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Thermodynamics of maltose binding protein unfolding
Author(s) -
Novokhatny Valery,
Ingham Kenneth
Publication year - 1997
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560060116
Subject(s) - maltose binding protein , chemistry , crystallography , ligand (biochemistry) , differential scanning calorimetry , maltose , enthalpy , biophysics , biochemistry , fusion protein , recombinant dna , thermodynamics , biology , receptor , enzyme , physics , gene
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 Δ H cal / Δ H vH 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 T M 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 Δ H cal / Δ H vH 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 Δ H cal / Δ H 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 Å 2 /g—that becomes accessible to solvent in the unfolded state. The high value of Δ C P determines a very steep Δ G 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.