Cox J A, Durussel I, Scott D J, Berchtold M W
Department of Biochemistry, University of Geneva, Switzerland.
Eur J Biochem. 1999 Sep;264(3):790-9. doi: 10.1046/j.1432-1327.1999.00650.x.
Parvalbumin (PV) and the homologous protein oncomodulin (OM) contain three EF-hand motifs, but the first site (AB) cannot bind Ca2+. Here we aimed to recreate the putative ancestral proteins [D19-28E]PV and [D19-28E]OM by replacing the 10-residue-long nonfunctional loop in the AB site by a 12-residue canonical loop. To create an optical conformational probe we also expressed the homologs with a F102W replacement. Unexpectedly, in none of the proteins did the mutation reactivate the AB site. The AB-remodeled parvalbumins bind two Ca2+ ions with strong positive cooperativity (nH = 2) and moderate affinity ([Ca2+]0.5 = 2 microM), compared with [Ca2+]0.5 = 37 nM and nH = 1 for the wild-type protein. Increasing Mg2+ concentrations changed nH from 2 to 0.65, but without modification of the [Ca2+]0. 5-value. CD revealed that the Ca2+ and Mg2+ forms of the remodeled parvalbumins lost one-third of their alpha helix content compared with the Ca2+ form of wild-type parvalbumin. However, the microenvironment of single Trp residues in the hydrophobic cores, monitored using intrinsic fluorescence and difference optical density, is the same. The metal-free remodeled parvalbumins possess unfolded conformations. The AB-remodeled oncomodulins also bind two Ca2+ with [Ca2+]0.5 = 43 microM and nH = 1.45. Mg2+ does not affect Ca2+ binding. Again the Ca2+ forms display two-thirds of the alpha-helical content in the wild-type, while their core is still strongly hydrophobic as monitored by Trp and Tyr fluorescence. The metal-free oncomodulins are partially unfolded and seem not to possess a hydrophobic core. Our data indicate that AB-remodeled parvalbumin has the potential to regulate cell functions, whereas it is unlikely that [D19-28E]OM can play a regulatory role in vivo. The predicted evolution of the AB site from a canonical to an abortive EF-hand may have been dictated by the need for stronger interaction with Mg2+ and Ca2+, and a high conformational stability of the metal-free forms.
小白蛋白（PV）和同源蛋白癌调蛋白（OM）含有三个EF手基序，但第一个位点（AB）不能结合Ca2+。在这里，我们旨在通过用一个12个残基的典型环取代AB位点中10个残基长的无功能环来重建假定的祖先蛋白[D19-28E]PV和[D19-28E]OM。为了创建一个光学构象探针，我们还表达了替换了F102W的同源物。出乎意料的是，在所有蛋白质中，该突变均未重新激活AB位点。与野生型蛋白的[Ca2+]0.5 = 37 nM和nH = 1相比，AB重塑的小白蛋白以强正协同性（nH = 2）和中等亲和力（[Ca2+]0.5 = 2 microM）结合两个Ca2+离子。增加Mg2+浓度使nH从2变为0.65，但未改变[Ca2+]0.5值。圆二色性显示，与野生型小白蛋白的Ca2+形式相比，重塑的小白蛋白的Ca2+和Mg2+形式的α螺旋含量损失了三分之一。然而，使用固有荧光和差示光密度监测的疏水核心中单个色氨酸残基的微环境是相同的。无金属的重塑小白蛋白具有未折叠的构象。AB重塑的癌调蛋白也以[Ca2+]0.5 = 43 microM和nH = 1.45结合两个Ca2+。Mg2+不影响Ca2+结合。同样，Ca2+形式在野生型中显示出三分之二的α螺旋含量，而通过色氨酸和酪氨酸荧光监测，其核心仍然具有很强的疏水性。无金属的癌调蛋白部分未折叠，似乎不具有疏水核心。我们的数据表明，AB重塑的小白蛋白具有调节细胞功能的潜力，而[D19-28E]OM在体内不太可能发挥调节作用。AB位点从典型的EF手向无效EF手的预测进化可能受与Mg2+和Ca2+更强相互作用以及无金属形式的高构象稳定性需求的支配。
