| 1. |
- Kulkarni, Yashraj S., et al.
(författare)
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Enzyme Architecture : Modeling the Operation of a Hydrophobic Clamp in Catalysis by Triosephosphate Isomerase
- 2017
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 139:30, s. 10514-10525
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Tidskriftsartikel (refereegranskat)abstract
- Triosephosphate isomerase (TIM) is a proficient catalyst of the reversible isomerization of dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde phosphate (GAP), via general base catalysis by E165. Historically, this enzyme has been an extremely important model system for understanding the fundamentals of biological catalysis. TIM is activated through an energetically demanding conformational change, which helps position the side chains of two key hydrophobic residues (1170 and L230), over the carboxylate side chain of E165. This is critical both for creating a hydrophobic pocket for the catalytic base and for maintaining correct active site architecture. Truncation of these residues to alanine causes significant falloffs in TIM's catalytic activity, but experiments have failed to provide a full description of the action of this clamp in promoting substrate deprotonation. We perform here detailed empirical valence bond calculations of the TIM-catalyzed deprotonation of DHAP and GAP by both wild type TIM and its 1170A, L230A, and 1170A/L230A mutants, obtaining exceptional quantitative agreement with experiment. Our calculations provide a linear free energy relationship, with slope 0.8, between the activation barriers and Gibbs free energies for these TIM-catalyzed reactions. We conclude that these clamping side chains minimize the Gibbs free energy for substrate deprotonation, and that the effects on reaction driving force are largely expressed at the transition state for proton transfer. Our combined analysis of previous experimental and current computational results allows us to provide an overview of the breakdown of ground-state and transition state effects in enzyme catalysis in unprecedented detail, providing a molecular description of the operation of a hydrophobic clamp in triosephosphate isomerase.
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| 2. |
- Shen, Ruidan, et al.
(författare)
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Insights into the importance of WPD-loop sequence for activity and structure in protein tyrosine phosphatases
- 2022
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Ingår i: Chemical Science. - : Royal Society of Chemistry. - 2041-6520 .- 2041-6539. ; 13:45, s. 13524-13540
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Tidskriftsartikel (refereegranskat)abstract
- Protein tyrosine phosphatases (PTPs) possess a conserved mobile catalytic loop, the WPD-loop, which brings an aspartic acid into the active site where it acts as an acid/base catalyst. Prior experimental and computational studies, focused on the human enzyme PTP1B and the PTP from Yersinia pestis, YopH, suggested that loop conformational dynamics are important in regulating both catalysis and evolvability. We have generated a chimeric protein in which the WPD-loop of YopH is transposed into PTP1B, and eight chimeras that systematically restored the loop sequence back to native PTP1B. Of these, four chimeras were soluble and were subjected to detailed biochemical and structural characterization, and a computational analysis of their WPD-loop dynamics. The chimeras maintain backbone structural integrity, with somewhat slower rates than either wild-type parent, and show differences in the pH dependency of catalysis, and changes in the effect of Mg2+. The chimeric proteins' WPD-loops differ significantly in their relative stability and rigidity. The time required for interconversion, coupled with electrostatic effects revealed by simulations, likely accounts for the activity differences between chimeras, and relative to the native enzymes. Our results further the understanding of connections between enzyme activity and the dynamics of catalytically important groups, particularly the effects of non-catalytic residues on key conformational equilibria.
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| 3. |
- Shen, Ruidan, et al.
(författare)
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Single Residue on the WPD-Loop Affects the pH Dependency of Catalysis in Protein Tyrosine Phosphatases
- 2021
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Ingår i: JACS Au. - : American Chemical Society (ACS). - 2691-3704. ; 1:5, s. 646-659
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Tidskriftsartikel (refereegranskat)abstract
- Catalysis by protein tyrosine phosphatases (PTPs) relies on the motion of a flexible protein loop (the WPD-loop) that carries a residue acting as a general acid/base catalyst during the PTP-catalyzed reaction. The orthogonal substitutions of a noncatalytic residue in the WPD-loops of YopH and PTP1B result in shifted pH-rate profiles from an altered kinetic pKa of the nucleophilic cysteine. Compared to wild type, the G352T YopH variant has a broadened pH-rate profile, similar activity at optimal pH, but significantly higher activity at low pH. Changes in the corresponding PTP1B T177G variant are more modest and in the opposite direction, with a narrowed pH profile and less activity in the most acidic range. Crystal structures of the variants show no structural perturbations but suggest an increased preference for the WPD-loop-closed conformation. Computational analysis confirms a shift in loop conformational equilibrium in favor of the closed conformation, arising from a combination of increased stability of the closed state and destabilization of the loop-open state. Simulations identify the origins of this population shift, revealing differences in the flexibility of the WPD-loop and neighboring regions. Our results demonstrate that changes to the pH dependency of catalysis by PTPs can result from small changes in amino acid composition in their WPD-loops affecting only loop dynamics and conformational equilibrium. The perturbation of kinetic pKa values of catalytic residues by nonchemical processes affords a means for nature to alter an enzyme’s pH dependency by a less disruptive path than altering electrostatic networks around catalytic residues themselves.
