Title | Thermodynamics of Nanobody Binding to Lactose Permease. |
Publication Type | Journal Article |
Year of Publication | 2016 |
Authors | Hariharan, P., M. Andersson, X. Jiang, E. Pardon, J. Steyaert, R. H Kaback, and L. Guan |
Journal | Biochemistry |
Volume | 55 |
Issue | 42 |
Pagination | 5917-5926 |
Date Published | 2016 Oct 25 |
ISSN | 1520-4995 |
Keywords | Calorimetry, Escherichia coli, Membrane Transport Proteins, Molecular Dynamics Simulation, Single-Domain Antibodies, Substrate Specificity, Thermodynamics |
Abstract | Camelid nanobodies (Nbs) raised against the outward-facing conformer of a double-Trp mutant of the lactose permease of Escherichia coli (LacY) stabilize the permease in outward-facing conformations. Isothermal titration calorimetry is applied herein to dissect the binding thermodynamics of two Nbs, one that markedly improves access to the sugar-binding site and another that dramatically increases the affinity for galactoside. The findings presented here show that both enthalpy and entropy contribute favorably to binding of the Nbs to wild-type (WT) LacY and that binding of Nb to double-Trp mutant G46W/G262W is driven by a greater enthalpy at an entropic penalty. Thermodynamic analyses support the interpretation that WT LacY is stabilized in outward-facing conformations like the double-Trp mutant with closure of the water-filled cytoplasmic cavity through conformational selection. The LacY conformational transition required for ligand binding is reflected by a favorable entropy increase. Molecular dynamics simulations further suggest that the entropy increase likely stems from release of immobilized water molecules primarily from the cytoplasmic cavity upon closure. |
DOI | 10.1021/acs.biochem.6b00826 |
Alternate Journal | Biochemistry |
PubMed ID | 27686537 |
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