Engineered synthetic antibodies as probes to quantify the energetic contributions of ligand binding to conformational changes in proteins.

Conformational changes in proteins due to ligand binding are ubiquitous in biological processes and are integral to many biological systems. However, it is often challenging to link ligand-induced conformational changes to a resulting biological function because it is difficult to distinguish between the energetic components associated with ligand binding and those due to structural rearrangements. Here, we used a unique approach exploiting conformation-specific and regio-specific synthetic antibodies (sABs) to probe the energetic contributions of ligand binding to conformation changes. Using maltose-binding protein (MBP) as a model system, customized phage-display selections were performed to generate sABs that stabilize MBP in different conformational states, modulating ligand-binding affinity in competitive, allosteric, or peristeric manners. We determined that the binding of a closed conformation-specific sAB (sAB-11M) to MBP in the absence of maltose is entropically driven, providing new insight into designing antibody-stabilized protein interactions. Crystal structures of sABs bound to MBP, together with biophysical data, delineate the basis of free energy differences between different conformational states and confirm the use of the sABs as energy probes for dissecting enthalpic and entropic contributions to conformational transitions. Our work provides a foundation for investigating the energetic contributions of distinct conformational dynamics to specific biological outputs. We anticipate that our approach also may be valuable for analyzing the energy landscapes of regulatory proteins controlling biological responses to environmental changes.

Structural and kinetic studies on metallo-ß-lactamase IMP-1.

In an effort to probe for metal binding to metallo-ß-lactamase (MßL) IMP-1, the enzyme was overexpressed, purified, and characterized. The resulting enzyme was shown to bind 2 equiv of Zn(II), exhibit significant catalytic activity, and yield EXAFS results similar to crystallographic data previously reported. Rapid kinetic studies showed that IMP-1 does not stabilize a nitrocefin-derived reaction intermediate; rather, the enzyme follows a simple Michaelis mechanism to hydrolyze nitrocefin. Metal-substituted and metal-reconstituted analogues of IMP-1 were prepared by directly adding metal ion stocks to metal-free enzyme, which was generated by dialysis versus EDTA. UV-vis studies on IMP-1 containing 1 equiv of Co(II) showed a strong ligand-to-metal charge transition at 340 nm, and the intensity of this feature increased when the second equivalent of Co(II) was added to the enzyme. EXAFS fits on IMP-1 containing 1 equiv of Co(II) strongly suggest the presence of a metal-metal interaction, and EPR spectra of the IMP-1 containing 1 and 2 equiv of Co(II) are very similar. Taken together, steady-state kinetic and spectroscopic studies suggest that metal binding to metal-free IMP-1 follows a positive-cooperative mode.