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| 4. |
- Österlund, Nicklas, et al.
(författare)
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Amyloid-beta Peptide Interactions with Amphiphilic Surfactants : Electrostatic and Hydrophobic Effects
- 2018
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Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 9:7, s. 1680-1692
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Tidskriftsartikel (refereegranskat)abstract
- The amphiphilic nature of the amyloid-beta (A beta) peptide associated with Alzheimer's disease facilitates various interactions with biomolecules such as lipids and proteins, with effects on both structure and toxicity of the peptide. Here, we investigate these peptide-amphiphile interactions by experimental and computational studies of A beta(1-40) in the presence of surfactants with varying physicochemical properties. Our findings indicate that electrostatic peptide-surfactant interactions are required for coclustering and structure induction in the peptide and that the strength of the interaction depends on the surfactant net charge. Both aggregation-prone peptide-rich coclusters and stable surfactant-rich coclusters can form. Only A beta(1-40) monomers, but not oligomers, are inserted into surfactant micelles in this surfactant-rich state. Surfactant headgroup charge is suggested to be important as electrostatic peptide-surfactant interactions on the micellar surface seems to be an initiating step toward insertion. Thus, no peptide insertion or change in peptide secondary structure is observed using a nonionic surfactant. The hydrophobic peptide-surfactant interactions instead stabilize the A beta monomer, possibly by preventing self-interaction between the peptide core and C terminus, thereby effectively inhibiting the peptide aggregation process. These findings give increased understanding regarding the molecular driving forces for A beta aggregation and the peptide interaction with amphiphilic biomolecules.
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| 5. |
- Ben-David, Moshe, et al.
(författare)
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Enzyme Evolution An Epistatic Ratchet versus a Smooth Reversible Transition
- 2020
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Ingår i: Molecular biology and evolution. - : Oxford University Press (OUP). - 0737-4038 .- 1537-1719. ; 37:4, s. 1133-1147
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Tidskriftsartikel (refereegranskat)abstract
- Evolutionary trajectories are deemed largely irreversible. In a newly diverged protein, reversion of mutations that led to the functional switch typically results in loss of both the new and the ancestral functions. Nonetheless, evolutionary transitions where reversions are viable have also been described. The structural and mechanistic causes of reversion compatibility versus incompatibility therefore remain unclear. We examined two laboratory evolution trajectories of mammalian paraoxonase-1, a lactonase with promiscuous organophosphate hydrolase (OPH) activity. Both trajectories began with the same active-site mutant, His115Trp, which lost the native lactonase activity and acquired higher OPH activity. A neo-functionalization trajectory amplified the promiscuous OPH activity, whereas the re-functionalization trajectory restored the native activity, thus generating a new lactonase that lacks His115. The His115 revertants of these trajectories indicated opposite trends. Revertants of the neo-functionalization trajectory lost both the evolved OPH and the original lactonase activity. Revertants of the trajectory that restored the original lactonase function were, however, fully active. Crystal structures and molecular simulations show that in the newly diverged OPH, the reverted His115 and other catalytic residues are displaced, thus causing loss of both the original and the new activity. In contrast, in the re-functionalization trajectory, reversion compatibility of the original lactonase activity derives from mechanistic versatility whereby multiple residues can fulfill the same task. This versatility enables unique sequence-reversible compositions that are inaccessible when the active site was repurposed toward a new function.
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| 6. |
- Enugala, Thilak Reddy, et al.
