| 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. ; 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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| 11. |
- Kamerlin, Shina C. L., 1981-, et al.
(författare)
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Managing Coronavirus Disease 2019 Spread With Voluntary Public Health Measures : Sweden as a Case Study for Pandemic Control
- 2020
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Ingår i: Clinical Infectious Diseases. - : Oxford University Press (OUP). - 1058-4838 .- 1537-6591. ; 71:12, s. 3174-3181
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundThe coronavirus disease 19 (COVID-19) pandemic has spread globally, causing extensive illness and mortality. In advance of effective antiviral therapies, countries have applied different public health strategies to control spread and manage healthcare need. Sweden has taken a unique approach of not implementing strict closures, instead urging personal responsibility. We analyze the results of this and other potential strategies for pandemic control in Sweden.MethodsWe implemented individual-based modeling of COVID-19 spread in Sweden using population, employment, and household data. Epidemiological parameters for COVID-19 were validated on a limited date range; where substantial uncertainties remained, multiple parameters were tested. The effects of different public health strategies were tested over a 160-day period, analyzed for their effects on intensive care unit (ICU) demand and death rate, and compared with Swedish data for April 2020.ResultsSwedish mortality rates are intermediate between rates for European countries that quickly imposed stringent public health controls and those for countries that acted later. Models most closely reproducing reported mortality data suggest that large portions of the population voluntarily self-isolate. Swedish ICU use rates remained lower than predicted, but a large fraction of deaths occurred in non-ICU patients. This suggests that patient prognosis was considered in ICU admission, reducing healthcare load at a cost of decreased survival in patients not admitted.ConclusionsThe Swedish COVID-19 strategy has thus far yielded a striking result: mild mandates overlaid with voluntary measures can achieve results highly similar to late-onset stringent mandates. However, this policy causes more healthcare demand and more deaths than early stringent control and depends on continued public will.
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| 12. |
- Kamerlin, Shina C. L., 1981-, et al.
(författare)
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Multiscale modeling of biological functions
- 2011
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Ingår i: Physical Chemistry, Chemical Physics - PCCP. - 1463-9076 .- 1463-9084. ; 13:22, s. 10401-10411
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Tidskriftsartikel (refereegranskat)abstract
- Recent years have witnessed a tremendous explosion in computational power, which in turn has resulted in great progress in the complexity of the biological and chemical problems that can be addressed by means of all-atom simulations. Despite this, however, our computational time is not infinite, and in fact many of the key problems of the field were resolved long before the existence of the current levels of computational power. This review will start by presenting a brief historical overview of the use of multiscale simulations in biology, and then present some key developments in the field, highlighting several cases where the use of a physically sound simplification is clearly superior to a brute-force approach. Finally, some potential future directions will be discussed.
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| 13. |
- Kamerlin, Shina C. L., 1981-, et al.
(författare)
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The effect of leaving group on mechanistic preference in phosphate monoesterhydrolysis
- 2011
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Ingår i: Organic and biomolecular chemistry. - : Royal Society of Chemistry (RSC). - 1477-0520 .- 1477-0539. ; 9:15, s. 5394-5406
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Tidskriftsartikel (refereegranskat)abstract
- We present 2-dimensional potential energy surfaces and transition states (TS) for water attack on a series of substituted phosphate monoester monoanions at the DFT level of theory, comparing a standard 6-31++g(d,p) basis set with a larger triple-zeta (augmented cc-pVTZ) basis set. Small fluorinated model compounds are used to simulate increasing leaving group stability without adding further geometrical complexity to the system. We demonstrate that whilst changing the leaving group causes little qualitative change in the potential energy surfaces (with the exception of the system with the most electron withdrawing leaving group, CF3O-, in which the associative pathway changes from a stepwise AN + DN pathway to a concerted ANDN pathway), there is a quantitative change in relative gas-phase and solution barriers for the two competing pathways. In line with previous studies, in the case of OCH3, the barriers for the associative and dissociative pathways are similar in solution, and the two pathways are equally viable and indistinguishable in solution. However, significantly increasing the stability of the leaving group (decreasing proton affinity, PA) results in the progressive favouring of a stepwise dissociative, DN + AN, mechanism over associative mechanisms.
