The energetic cost of domain reorientation in maltose-binding protein as studied by NMR and fluorescence spectroscopy
- † Departments of Biochemistry, Medical Genetics and Microbiology, and Chemistry, and Protein Engineering Network Centers of Excellence, University of Toronto, Toronto, ON, Canada M5S 1A8; and ‡ Programme in Structural Biology and Biochemistry, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8
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Edited by Adriaan Bax, National Institutes of Health, Bethesda, MD, and approved August 21, 2003 (received for review July 10, 2003)
Abstract
Maltose-binding protein (MBP) is a two-domain protein that undergoes a ligand-mediated conformational rearrangement from an “open” to a “closed” structure on binding to maltooligosaccharides. To characterize the energy landscape associated with this transition, we have generated five variants of MBP with mutations located in the hinge region of the molecule. Residual dipolar couplings, measured in the presence of a weak alignment medium, have been used to establish that the average structures of the mutant proteins are related to each other by domain rotation about an invariant axis, with the rotation angle varying from 5° to 28°. Additionally, the domain orientations observed in the wild-type apo and ligand-bound (maltose, maltotriose, etc.) structures are related through a rotation of 35° about the same axis. Remarkably, the free energy of unfolding, measured by equilibrium denaturation experiments and monitored by fluorescence spectroscopy, shows a linear correlation with the rotation angle, with the stability of the (apo)protein decreasing with domain closure by 212 ± 16 cal·mol–1 per degree of rotation. The apparent binding energy for maltose also shows a similar correlation with the interdomain angle, suggesting that the mutations, as they relate to binding, affect predominantly the ligand-free structure. The linearity of the energy change is interpreted in terms of an increase in the extent of hydrophobic surface that becomes solvent accessible on closure. The combination of structural, stability, and binding data allows separation of the energetics of domain reorientation from ligand binding. This work presents a near quantitative structure-energy-binding relationship for a series of mutants of MBP, illustrating the power of combined studies involving protein engineering and solution NMR spectroscopy.
