Cold-sensitive assembly of a mutant manganese-stabilizing protein caused by a Val to Ala replacement

被引:30
作者
Betts, SD
Ross, JR
Pichersky, E
Yocum, CF
机构
[1] UNIV MICHIGAN,DEPT BIOL,ANN ARBOR,MI 48109
[2] UNIV MICHIGAN,DEPT CHEM,ANN ARBOR,MI 48109
关键词
D O I
10.1021/bi953066t
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Photosystem II (PSII) is a multisubunit transmembrane protein complex that oxidizes water and evolves O-2. A tetranuclear manganese cluster associated with integral membrane subunits of PSII catalyzes water oxidation. The 33-kDa water-soluble PSII subunit, or manganese-stabilizing protein (MSP), stabilizes the O-2-evolving manganese cluster and accelerates O-2 evolution. Spinach PSII can be depleted of native MSP under conditions which retain a functional manganese cluster. Reconstition of MSP-depleted PSII with recombinant MSP was equally efficient at 4 and 22 degrees C. Replacement of Val235 (a conserved residue near the C-terminus of MSP) with Ala inhibited assembly of MSP at 4 degrees C, but not at 22 degrees C. Once assembled, [V235A]MSP remained bound to PSII even at 4 degrees C and in the presence of low concentrations of urea. Results from far-UV circular dichroism spectrometry indicated that [V235A]-MSP was destabilized by low temperature to a greater extent than the wild-type protein. However, the effect of temperature on the secondary structure of both the mutant and wild-type proteins was small compared to the temperature-independent destabilization of secondary structure induced by the mutation. These results demonstrate that the V235A mutation introduces an activation energy barrier for assembly of MSP into PSII, and it is suggested that the mutation acts by inhibiting isomerization of one or more prolyl peptide bonds required for assembly.
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页码:6302 / 6307
页数:6
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