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| 14. |
- Kamerlin, Shina C. L., 1981-, et al.
(författare)
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The empirical valence bond model : theory and applications
- 2011
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Ingår i: WIREs Computational Molecular Science. - : Wiley. - 1759-0876 .- 1759-0884. ; 1:1, s. 30-45
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Tidskriftsartikel (refereegranskat)abstract
- Recent years have seen an explosion in computer power, allowing for the examination of ever more challenging problems. For instance, a recent simulation study, which was the first of its kind, was able to actually explore the dynamical nature of enzyme catalysis on a millisecond timescale (Pisliakov AV, Cao J, Kamerlin SCL, Warshel A. Proc Natl Acad Sci U S A 2009, 106:17359.), something that as recently as a year or two ago would have been considered impossible. However, the questions that need addressing are nevertheless very complex, and experimental approaches can unfortunately often be inconclusive (Åqvist J, Kolmodin K, Florián J, Warshel A, Chem Biol 1999, 6:R71.) in answering them. Therefore, it is essential to have an approach that is both reliable and able to capture complex systems in order to resolve long-standing controversies [particularly with regards to questions such as the origin of enzyme catalysis, where the relevant energy contributions cannot be separated without some computational models (Warshel A, Sharma PK, Kato M, Xiang Y, Liu H, Olsson MHM, Chem Rev 2006, 106:3210.)]. Herein, we will present the empirical valence bond (EVB) approach, which, at present, is arguably the most powerful tool for examining chemical reactivity in the condensed phase. We will illustrate the effectiveness of the EVB method when evaluating, for instance, catalytic effects and demonstrate that it is currently the optimal tool for elucidating challenging problems such as understanding the catalytic power of enzymes. Finally, the increasing appreciation of this approach can maybe best illustrated not only by its proliferation but also by attempts to capture its basic chemistry under a different name, as will be discussed in this work.
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| 15. |
- Kulkarni, Yashraj S., et al.
(författare)
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Role of Ligand-Driven Conformational Changes in Enzyme Catalysis : Modeling the Reactivity of the Catalytic Cage of Triosephosphate Isomerase
- 2018
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 140:11, s. 3854-3857
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Tidskriftsartikel (refereegranskat)abstract
- We have previously performed empirical valence bond calculations of the kinetic activation barriers, Delta G(calc) double dagger, for the deprotonation of complexes between TIM and the whole substrate glyceraldehyde-3-phosphate (GAP, Kulkarni et al. J. Am. Chem. Soc. 2017, 139, 10514-10525). We now extend this work to also study the deprotonation of the substrate pieces glycolaldehyde (GA) and GA.HPi [HPi = phosphite dianion]. Our combined calculations provide activation barriers, Delta G(calc)(double dagger) for the TIM-catalyzed deprotonation of GAP (12.9 +/- 0.8 kcal.mol(-1)), of the substrate piece GA (15.0 +/- 2.4 kcal.mol(-1)), and of the pieces GA.HP, (15.5 +/- 3.5 kcal.mol(-1)). The effect of bound dianion on Delta G(calc) double dagger is small (<= 2.6 kcal.mol(-1)), in comparison to the much larger 12.0 and 5.8 kcal.mol(-1) intrinsic phosphodianion and phosphite dianion binding energy utilized to stabilize the transition states for TIM-catalyzed deprotonation of GAP and GA. HP, respectively. This shows that the dianion binding energy is essentially fully expressed at our protein model for the Michaelis complex, where it is utilized to drive an activating change in enzyme conformation. The results represent an example of the synergistic use of results from experiments and calculations to advance our understanding of enzymatic reaction mechanisms.
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| 16. |
- Kulkarni, Yashraj, et al.
