[Point amino acid substitutions in Ca2+-binding centers of recoverin. III. Mutant with the fourth reconstructed Ca2+-binding site].
Unlike wild type recoverin with only two (the second and the third) functioning Ca(2+)-binding sites out of four potential ones, the +EF4 mutant contains a third active Ca(2+)-binding site. This site was reconstructed from the fourth potential Ca(2+)-binding domain by the introduction of several amino acid substitutions in it by site-directed mutagenesis. The effect of these mutations in the fourth potential Ca(2+)-binding site of myristoylated recoverin on the structural features and conformational stability of the protein was studied by fluorimetry and circular dichroism. The apoform of the resulting mutant (free of Ca2+ ions) was shown to have a higher calcium capacity, significantly lower thermal stability, and noticeably different secondary and tertiary structures as compared with the apoform of wild type recoverin.
Permiakov
S E
SE
Institute for Biological Instrument Making, Russian Academy of Sciences, Pushchino, Moscow oblast, Russia. permyakov@ibp.serpukhov.su
Uverskiĭ
V N
VN
Cherskaia
A M
AM
Shul'ga-Morskoĭ
S V
SV
Zinchenko
D V
DV
Alekseev
A M
AM
Zerniĭ
E Iu
EIu
Zargarov
A A
AA
Senin
I I
II
Lipkin
V M
VM
Filippov
P P
PP
Permiakov
E A
EA
D019943:Amino Acid Substitution; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020832:EF Hand Motifs; D005136:Eye Proteins; D051597:Hippocalcin; D008074:Lipoproteins; D009419:Nerve Tissue Proteins; D017354:Point Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051603:Recoverin
D002135:Calcium-Binding Proteins; D005136:Eye Proteins; D008074:Lipoproteins; D009419:Nerve Tissue Proteins; D051603:Recoverin; D051597:Hippocalcin; D002118:Calcium
Tochechnye aminokislotnye zameny v Ca2+-sviazyvaiushchikh tsentrakh rekoverina. III. Mutant s chetvertym rekonstruirovannym Ca2+-sviazyvaiushchim tsentrom.