Maltose-binding protein from the hyperthermophilic bacterium Thermotoga maritima: Stability and binding properties

Recombinant maltose-binding protein from Thermotoga maritima (TmMBP) was ex pressed in Escherichia coli and purified to homogeneity, applying heat incu bation of the crude extract at 75 degrees C. As taken from the spectral, ph ysicochemical and binding properties, the recombinant protein is indistingu ishable from the natural protein isolated from the periplasm of Thermotoga maritima. At neutral pH, TmMBP exhibits extremely high intrinsic stability with a thermal transition >105 degrees C. Guanidinium chloride-induced equi librium unfolding transitions at varying temperatures result in a stability maximum at approximate to 40 degrees C. At room temperature, the thermodyn amic analysis of the highly cooperative unfolding equilibrium transition yi elds Delta G(N) --> (U) 100(+/-5) kJ mol(-1) for the free energy of stabili zation. Compared to mesophilic MBP from E. coli as a reference, this value is increased by about 60 kJ mol(-1). At temperatures around the optimal gro wth temperature of T. maritima (t(opt) approximate to 80 degrees C), the yi eld of refolding does not exceed 80%; the residual 20% are misfolded, as in dicated by ii decrease in stability as well as loss of the maltose-binding capacity. TmMBP is able to bind maltose, maltotriose and trehalose with dis sociation constants in the nanomolar to micromolar range, combining the sub strate specificities of the homologs from the mesophilic bacterium E.coli a nd the hyperthermophilic archaeon Thermococcus litoralis. Fluorescence quen ch experiments allowed the dissociation constants of Ligand binding to be q uantified. Binding of maltose was found to be endothermic and entropy-drive n, with Delta H-b = + 47 kJ mol(-1) and Delta S-b = + 257 J mol(-1) K-1. Ex trapolation of the linear vant 'Hoff plot to t(opt) resulted in K-d approxi mate to 0.3 mu M. This result is in agreement with data reported for: the M BPs from E, coli and T, litoralis at their respective optimum growth temper atures, corroborating the general observation that proteins under their spe cific physiological conditions are in corresponding states. (C) 2000 Academ ic Press.