| 1. |
- Amrein, Beat Anton, et al.
(författare)
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CADEE : Computer-Aided Directed Evolution of Enzymes
- 2017
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Ingår i: IUCrJ. - 2052-2525. ; 4:1, s. 50-64
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Tidskriftsartikel (refereegranskat)abstract
- The tremendous interest in enzymes as biocatalysts has led to extensive work in enzyme engineering, as well as associated methodology development. Here, a new framework for computer-aided directed evolution of enzymes (CADEE) is presented which allows a drastic reduction in the time necessary to prepare and analyze in silico semi-automated directed evolution of enzymes. A pedagogical example of the application of CADEE to a real biological system is also presented in order to illustrate the CADEE workflow.
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| 2. |
- Kulkarni, Yashraj
(författare)
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Computational Modeling of the Structure, Function and Dynamics of Biomolecular Systems
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are a structurally diverse and functionally versatile class of biomolecules. They perform a variety of life-sustaining biological processes with utmost efficiency. A profound understanding of protein function requires knowledge of its structure. Experimentally determined protein structures can serve as a starting point for computer simulations in order to study their dynamic behavior at a molecular level. In this thesis, computational methods have been used to understand structure-function relationships in two classes of proteins - intrinsically disordered proteins (IDP) and enzymes.Misfolding and subsequent aggregation of the amyloid beta (Aβ) peptide, an IDP, is associated with the progression of Alzheimer’s disease. Besides enriching our understanding of structural dynamics, computational studies on a medically relevant IDP such as Aβ can potentially guide therapeutic development. In the present work, binding interactions of the monomeric form of this peptide with biologically relevant molecular species such as divalent metal ions (Zn2+, Cu2+, Mn2+) and amphiphilic surfactants were characterized using long timescale molecular dynamics (MD) simulations. Among the metal ions, while Zn2+ and Cu2+ maintained coordination to a well-defined binding site in Aβ, Mn2+-binding was observed to be comparatively weak and transient. Surfactants with charged headgroups displayed strong binding interaction with Aβ. Complemented by biophysical experiments, these studies provided a multifaceted perspective of Aβ interactions with the partner molecules.Triosephosphate isomerase (TIM), a highly evolved and catalytically proficient enzyme, was studied using empirical valence bond (EVB) calculations to obtain deeper insights into the catalytic reaction mechanism. Multiple structural features of TIM such as the flexible loop and preorganized active site residues were investigated for their role in enzyme catalysis. The effect of substrate binding was also studied using truncated substrates. Finally, using enhanced sampling methods, dynamic behavior of the catalytically important loop 6 was characterized. The importance of structural stability and flexibility on protein function was illustrated by the work presented in this thesis, thus furthering our scientific understanding of proteins at a molecular level.
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| 3. |
- Kulkarni, Yashraj, et al.
(författare)
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Computational physical organic chemistry using the empirical valence bond approach
- 2019
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Ingår i: Advances in Physical Organic Chemistry. - : Elsevier. - 0065-3160 .- 2162-5921. ; 53, s. 69-104
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Tidskriftsartikel (refereegranskat)abstract
- There has been growing interest in applying the empirical valence bond approach to a range of (bio)chemical problems, primarily to study enzymatic and non-enzymatic catalysis, but also to studying other processes such as excited state chemistry and reaction dynamics. Despite its apparent theoretical simplicity, this approach is a powerful computational tool that can be used to reproduce and rationalize a wide range of experimental observables, such as linear free energy relationships, kinetic isotope effects, and temperature effects on reaction rates. We provide here both a theoretical background for this approach, as well as highlighting several of its broad applications in computational physical organic chemistry.
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| 4. |
- 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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| 5. |
- 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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| 6. |
- 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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| 7. |
- Liao, Qinghua, et al.
(författare)
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Loop Motion in Triosephosphate Isomerase Is Not a Simple Open and Shut Case
- 2018
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 140:46, s. 15889-15903
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Tidskriftsartikel (refereegranskat)abstract
- Conformational changes are crucial for the catalytic action of many enzymes. A prototypical and well-studied example is loop opening and closure in triosephosphate isomerase (TIM), which is thought to determine the rate of catalytic turnover in many circumstances. Specifically, TIM loop 6 “grips” the phosphodianion of the substrate and, together with a change in loop 7, sets up the TIM active site for efficient catalysis. Crystal structures of TIM typically show an open or a closed conformation of loop 6, with the tip of the loop moving ∼7 Å between conformations. Many studies have interpreted this motion as a two-state, rigid-body transition. Here, we use extensive molecular dynamics simulations, with both conventional and enhanced sampling techniques, to analyze loop motion in apo and substrate-bound TIM in detail, using five crystal structures of the dimeric TIM from Saccharomyces cerevisiae. We find that loop 6 is highly flexible and samples multiple conformational states. Empirical valence bond simulations of the first reaction step show that slight displacements away from the fully closed-loop conformation can be sufficient to abolish most of the catalytic activity; full closure is required for efficient reaction. The conformational change of the loops in TIM is thus not a simple “open and shut” case and is crucial for its catalytic action. Our detailed analysis of loop motion in a highly efficient enzyme highlights the complexity of loop conformational changes and their role in biological catalysis.
