Measurements of ATP binding on the large cytoplasmic loop of the sarcoplasmic reticulum Ca(2+)-ATPase overexpressed in Escherichia coli.
The large cytoplasmic loop of the sarcoplasmic reticulum Ca(2+)-ATPase (LCL), situated between Lys329 and Phe740, is believed to contain both its phosphorylation and ATP binding domains. A cDNA fragment coding for this amino acid sequence was generated in vitro and cloned in vector pQE8 which allowed the overexpression in Escherichia coli of this Ca(2+)-ATPase domain fused with a cluster of 6 histidines at its NH2 terminus. The fusion protein produced in an insoluble form within bacteria was solubilized in 4 M urea, purified on immobilized Ni2+, and then renatured by elimination of urea. More than 4 mg of purified renatured fusion protein was obtained from 500 ml of culture. ATP binding on the refolded protein was demonstrated by two methods: 1) detection of ATP-induced intrinsic fluorescence change and 2) binding of the fluorescent ATP analogue 2',3'-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate (TNP-ATP) and its chase by ATP. It is shown that the LCL protein has one single TNP-ATP binding site having a dissociation constant (Kd) of 1.6-1.9 microM. Both methods yielded a Kd for ATP around 200 microM. Binding of other nucleotides was detected with a sequence of Kd identical to that found for native Ca(2+)-ATPase: ATP < ADP < GTP < AMP < ITP. A Mg2+ binding site was also found on the LCL protein (Kd = 100 microM at pH 7.2). The fluorescence of bound TNP-ATP was found to be highly dependent on Mg2+ binding on this site.
Moutin
M J
MJ
Laboratoire de Biophysique Moléculaire et Cellulaire, URA 520 du Centre National de la Recherche Scientifique, Grenoble, France.
Cuillel
M
M
Rapin
C
C
Miras
R
R
Anger
M
M
Lompré
A M
AM
Dupont
Y
Y
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D000252:Calcium-Transporting ATPases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004926:Escherichia coli; D007700:Kinetics; D008239:Lysine; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D010649:Phenylalanine; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012265:Ribonucleotides / Q000378:metabolism / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D012265:Ribonucleotides; D010649:Phenylalanine; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D008239:Lysine