Marchenko N Iu, Marchenkov V V, Kaĭsheva A L, Kashparov I A, Kotova N V, Kaliman P A, Semisotnov G V
Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia.
Biochemistry (Mosc). 2006 Dec;71(12):1357-64. doi: 10.1134/s000629790612011x.
The chaperonin GroEL of the heat shock protein family from Escherichia coli cells can bind various polypeptides lacking rigid tertiary structure and thus prevent their nonspecific association and provide for acquisition of native conformation. In the present work we studied the interaction of GroEL with six denatured proteins (alpha-lactalbumin, ribonuclease A, egg lysozyme in the presence of dithiothreitol, pepsin, beta-casein, and apocytochrome c) possessing negative or positive total charge at neutral pH values and different in hydrophobicity (affinity for a hydrophobic probe ANS). To prevent the influence of nonspecific association of non-native proteins on their interaction with GroEL and make easier the recording of the complexing, the proteins were covalently attached to BrCN-activated Sepharose. At low ionic strength (lower than 60 mM), tight binding of the negatively charged denatured proteins with GroEL (which is also negatively charged) needed relatively low concentrations (approximately 10 mM) of bivalent cations Mg2+ or Ca2+. At the high ionic strength (approximately 600 mM), a tight complex was produced also in the absence of bivalent cations. In contrast, positively charged denatured proteins tightly interacted with GroEL irrespectively of the presence of bivalent cations and ionic strength of the solution (from 20 to 600 mM). These features of GroEL interaction with positively and negatively charged denatured proteins were confirmed by polarized fluorescence (fluorescence anisotropy). The findings suggest that the affinity of GroEL for denatured proteins can be determined by the balance of hydrophobic and electrostatic interactions.
来自大肠杆菌细胞的热休克蛋白家族伴侣蛋白GroEL可以结合各种缺乏刚性三级结构的多肽，从而防止它们发生非特异性缔合，并促使其获得天然构象。在本研究中，我们研究了GroEL与六种变性蛋白质（α-乳白蛋白、核糖核酸酶A、在二硫苏糖醇存在下的卵溶菌酶、胃蛋白酶、β-酪蛋白和脱辅基细胞色素c）的相互作用，这些蛋白质在中性pH值下带有负电荷或正电荷，并且疏水性（对疏水探针ANS的亲和力）各不相同。为了防止非天然蛋白质的非特异性缔合对其与GroEL相互作用的影响，并便于记录络合过程，将这些蛋白质共价连接到溴化氰活化的琼脂糖上。在低离子强度（低于60 mM）下，带负电荷的变性蛋白质与同样带负电荷的GroEL紧密结合需要相对较低浓度（约10 mM）的二价阳离子Mg2+或Ca2+。在高离子强度（约600 mM）下，即使没有二价阳离子也会形成紧密的复合物。相比之下，带正电荷的变性蛋白质与GroEL紧密相互作用，而与二价阳离子的存在和溶液的离子强度（20至600 mM）无关。GroEL与带正电荷和带负电荷的变性蛋白质相互作用的这些特征通过偏振荧光（荧光各向异性）得到了证实。这些发现表明，GroEL对变性蛋白质的亲和力可以由疏水相互作用和静电相互作用的平衡来决定。
