Abstract
This paper describes a biophysical investigation of residual mobility in complexes of bovine carbonic anhydrase II (BCA) and para-substituted benzenesulfonamide ligands with chains of 1–5 glycine subunits, and explains the previously observed increase in entropy of binding with chain length. The reported results represent the first experimental demonstration that BCA is not the rigid, static globulin that has been typically assumed, but experiences structural fluctuations upon binding ligands. NMR studies with 15N-labeled ligands demonstrated that the first glycine subunit of the chain binds without stabilization or destabilization by the more distal subunits, and suggested that the other glycine subunits of the chain behave similarly. These data suggest that a model based on ligand mobility in the complex cannot explain the thermodynamic data. Hydrogen/deuterium exchange studies provided a global estimate of protein mobility and revealed that the number of exchanged hydrogens of BCA was higher when the protein was bound to a ligand with five glycine subunits than when bound to a ligand with only one subunit, and suggested a trend of increasing number of exchanged hydrogens with increasing chain length of the BCA-bound ligand, across the series. These data support the idea that the glycine chain destabilizes the structure of BCA in a length-dependent manner, causing an increase in BCA mobility. This study highlights the need to consider ligand-induced mobility of even “static” proteins in studies of protein-ligand binding, including rational ligand design approaches.
Highlights
Understanding the driving forces for the non-covalent association of a protein with a small-molecule ligand–e.g., interactions based on electrostatics, hydrophobicity, and solvation–is an area of high interest for chemistry, biology, and medicine
The hydrogen/deuterium exchange studies suggest that the effect of the oligoglycine chain on bovine carbonic anhydrase II (BCA) mobility is the origin of the thermodynamic profile of these ligands, with less unfavorable entropy resulting from the increased protein mobility and the less favorable enthalpy from fewer ordered internal hydrogen bonds; the net effect being enthalpy/entropy compensation across the series
We have used a combination of biophysical studies (NMR and hydrogen/deuterium exchange) to determine the origin of the perplexing trend of increasingly less unfavorable entropy, and increasingly less favorable enthalpy, in the association of a model protein with a series of psubstituted benzenesulfonamides with oligoglycine chains of variable length (1–5 subunits)
Summary
Understanding the driving forces for the non-covalent association of a protein with a small-molecule ligand–e.g., interactions based on electrostatics, hydrophobicity, and solvation–is an area of high interest for chemistry, biology, and medicine. While significant progress has been made in understanding noncovalent association, a number of challenges still remain [2,3,4,5] Two of these challenges are: (i) developing an understanding of the role of water in the binding process that goes beyond the low level of resolution offered by many theories of the so-called ‘‘hydrophobic effect’’ [6,7,8,9,10], and (ii) quantifying the conformational mobility for the macromolecule and the ligand in the complex [3,11,12,13].
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