| 1. |
- Fagerström, Alexandra, et al.
(författare)
-
Chiral recognition mechanism of cellobiohydrolase Cel7A for ligands based on the β‐blocker propranolol : The effect of explicit water molecules on binding and selectivities
- 2023
-
Ingår i: Natural Sciences. - : John Wiley & Sons. - 2698-6248 .- 2698-6248. ; 3:3
-
Tidskriftsartikel (refereegranskat)abstract
- Proteins are useful chiral selectors. In order to understand the recognition mechanism and chiral discrimination, the binding of the (R)- and (S)-enantiomers of a series of designed amino alcohol inhibitors based on propranolol to cellobiohydrolase Cel7A (Trichoderma reesei) has been studied more closely. X-ray crystal structures were determined of the protein complex with the (R)- and (S)-enantiomers of the strongest binding propranolol analogue. The combination of the structural data, thermodynamic data from capillary electrophoresis and microcalorimetry experiments and computational modelling give a clearer insight into the origin of the enantioselectivity and its opposite thermodynamic signature. The new crystal structures were used in computational molecular flexible dockings of the propranolol analogues using the program Glide. The results indicated that several water molecules in the active site were essential for the docking of the (R)-enantiomers but not for the (S)-enantiomers. The results are discussed in relation to the enantiomeric discrimination of the enzyme. Both dissociation constants (Kd values) and thermodynamical data are included to show the effects of the structural modifications in the ligand on enthalpy and entropy in relation to the enantioselectivity.
|
|
| 2. |
- Fagerström, Alexandra
(författare)
-
Enantioselective Binding of Designed Propranolol Analogues to the Cellobiohydrolase Cel7A
- 2006
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes the affinity and enantiomeric selectivity of the cellobiohydrolase Cel7A (isolated from Hypocrea jercorina). The chiral recognition of the enzyme has been studied with respect to amino alcohols and new ligands have been designed, based on the structure of the common ?-blocker propranolol. Capillary electrophoresis have been used to determine the dissociation constants of the ligand binding at pH 5.0 and 7.0 to obtain information about the effects of the protonation state of the enzyme. A decrecased selectivity was shown at pH 7, despite an increase in affinity. The structural modifications in the ligands have also been related to the affinity and selectivity obtained. An expantion of the aromatic ring system of propranolol in combination with additional hydroxyl groups gave an increase in both affinity and selectivity. This resulted in an improved inhibitor of the enzyme. The energies involved in complexation, as in enthalpy and entropy, have been determined for a selected number of ligands and the entropy is the driving force of the selectivity, indicating the importnace of water displacement upon ligand binding. Also, new X-ray crystal structures have been obtained and structures of both enantiomers of the strongest binding ligand are now available. These structures have been used in computational calculations to study the ligand binding. The water molecules in the active site have been shown to have a much greater effect on the binding of the (R)-enantiomers, than for the (S)-enantiomers, which gives rise to the chiral selectivity of Cel7A.
|
|
| 3. |
|
|
| 4. |
- Fagerström, Alexandra, et al.
(författare)
-
New propranolol analogues: binding and chiral discrimination by cellobiohydrolase Cel7A
- 2006
-
Ingår i: Organic and Biomolecular Chemistry. - : Royal Society of Chemistry (RSC). - 1477-0539. ; 4:16, s. 3067-3076
-
Tidskriftsartikel (refereegranskat)abstract
- Novel propranolol analogues have been designed and synthesised and their enantioselective binding to the cellulose degrading enzyme, Cel7A, has been evaluated. Affinity and enantioselectivity have been determined by capillary electrophoresis experiments. Ligands with significantly improved affinity and selectivity have been obtained and an analysis of the results has led to insights concerning the relation between the changes in ligand structure and selectivity as well as affinity to the protein.
|
|
| 5. |
- Fagerström, Alexandra, et al.
