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(pH=6.5) and 1.0 M sodium acetate) forms the domain swapped dimer. Most of V57Pcrystals did not diffract well (resolution above 5 Å). Data indexing resulted in a highsymmetry space group P622 with enormous cell parameter (a=246 Å, b=246 Å and c=375Å). It indicates presence of more than 45 copies of the molecule in the asymmetric unit andpresumably oligomeric state in the crystal. We were able to get good quality data only forcrystals grown in 0.1 M Imidazole (pH=6.5) and 1.0 M Sodium acetate. This form has thedomain swapped dimer in the asymmetric unit.Fig. 2. Crystal structures of hCC mutants.The secondary structure of the presented hCC mutants can be summarized as follows:(N)- 1- - 2-L1- 3-(AS)- 4-L2- 5-(C). Similar to other proteins belonging to the cystatinfamily, for which the crystal structure was determined (chicken cystatin, dimeric wt hCC),no electron density was observed for the N-terminal region comprising the first elevenresidues, probably because of its flexibility.The experimental structure of human cystatin C V57N mutant illustrates the fold ofmonomeric cystatin C and defines conformation of loop L1, which is essential for theinhibition of papain-like cysteine proteases. InhCCV57N the electron density in the L1 area ofmolecules A and B is of very good quality (Figure 3),allowing modeling of the backbone and side chainconformations without ambiguity.In summary, by applying rational mutagenesisapproach we were able to obtain hCC variants stablein the monomeric form both in solution and in thecrystal (V57N), monomeric in solution but dimeric inthe crystal (V57D) and dimeric in solution andpresumably oligomeric in the crystal (V57P). Fig. 3. Conformation of loop L1.AcknowledgmentsThis work was supported by a grant of Polish Ministry of Science and Higher Education No2739/B/H03/2010/38 and DS/8440-4-0172-0.References1. Olafsson, I., Grubb, A. Amyloid Int. J. Exp. Clin. Invest. 7, 70-79 (2000).2. Janowski, R., Kozak, M., Jankowska, E., Grzonka, Z., Grubb, A., Abrahamson, M., Jaskólski, M.Nat. Struct. Biol. 8, 316-320 (2001).3. Engh, R.A., Dieckmann, T., Bode, W., Aueswald, E.A., Turk, V., Huber, R., Oschkinat, H. J. Mol.Biol. 234, 1060-1069, (1993).4. Martin, J.R., Craven, C.J., Jerala, R., Kroonzitko, L., Żerovnik, E., Turk, V., Waltho, J.P. J. Mol.Biol. 246, 331-343 (1995).5. Ding, F., Prutzman, K.C., Campbell, S.L., Dokholyan, N.V. Structure 14, 5-14 (2006).6. Rodziewicz-Motowidło, S., Iwaszkiewicz, J., Sosnowska, R., Czaplewska, P., Sobolewski, E.,Szymańska, A., Stachowiak, K., Liwo, A. Biopolymers 91,373-83 (2009).7. Szymańska, A., Radulska, A., Czaplewska, P., Grubb, A., Grzonka, Z., Rodziewicz-Motowidło, S.Acta Biochim. Pol. 56, 455-463 (2009).8. Orlikowska, M., Jankowska, E., Kołodziejczyk, R., Jaskólski, M., Szymańska, A., sent to J. Struct.Biol.281