TY - JOUR
T1 - Crystallization and preliminary X-ray diffraction analysis of Val57 mutants of the amyloidogenic protein human cystatin C
AU - Orlikowska, Marta
AU - Jankowska, Elzbieta
AU - Borek, Dominika
AU - Otwinowski, Zbyszek
AU - Skowron, Piotr
AU - Szymaska, Aneta
PY - 2011/12
Y1 - 2011/12
N2 - 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.
AB - 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.
KW - cysteine protease inhibitors
KW - human cystatin C
KW - single-point mutations
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U2 - 10.1107/S1744309111039741
DO - 10.1107/S1744309111039741
M3 - Article
C2 - 22139178
AN - SCOPUS:83055165569
SN - 1744-3091
VL - 67
SP - 1608
EP - 1611
JO - Acta Crystallographica Section F: Structural Biology and Crystallization Communications
JF - Acta Crystallographica Section F: Structural Biology and Crystallization Communications
IS - 12
ER -