title: Crystallization and preliminary X-ray diffraction analysis of Val57 mutants of the amyloidogenic protein human cystatin C.
abstract: Human cystatin C (hCC) is a low-molecular-mass protein (120 amino-acid residues, 13 343 Da) found in all nucleated cells. Its main physiological role is regulation of the activity of cysteine proteases. Biologically active hCC is a monomeric protein, but all crystallization efforts have resulted in a dimeric domain-swapped structure. Recently, two monomeric structures were reported for cystatin C variants. In one of them stabilization was achieved by abolishing the possibility of domain swapping by the introduction of an additional disulfide bridge connecting the two protein domains (Cys47-Cys69). In the second structure, reported by this group, the monomeric hCC fold was preserved by stabilization of the conformationally constrained loop (L1) by a single-amino-acid substitution (V57N). To further assess the influence of changes in the sequence and properties of loop L1 on the dimerization propensity of cystatin C, two additional hCC mutants were obtained: one with a residue favoured in β-turns (V57D) and another with proline (V57P), a residue that is known to be a structural element that can rigidify but also broaden turns. Here, the expression, purification and crystallization of V57D and V57P variants of recombinant human cystatin C are described. Crystals were grown by the vapour-diffusion method. Several diffraction data sets were collected using a synchrotron source at the Advanced Photon Source, Argonne National Laboratory, Chicago, USA.
authors.affiliation: Department of Medicinal Chemistry, Faculty of Chemistry, University of Gdansk, Gdansk, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Orlikowska
authors.forename: Elzbieta
authors.initials: E
authors.lastname: Jankowska
authors.forename: Dominika
authors.initials: D
authors.lastname: Borek
authors.forename: Zbyszek
authors.initials: Z
authors.lastname: Otwinowski
authors.forename: Piotr
authors.initials: P
authors.lastname: Skowron
authors.forename: Aneta
authors.initials: A
authors.lastname: Szymańska
mesh_terms: D003460:Crystallization; D018360:Crystallography, X-Ray; D055316:Cystatin C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D014633:Valine / Q000235:genetics / Q000378:metabolism
chemical_list: D055316:Cystatin C; D014633:Valine