(författare)
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Uncovering the Role of Key Active-Site Side Chains in Catalysis : An Extended Brønsted Relationship for Substrate Deprotonation Catalyzed by Wild-Type and Variants of Triosephosphate Isomerase
- 2019
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 141:40, s. 16139-16150
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Tidskriftsartikel (refereegranskat)abstract
- We report results of detailed empirical valence bond simulations that model the effect of several amino acid substitutions on the thermodynamic (ΔG°) and kinetic activation (ΔG⧧) barriers to deprotonation of dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (GAP) bound to wild-type triosephosphate isomerase (TIM), as well as to the K12G, E97A, E97D, E97Q, K12G/E97A, I170A, L230A, I170A/L230A, and P166A variants of this enzyme. The EVB simulations model the observed effect of the P166A mutation on protein structure. The E97A, E97Q, and E97D mutations of the conserved E97 side chain result in ≤1.0 kcal mol–1 decreases in the activation barrier for substrate deprotonation. The agreement between experimental and computed activation barriers is within ±1 kcal mol–1, with a strong linear correlation between ΔG⧧ and ΔG° for all 11 variants, with slopes β = 0.73 (R2 = 0.994) and β = 0.74 (R2 = 0.995) for the deprotonation of DHAP and GAP, respectively. These Brønsted-type correlations show that the amino acid side chains examined in this study function to reduce the standard-state Gibbs free energy of reaction for deprotonation of the weak α-carbonyl carbon acid substrate to form the enediolate phosphate reaction intermediate. TIM utilizes the cationic side chain of K12 to provide direct electrostatic stabilization of the enolate oxyanion, and the nonpolar side chains of P166, I170, and L230 are utilized for the construction of an active-site cavity that provides optimal stabilization of the enediolate phosphate intermediate relative to the carbon acid substrate.
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| 17. |
- Pabis, Anna, et al.
(författare)
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Simulating the reactions of substituted pyridinio-N-phosphonates with pyridine as a model for biological phosphoryl transfer
- 2017
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Ingår i: Organic and biomolecular chemistry. - : Royal Society of Chemistry. - 1477-0520 .- 1477-0539. ; 15:35, s. 7308-7316
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Tidskriftsartikel (refereegranskat)abstract
- Phosphoryl transfer reactions can proceed through several plausible mechanisms, and the potential for both solvent and substrate-assisted pathways (involving proton transfer to the phosphoryl oxygens) complicates both experimental and computational interpretations. To avoid this problem, we have used electronic structure calculations to probe the mechanisms of the reactions of pyridinio-N-phosphonates with pyridine. These compounds avoid the additional complexity introduced by proton transfer between the nucleophile and the leaving group, while also serving as a valuable model for biological P-N cleavage. Through a comparative study of a range of substrates of varying basicity, we demonstrate a unified concerted mechanism for the phosphoryl transfer reactions of these model compounds, proceeding through a dissociative transition state. Finally, a comparison of these transition states with previously characterized transition states for related compounds provides a more complete model for non-enzymatic phosphoryl transfer, which is a critical stepping stone to being able to fully understand phosphoryl transfer in biology.
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| 18. |
- Plotnikov, Nikolay V., et al.
