P 40 ISSRNS 2012: Abstracts / Synchrotron Radiation in Natural Science Vol. 11, No 1 – 2 (2012)
SMALL ANGLE X-RAY SCATTERING (SAXS) STUDIES OF MONOMERIC HUMAN CYSTATIN C IN SOLUTION
M. Murawska1, A. Grubb2, and M. Kozak1∗
1Department of Macromolecular Physics, A. Mickiewicz University, Umultowska 85, 61-614 Pozna´n, Poland
2Department of Clinical Chemistry, Lund University, Lund, Sweden.
Keywords: BioSAXS, human cystatin C
∗e-mail : mkozak@amu.edu.pl
Human cystatin C (HCC) is an inhibitor of cys- teine proteases. This protein is present in many body fluids like blood, urine, saliva, cerebrospinal fluid, pleural fluid and in tissues such as cerebral cortex. Cystatin C is used as an important marker of kidney function with better correlation with mor- tality and cardiovascular problems then the other common markers like creatinine or glomerular fil- tration rate. This protein was observed as coprecip- itate of pathological amyloid fibrils in the brains of patients with Alzheimer’s disease. Especially a lot of cystatin C was present in the cerebrospinal fluid.
For correct functioning in this role cystatin should occur in the form of monomers.
In the crystal, native HCC forms dimers via the domain swapping mechanism. This mechanism can be directly related to the cystatin tendency to- wards formation of amyloid deposits. Rarely, a nat- urally occurring mutation HCC (Leu68Gln) results in massive amyloidosis, cerebral haemorrhage and ultimately to death at a young age.
The study presented was aimed at developing low-resolution structure of monomeric form of hu- man cystatin C in solution, stabilized by disulfide bonds against domain swapping, and comparing this structure with the crystal structure of monomeric human cystatin C (PDB: 3GAX).
The X-ray scattering data were obtained using synchrotron radiation and SAXS camera (beam line BLi911-4 [4], MAXII storage ring of the MAX-Lab Lund, Sweden; λ = 0.091 nm). Low-resolution structure of the monomeric human cystatin C in so- lution was restored by a computer simulation in pro- gram DAMMIN [5]. Independently, the SAXS data were directly compared using CRYSOL [6] with the theoretical scattering curve obtained on the basis of
crystal structure. This comparison clearly indicated that the preferred conformation of HCC occurring in solution is almost identical with the crystal struc- ture.
Acknowledgments: The present study was carried out with financial support from the Ministry of Science and Higher Education (grant nr N N202 127237).
References
[1] R. Janowski, M. Kozak, E. Jankowska, Z. Grzonka, A. Grubb, M. Abrahamson, M. Jaskolski, “Hu- man cystatin C, an amyloidogenic protein, dimer- izes through three dimensional domain swapping,”
Nature Struct. Biol. 8 (2001) 316 – 320.
[2] M. Orlikowska, E. Jankowska, R. Ko lodziejczyk, M.
Jaskolski, A. Szyma´nska, “Hinge-loop mutation can be used to control 3D domain swapping and amy- loidogenesis of human cystatin C,” J. Struct. Biol.
173 (2011) 406 – 413.
[3] A. Grubb, “Cystatin C — properties and use as diagnostic marker,” Adv. Clin. Chem. 35 (2000) 63 – 99.
[4] C.B. Mammen, T. Ursby, Y. Cerenius, M.
Thunnissen, J. Als-Nielsen, S. Larsen, and A. Liljas,
“Design of a 5-station macromolecular crystallogra- phy beamline at MAX-lab,” Acta Phys. Pol. A 101 (2002) 595 – 602.
[5] D.I. Svergun, “Restoring low resolution structure of biological macromolecules from solution scatter- ing using simulated annealing,” Biophys. J. (1999) 2879 – 2886.
[6] D.I. Svergun, C. Barberato, and M.H.J. Koch,
“CRYSOL — a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates”, J. Appl. Cryst. 28 (1995) 768 – 773.
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