Factory Activity Report 2016 #34 (2017)

BL-6A/2016G682

SAXS study of Bence-Jones protein BIF at different ionic conditions.

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

Experimental
We obtained recombinant protein BIF (Mw=25 kDa) andstudied the process of fibril formation in two buffer
systems reflecting environments within the nephron. Buffer
1: 50 mM Na-P, pH 7.2, 0.1 M NaCl. Buffer 2: buffer 1+
20 mM DTT (dithiothreitol). The protein concentration
was around 0.35 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.
Results
SAXS experiments were carried out at small protein
concentration due to high amyloidogenic properties of BIF.
Therefore one can see the overall dimensions and shape ofmolecules. The obtained radii of gyration wereRg=(55.2±1.0) Å (for BIF in buffer 1) andRg=(114.7±1.0) Å (for BIF in buffer 2). After incubationof BIF during 1 hour in buffer 2 at 37oC the value of Rg
was (121.3±1.8) Å. It means that in both cases protein isoligomeric. We investigated the shape of oligomers
plotting dependence logI-logQ (Fig. 1). One can see thatin both cases we have linear dependence with incline 1.02
(correlation coefficient r=0.978) for buffer 1 and incline
3.42 (r=0.916) for buffer 2 at room temperature and 3.68(r=0.965) at 37oC. It means that in buffer 1 shape ofoligomer is rod-like one and granular one in buffer 2.
Perhaps the presence of DTT results in the destruction offilaments into granules. Incubation of protein with DTT at37oC practically does not change the dimensions and shapeof granular particles. These data are in agreement with ouratomic force microscopy observations where we registeredrod-like structure in buffer 1, and granular multimers in
buffer 2.
-0.8

-1.2

-1.6

log I

Introduction
Multiple myeloma (MM) is one of the incurable diseases.
One of the severe complications 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-Jonesprotein BIF was found 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 in
amyloid deposits formation. Here we present SAXS patternof BIF at different ionic conditions simulating native
pathway of this protein in kidney.

-2.0-2.4

-2.8
-2.1

-2.0

-1.9-1.8-1.7-1.6-1.5log QFig.1
The dependence log I versus log Q for BIF 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