Crystallization and preliminary X-ray studies of mouse centrin1.
Centrins belong to a family of Ca2+-binding EF-hand proteins that play a fundamental role in centrosome duplication and the function of cilia. To shed light on the structure-function relationship of these proteins, mouse centrin1 has been crystallized. The mouse centrin1 has been expressed in Escherichia coli as a GST-centrin fusion protein containing a thrombin protease cleavage site between the fusion partners. Two constructs with different linking-sequence lengths were expressed and purified. Thrombin cleavage yielded functional centrin1 and N-terminally extended centrin1 containing 25 additional residues upstream of its N-terminus. Only N-terminally extended centrin1 (MW approximately 22 240 Da) could be crystallized at room temperature, using 20-25%(w/v) PEG 1500, 5-10%(v/v) ethylene glycol and 1-2%(v/v) dioxane. Crystals were suitable for X-ray analysis, diffracting to 2.9 A at 295 K using a rotating-anode X-ray source. They belong to space group C2, with unit-cell parameters a = 60.7, b = 59.6, c = 58.3 A, beta = 109.4 degrees. Assuming the asymmetric cell to be occupied by one centrin1 molecule of 22.2 kDa, the unit cell contains 45% solvent with a crystal volume per protein weight, VM, of 2.2 A3 Da(-1).
Park
Jung Hee
JH
Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Ziegelstrasse 5-9, D-10098 Berlin, Germany.
Krauss
Norbert
N
Pulvermüller
Alexander
A
Scheerer
Patrick
P
Höhne
Wolfgang
W
Giessl
Andreas
A
Wolfrum
Uwe
U
Hofmann
Klaus Peter
KP
Ernst
Oliver Peter
OP
Choe
Hui-Woog
HW
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D002135:Calcium-Binding Proteins; D002868:Chromosomal Proteins, Non-Histone; D011994:Recombinant Proteins; C056608:caltractin