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| 8. |
- Parkash, Vimal, et al.
(författare)
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A sensor complements the steric gate when DNA polymerase ε discriminates ribonucleotides
- 2023
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 51:20, s. 11225-11238
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Tidskriftsartikel (refereegranskat)abstract
- The cellular imbalance between high concentrations of ribonucleotides (NTPs) and low concentrations of deoxyribonucleotides (dNTPs), is challenging for DNA polymerases when building DNA from dNTPs. It is currently believed that DNA polymerases discriminate against NTPs through a steric gate model involving a clash between a tyrosine and the 2 '-hydroxyl of the ribonucleotide in the polymerase active site in B-family DNA polymerases. With the help of crystal structures of a B-family polymerase with a UTP or CTP in the active site, molecular dynamics simulations, biochemical assays and yeast genetics, we have identified a mechanism by which the finger domain of the polymerase sense NTPs in the polymerase active site. In contrast to the previously proposed polar filter, our experiments suggest that the amino acid residue in the finger domain senses ribonucleotides by steric hindrance. Furthermore, our results demonstrate that the steric gate in the palm domain and the sensor in the finger domain are both important when discriminating NTPs. Structural comparisons reveal that the sensor residue is conserved among B-family polymerases and we hypothesize that a sensor in the finger domain should be considered in all types of DNA polymerases.
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| 9. |
- Parkash, Vimal, et al.
(författare)
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Structural consequence of the most frequently recurring cancer-associated substitution in DNA polymerase epsilon
- 2019
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- The most frequently recurring cancer-associated DNA polymerase epsilon (Pol epsilon) mutation is a P286R substitution in the exonuclease domain. While originally proposed to increase genome instability by disrupting exonucleolytic proofreading, the P286R variant was later found to be significantly more pathogenic than Pol epsilon proofreading deficiency per se. The mechanisms underlying its stronger impact remained unclear. Here we report the crystal structure of the yeast orthologue, Pol epsilon-P301R, complexed with DNA and an incoming dNTP. Structural changes in the protein are confined to the exonuclease domain, with R301 pointing towards the exonuclease site. Molecular dynamics simulations suggest that R301 interferes with DNA binding to the exonuclease site, an outcome not observed with the exonuclease-inactive Pol epsilon-D290A, E292A variant lacking the catalytic residues. These results reveal a distinct mechanism of exonuclease inactivation by the P301R substitution and a likely basis for its dramatically higher mutagenic and tumorigenic effects.
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| 10. |
- Wallin, Cecilia, et al.
(författare)
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Characterization of Mn(II) ion binding to the amyloid-beta peptide in Alzheimer's disease
- 2016
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Ingår i: Journal of Trace Elements in Medicine and Biology. - : Elsevier BV. - 0946-672X .- 1878-3252. ; 38, s. 183-193
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Tidskriftsartikel (refereegranskat)abstract
- Growing evidence links neurodegenerative diseases to metal exposure. Aberrant metal ion concentrations have been noted in Alzheimer's disease (AD) brains, yet the role of metals in AD pathogenesis remains unresolved. A major factor in AD pathogenesis is considered to be aggregation of and amyloid formation by amyloid-beta (A beta) peptides. Previous studies have shown that A beta displays specific binding to Cu(II) and Zn(II) ions, and such binding has been shown to modulate A beta aggregation. Here, we use nuclear magnetic resonance (NMR) spectroscopy to show that Mn(II) ions also bind to the N-terminal part of the A beta(1-40) peptide, with a weak binding affinity in the milli- to micromolar range. Circular dichroism (CD) spectroscopy, solid state atomic force microscopy (AFM), fluorescence spectroscopy, and molecular modeling suggest that the weak binding of Mn(II) to A beta may not have a large effect on the peptide's aggregation into amyloid fibrils. However, identification of an additional metal ion displaying A beta binding reveals more complex AD metal chemistry than has been previously considered in the literature.
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| 11. |
- 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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