Maltose-binding protein (MBP) is in a molten globule state at pH 3 as characterized by ANS binding. DEER measurements of seven spin-labeled double mutants as in the native state at pH 7 had shown excellent agreement with X-ray data. At pH 3 corresponding DEER measurements of all the mutants yield a broad distribution of distances. This was to be expected if there is no defined tertiary structure and the individual helices pointing into all possible directions. However, as MBP still binds maltose as molten globule although more weakly, the native structure must be retained at or near the active site. This is now being investigated with a new set of mutants.
W208 and W237 ]n erdcr to confinn the cnLrgy homo transfer m PAP recombmant WT, W208F and W237FPAP wLre prcpared in 20 mM phosphate butter (pH 7 O) at!erthe expresgion and purtfication The fluorescencL poldrizatien on the exLitduon dt 31O nm vLas higher than that exc]ted at 295 nni and the energy transfor etfiLiLncy bLlwLLn to Tipb Li aludtLd by thL absorption co efTicienc} and fiuorescence quantum yield ofild typL and mutant PAP was about 60 % The energy trangter bet" een W208 and W237-as a]so examined
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