(författare)
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The Role of Substrate-Coenzyme Crosstalk in Determining Turnover Rates in Rhodococcus ruber Alcohol Dehydrogenase
- 2020
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Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435 .- 2155-5435. ; 10:16, s. 9115-9128
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Tidskriftsartikel (refereegranskat)abstract
- Eight related alcohol dehydrogenases that had been originally isolated by laboratory evolution of ADH-A from Rhodococcus ruber DSM44541 for modified substrate scopes, were together with their parent wild-type, subjected to biochemical characterization of possible activities with a panel of chiral alcohols and pro-chiral ketones. Determinations of rates of catalyzed alcohol oxidations and ketone reductions, and of cofactor release, pointed out to the role of a W295A substitution as being decisive in steering enantioselectivity in the oxidation of arylated 1-methyl substituted alcohols. Molecular dynamics simulations of enzyme-substrate interactions in the Michaelis complexes of wild-type and a Y294F/W295A double mutant could rationalize the experimentally observed shift in enantioselectivity and differences in catalytic activity with 4-phenyl-2-butanol. Finally, we present herein evidence for apparent inter-dependency between substrate/product and the cofactor in the ternary complex, that directly affects the NADH dissociation rates, and thus that this substrate-coenzyme crosstalk plays a direct role in determining the turnover rates.
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| 7. |
- Fisher, Gemma, et al.
(författare)
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Allosteric rescue of catalytically impaired ATP phosphoribosyltransferase variants links protein dynamics to active-site electrostatic preorganisation
- 2022
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- ATP phosphoribosyltransferase catalyses the first step of histidine biosynthesis and is controlled via a complex allosteric mechanism where the regulatory protein HisZ enhances catalysis by the catalytic protein HisGS while mediating allosteric inhibition by histidine. Activation by HisZ was proposed to position HisGS Arg56 to stabilise departure of the pyrophosphate leaving group. Here we report active-site mutants of HisGS with impaired reaction chemistry which can be allosterically restored by HisZ despite the HisZ:HisGS interface lying ~20 Å away from the active site. MD simulations indicate HisZ binding constrains the dynamics of HisGS to favour a preorganised active site where both Arg56 and Arg32 are poised to stabilise leaving-group departure in WT-HisGS. In the Arg56Ala-HisGS mutant, HisZ modulates Arg32 dynamics so that it can partially compensate for the absence of Arg56. These results illustrate how remote protein-protein interactions translate into catalytic resilience by restoring damaged electrostatic preorganisation at the active site.
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| 8. |
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| 9. |
- Hong, Nan-Sook, et al.
(författare)
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The evolution of multiple active site configurations in a designed enzyme
- 2018
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Developments in computational chemistry, bioinformatics, and laboratory evolution have facilitated the de novo design and catalytic optimization of enzymes. Besides creating useful catalysts, the generation and iterative improvement of designed enzymes can provide valuable insight into the interplay between the many phenomena that have been suggested to contribute to catalysis. In this work, we follow changes in conformational sampling, electrostatic preorganization, and quantum tunneling along the evolutionary trajectory of a designed Kemp eliminase. We observe that in the Kemp Eliminase KE07, instability of the designed active site leads to the emergence of two additional active site configurations. Evolutionary conformational selection then gradually stabilizes the most efficient configuration, leading to an improved enzyme. This work exemplifies the link between conformational plasticity and evolvability and demonstrates that residues remote from the active sites of enzymes play crucial roles in controlling and shaping the active site for efficient catalysis.
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| 10. |
- Kamerlin, Shina C. L., 1981-, et al.
(författare)
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Coarse-grained (multiscale) simulations in studies of biophysical and chemical systems
- 2011
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Ingår i: Annual review of physical chemistry (Print). - : Annual Reviews. - 0066-426X .- 1545-1593. ; 62, s. 41-64
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Tidskriftsartikel (refereegranskat)abstract
- Recent years have witnessed an explosion in computational power, leading toattempts to model ever more complex systems. Nevertheless, there remain cases for which the use of brute-force computer simulations is clearly not the solution. In such cases, great benefit can be obtained from the use of physically sound simplifications. The introduction of such coarse graining can be traced back to the early usage of a simplified model in studies of proteins. Since then, the field has progressed tremendously. In this review,we cover both key developments in the field and potential future directions. Additionally, particular emphasis is given to two general approaches, namely the renormalization and reference potential approaches, which allow one to move back and forth between the coarse-grained (CG) and full models, as these approaches provide the foundation for CG modeling of complex systems.
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