Tretyachenko-Ladokhina V G, Ladokhin A S, Wang L, Steggles A W, Holloway P W
Department of Biochemistry, University of Virginia Health Sciences Center, Charlottesville 22908.
Biochim Biophys Acta. 1993 Dec 12;1153(2):163-9. doi: 10.1016/0005-2736(93)90401-k.
The structure-function relationships of the 43-amino-acid membrane-binding domain of cytochrome b5 have been examined in two mutant forms of the protein. In one mutant, two tryptophans in the membrane-binding domain, at positions 108 and 112, were replaced by leucines, and in the second mutant, in addition, aspartic acid 103 was also replaced by leucine. The fluorescence emission spectra of the three proteins and their degree of quenching by brominated lipids indicate that the mutations are not producing major conformational changes or allowing a deeper degree of penetration of the domain into the bilayer. The hydrophobicities of the three proteins were compared, by determining strengths of self-association and membrane affinities, and it was found that the protein with two additional leucines was much less hydrophobic and the one with three additional leucines was much more hydrophobic than the native cytochrome. It appears that small changes in amino acid composition, which produce no gross changes in the structure of the membrane-binding domain, will nevertheless produce very large changes in the strengths of self- and membrane-association. These differences in self-association had profound effects on the times required for membrane-association to reach equilibrium.
细胞色素b5的43个氨基酸的膜结合结构域的结构-功能关系已在该蛋白的两种突变形式中进行了研究。在一种突变体中，膜结合结构域中第108位和第112位的两个色氨酸被亮氨酸取代，在第二种突变体中，此外，第103位的天冬氨酸也被亮氨酸取代。这三种蛋白质的荧光发射光谱及其被溴化脂质淬灭的程度表明，这些突变不会产生主要的构象变化，也不会使该结构域更深地穿透双层膜。通过测定自缔合强度和膜亲和力比较了这三种蛋白质的疏水性，发现额外含有两个亮氨酸的蛋白质疏水性要低得多，而额外含有三个亮氨酸的蛋白质疏水性比天然细胞色素高得多。似乎氨基酸组成的微小变化，虽然不会在膜结合结构域的结构上产生总体变化，但仍会在自缔合和膜缔合强度上产生非常大的变化。这些自缔合的差异对膜缔合达到平衡所需的时间有深远影响。