(författare)
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Paradynamics : an effective and reliable model for ab initio QM/MM free-energy calculations and related tasks
- 2011
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 115:24, s. 7950-7962
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Tidskriftsartikel (refereegranskat)abstract
- Recent years have seen tremendous effort in the development of approaches with which to obtain quantum mechanics/molecular mechanics (QM/MM) free energies for reactions in the condensed phase. Nevertheless, there remain significant challenges to address, particularly, the high computational cost involved in performing proper configurational sampling and, in particular, in obtaining ab initio QM/MM (QM(ai)/MM) free-energy surfaces. One increasingly popular approach that seems to offer an ideal way to progress in this direction is the elegant metadynamics (MTD) approach. However, in the current work, we point out the subtle efficiency problems associated with this approach and illustrate that we have at hand what is arguably a more powerful approach. More specifically, we demonstrate the effectiveness of an updated version of our original idea of using a classical reference potential for QM(ai)/MM calculations [J. Phys. Chem. 1995, 99, 17516)], which we refer to as paradynamics (PD). This approach is based on the use of an empirical valence bond (EVB) reference potential, which is already similar to the real ab initio potential. The reference potential is fitted to the ab initio potential by an iterative and, to a great degree, automated refinement procedure. The corresponding free-energy profile is then constructed using the refined EVB potential, and the linear response approximation (LRA) is used to evaluate the QM(ai)/MM activation free-energy barrier. The automated refinement of the EVB surface (and thus the reduction of the difference between the reference and ab initio potentials) is a key factor in accelerating the convergence of the LRA approach. We apply our PD approach to a test reaction, namely, the SN2 reaction between a chloride ion and methyl chloride, and demonstrate that, at present, this approach is far more powerful and cost-effective than the metadynamics approach (at least in its current implementation). We also discuss the general features of the PD approach in terms of its ability to explore complex systems and clarify that it is not a specialized approach limited to only accelerating QM(ai)/MM calculations with proper sampling, but rather can be used in a wide variety of applications. In fact, we point out that the use of a reference (CG) potential coupled with its PD refinement, as well as our renormalization approach, provides very general and powerful strategies that can be used very effectively to explore any property that has been studied by the MTD approach.
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| 19. |
- Risso, Valeria A., et al.
(författare)
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Enhancing a de novo enzyme activity by computationally-focused ultra-low-throughput screening
- 2020
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Ingår i: Chemical Science. - : Royal Society of Chemistry (RSC). - 2041-6520 .- 2041-6539. ; 11:24, s. 6134-6148
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Tidskriftsartikel (refereegranskat)abstract
- Directed evolution has revolutionized protein engineering. Still, enzyme optimization by random library screening remains sluggish, in large part due to futile probing of mutations that are catalytically neutral and/or impair stability and folding. FuncLib is a novel approach which uses phylogenetic analysis and Rosetta design to rank enzyme variants with multiple mutations, on the basis of predicted stability. Here, we use it to target the active site region of a minimalist-designed, de novo Kemp eliminase. The similarity between the Michaelis complex and transition state for the enzymatic reaction makes this system particularly challenging to optimize. Yet, experimental screening of a small number of active-site variants at the top of the predicted stability ranking leads to catalytic efficiencies and turnover numbers (∼2 × 104 M−1 s−1 and ∼102 s−1) for this anthropogenic reaction that compare favorably to those of modern natural enzymes. This result illustrates the promise of FuncLib as a powerful tool with which to speed up directed evolution, even on scaffolds that were not originally evolved for those functions, by guiding screening to regions of the sequence space that encode stable and catalytically diverse enzymes. Empirical valence bond calculations reproduce the experimental activation energies for the optimized eliminases to within ∼2 kcal mol−1 and indicate that the enhanced activity is linked to better geometric preorganization of the active site. This raises the possibility of further enhancing the stability-guidance of FuncLib by computational predictions of catalytic activity, as a generalized approach for computational enzyme design.
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| 20. |
- Yehorova, Dariia, et al.
(författare)
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Key interaction networks : Identifying evolutionarily conserved non-covalent interaction networks across protein families
- 2024
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Ingår i: Protein Science. - : John Wiley & Sons. - 0961-8368 .- 1469-896X. ; 33:3
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Tidskriftsartikel (refereegranskat)abstract
- Protein structure (and thus function) is dictated by non-covalent interaction networks. These can be highly evolutionarily conserved across protein families, the members of which can diverge in sequence and evolutionary history. Here we present KIN, a tool to identify and analyze conserved non-covalent interaction networks across evolutionarily related groups of proteins. KIN is available for download under a GNU General Public License, version 2, from https://www.github.com/kamerlinlab/KIN. KIN can operate on experimentally determined structures, predicted structures, or molecular dynamics trajectories, providing insight into both conserved and missing interactions across evolutionarily related proteins. This provides useful insight both into protein evolution, as well as a tool that can be exploited for protein engineering efforts. As a showcase system, we demonstrate applications of this tool to understanding the evolutionary-relevant conserved interaction networks across the class A β-lactamases.
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