Thermodynamics of maltose binding protein unfolding

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Abstract
The maltose binding protein (MBP or MalE) of Escherichia coli is the periplasmic component of the transport system for malto-oligosaccharides. It is used widely as a carrier protein for the production of recombinant fusion proteins. The melting of recombinant MBP was studied by differential scanning and titration calorimetry and fluorescence spectroscopy under different solvent conditions. MBP exhibits a single peak of heat absorption with a ΔHcal/ΔHvH ratio in the range of 1.3–1.5, suggesting that the protein comprises two strongly interacting thermodynamic domains. Binding of maltose resulted in elevation of the TM by 8–15 °C, depending on pH. The presence of ligand at neutral pH, in addition to shifting the melting process to higher temperature, caused it to become more cooperative. The ΔHcal/ΔHvH ratio decreased to unity, indicating that the two domains melt together in a single two-state transition. This ligand-induced merging of the two domains appears to occur only at neutral pH, because at low pH maltose simply stabilized MBP and did not cause a decrease of the ΔHcal/ΔH vH ratio. Binding of maltose to MBP is characterized by very low enthalpy changes,∼-1 kcal/mol. The melting of MBP is accompanied by an exceptionally large change in heat capacity, 0.16 cal/K-g, which is consistent with the high amount of nonpolar surface—0.72 Å2/g—that becomes accessible to solvent in the unfolded state. The high value of ΔCP determines a very steep ΔG versus T profile for this protein and predicts that cold denaturation should occur above freezing temperatures. Evidence for this was provided by changes in fluorescence intensity upon cooling the protein. A sigmoidal cooperative transition with a midpoint near 5°C was observed when MBP was cooled at low pH. Analysis of the melting of several fusion proteins containing MBP illustrated the feasibility of assessing the folding integrity of recombinant products prior to separating them from the MBP carrier protein.
Funding Information
  • U.S. National Institutes of Health (H121791)