Binding of Solvent Molecules to a Protein Surface in Binary Mixtures Follows a Competitive Langmuir Model

Abstract

The binding of solvent molecules to a protein surface was modeled by molecular dynamics simulations of of Candida antarctica (C. antarctica) lipase B in binary mixtures of water, methanol, and toluene. Two models were analyzed: a competitive Langmuir model which assumes identical solvent binding sites with a different affinity toward water (KWat), methanol (KMet), and toluene (KTol) and a competitive Langmuir model with an additional interaction between free water and already bound water (KWatWat). The numbers of protein-bound molecules of both components of a binary mixture were determined for different compositions as a function of their thermodynamic activities in the bulk phase, and the binding constants were simultaneously fitted to the six binding curves (two components of three different mixtures). For both Langmuir models, the values of KWat, KMet, and KTol were highly correlated. The highest binding affinity was found for methanol, which was almost 4-fold higher than the binding affinities of water and toluene (KMet ≫ KWat ≈ KTol). Binding of water was dominated by the water-water interaction (KWatWat). Even for the three protein surface patches of highest water affinity, the binding affinity of methanol was 2-fold higher than water and 8-fold higher than toluene (KMet > KWat > KTol). The Langmuir model provides insights into the protein destabilizing mechanism of methanol which has a high binding affinity toward the protein surface. Thus, destabilizing solvents compete with intraprotein interactions and disrupt the tertiary structure. In contrast, benign solvents such as water or toluene have a low affinity toward the protein surface. Water is a special solvent: only few water molecules bind directly to the protein; most water molecules bind to already bound water molecules thus forming water patches. A quantitative mechanistic model of protein-solvent interactions that includes competition and miscibility of the components contributes a robust basis for solvent and protein engineering.

Understanding the activation mechanism of Thermomyces lanuginosus lipase using rational design and tryptophan‐induced fluorescence quenching

“…TLL and the constructed lid-variants were stable up to $70% ethanol (e ¼ 40). At higher ethanol concentrations, spectral shifts occurred and secondary structure was lost ascribed to a destabilizing effect of strong, unspecific binding of ethanol to the protein surface and stripping of the essential water layer around the enzyme [117][118][119]. Overall, these studies underline the structural robustness of TLL and indicate that any structural movements detected above e ¼ 40 are representative of local conformational changes occurring in the lid, not to a globular protein unfolding event ( Fig.…”

Cold-active extracellular lipase: Expression in Sf9 insect cells, purification, and catalysis

“…Binding of a thin layer of water molecules to the surface is essential for the enzyme protein to maintain its native conformation [ 37 ]. Water is a particular solvent type that shows lower affinity toward the protein surface in comparison to water-miscible organic solvents [ 48 ]. Water patches on the protein surface are formed by a limited number of directly-bound water molecules and also by water–water interactions.…”

Modelling of substrate access and substrate binding to cephalosporin acylases

“…A substrate molecule (CPC) was defined as bound to the protein surface close to the entrance to the binding pocket, if its center of mass was within 5 Å from any protein atom within 25 Å from the hydroxyl oxygen of β1 serine. The affinity of CPC for the enzyme was determined by fitting a Langmuir binding model 65 , assuming non-cooperative binding to a limited number of identical binding sites. The number of bound substrate molecules CPC b was determined by counting (GROMACS tool gmx trjorder ) the number of CPC molecules bound to the protein and by averaging over the simulation runs at the same substrate concentration.…”