(författare)
-
pH dependency of ligand binding to cellobiohydrolase 1 (Cel7A) - Affinity, selectivity and inhibition for designed propranolol analogues
- 2007
-
Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1138:1-2, s. 276-283
-
Tidskriftsartikel (refereegranskat)abstract
- The affinity and enantioselectivity have been determined for designed propranolol derivatives as ligands for Cel7A by capillary electrophoresis (CE) at pH 7.0. These results have been compared to measurements at pH 5.0. In agreement with previous studies, the affinity increased at the higher pH. However, the affinity was not as dependent of the ligand structure at pH 7.0 as at pH 5.0, and the selectivity was generally decreased. Instead, at pH 7.0, the changes in binding were mainly dependent on the presence of additional dihydroxyl groups, indicating an increased importance of the electrostatic interactions. To evaluate the pH dependent variations in binding, changes in both the ligand and in the enzyme had to be taken into account. To ensure that the ligands had the same charge in all measurements, pKa-values of all compounds were determined. The ligand-protein interaction has also been studied by inhibition experiments at both pHs to evaluate the specific binding to the active site when competing with the substrate p-nitrophenyl lactoside (pNPL). With support of docking computations we propose a hypothesis on the effect of the ligand structure and pH dependency of the binding and selectivity of amino alcohols to Cel7A. (c) 2006 Elsevier B.V. All rights reserved.
|
|
| 6. |
- Hamark, Christoffer, et al.
(författare)
-
Enantioselective Binding of Propranolol and Analogues Thereof to Cellobiohydrolase Cel7A
- 2018
-
Ingår i: Chemistry - A European Journal. - : Wiley. - 0947-6539 .- 1521-3765. ; 24:68, s. 17975-17985
-
Tidskriftsartikel (refereegranskat)abstract
- At the catalytic site for the hydrolysis of cellulose the enzyme cellobiohydrolase Cel7A binds the enantiomers of the adrenergic beta-blocker propranolol with different selectivity. Methyl-to-hydroxymethyl group modifications of propranolol, which result in higher affinity and improved selectivity, were herein studied by H-1,H-1 and H-1,C-13 scalar spin-spin coupling constants as well as utilizing the nuclear Overhauser effect (NOE) in conjunction with molecular dynamics simulations of the ligands per se, which showed the presence of all-antiperiplanar conformations, except for the one containing a vicinal oxygen-oxygen arrangement governed by the gauche effect. For the ligand-protein complexes investigated by NMR spectroscopy using, inter alia, transferred NOESY and saturation-transfer difference (STD) NMR experiments the S-isomers were shown to bind with a higher affinity and a conformation similar to that preferred in solution, in contrast to the R-isomer. The fact that the S-form of the propranolol enantiomer is pre-arranged for binding to the protein is also observed for a crystal structure of dihydroxy-(S)-propranolol and Cel7A presented herein. Whereas the binding of propranolol is entropy driven, the complexation with the dihydroxy analogue is anticipated to be favored also by an enthalpic term, such as for its enantiomer, that is, dihydroxy-(R)-propranolol, because hydrogen-bond donation replaces the corresponding bonding from hydroxyl groups in glucosyl residues of the natural substrate. In addition to a favorable entropy component, albeit lesser in magnitude, this represents an effect of enthalpy-to-entropy compensation in ligand-protein interactions.
|
|
| 7. |
- Nilsson, Mikael, et al.
(författare)
-
Thermodynamics studies of designed ligands binding to Cel7A using partial-filling capillary electrophoresis
- 2008
-
Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 29:2, s. 358-362
-
Tidskriftsartikel (refereegranskat)abstract
- A convenient experimental method for thermodynamical studies based on partial-filling affinity CE is presented. The advantages of this approach are the possibility to determine binding energies from relatively weak interactions as well as the small amounts of samples consumed. In order to explore the affinity and selectivity of the cellobiolrydrolase Cel7A, a number of propranolol analogues were recently designed. The affinities of a selection of these ligands were determined in the temperature interval 15-40 degrees C, and Delta G degrees, Delta H degrees and Delta S degrees were obtained by means of Van't Hoff plots. Through these experiments, the importance of the entropy contribution in the complexation between the ligands and Cel7A has been demonstrated.
|
|
