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  • 1.
    Andre, Ingemar
    et al.
    Lund University.
    Bjelic, Sinisa
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Computational assessment of folding energy landscapes in heterodimeric coiled coils2018In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 86, no 7, p. 790-801Article in journal (Refereed)
    Abstract [en]

    The coiled coil structural motif consists of alpha helices supercoiling around each other to form staggered knobs-into-holes packing. Such structures are deceptively simple, especially as they often can be described with parametric equations, but are known to exist in various conformations. Even the simplest systems, consisting of 2 monomers, can assemble into a wide range of states. They can form canonical as well as noncanonical coiled coils, be parallel or antiparallel, where helices associate with different degrees of shift, tilt, and rotation. Here, we investigate the energy landscape of heterodimeric coiled coils by carrying out de novo folding simulations starting from amino acid sequence. We folded a diverse set of 22 heterodimers and demonstrate that the approach is capable of identifying the atomic details in the experimental structure in the majority of cases. Our methodology also enables exploration of alternative states that can be accessible in solution beyond the experimentally determined structure. For many systems, we observe folding energy landscapes with multiple energy minima and several isoenergetic states. By comparing coiled coils from single domains and those extracted from larger proteins, we find that standalone coiled coils have deeper energy wells at the experimentally determined conformation. By folding the competing homodimeric states in addition to the heterodimers, we observe that the structural specificity towards the heteromeric state is often small. Taken together, our results demonstrate that de novo folding simulations can be a powerful tool to characterize structural specificity of coiled coils when coupled to assessment of energy landscapes.

  • 2.
    Bjelic, Sinisa
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Protein carriers for passage of the Blood-Brain Barrier2015In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 24, p. 177-177Article in journal (Other academic)
  • 3.
    Bjelic, Sinisa
    et al.
    Uppsala University.
    Aqvist, Johan
    Computational prediction of structure, substrate binding mode, mechanism, and rate for a malaria protease with a novel type of active site.2004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 46, p. 14521-14528Article in journal (Refereed)
    Abstract [en]

    The histo-aspartic protease (HAP) from the malaria parasite P. falciparum is one of several new promising targets for drug intervention. The enzyme possesses a novel type of active site, but its 3D structure and mechanism of action are still unknown. Here we use a combination of homology modeling, automated docking searches, and molecular dynamics/reaction free energy profile simulations to predict the enzyme structure, conformation of bound substrate, catalytic mechanism, and rate of the peptide cleavage reaction. We find that the computational tools are sufficiently reliable both for identifying substrate binding modes and for distinguishing between different possible reaction mechanisms. It is found that the favored pathway only involves direct participation by the catalytic aspartate, with the neighboring histidine providing critical stabilization (by a factor of approximately 10000) along the reaction. The calculated catalytic rate constant of about 0.1 s(-1) for a hexapeptide substrate derived from the alpha chain of human hemoglobin is in excellent agreement with experimental kinetic data for a similar peptide fragment.

  • 4.
    Bjelic, Sinisa
    et al.
    Uppsala University.
    Brandsdal, Bjørn O
    University of Tromsø, Norway.
    Åqvist, Johan
    Uppsala University.
    Cold adaptation of enzyme reaction rates2008In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 38, p. 10049-10057Article in journal (Refereed)
    Abstract [en]

    A major issue for organisms living at extreme temperatures is to preserve both stability and activity of their enzymes. Cold-adapted enzymes generally have a reduced thermal stability, to counteract freezing, and show a lower enthalpy and a more negative entropy of activation compared to mesophilic and thermophilic homologues. Such a balance of thermodynamic activation parameters can make the reaction rate decrease more linearly, rather than exponentially, as the temperature is lowered, but the structural basis for rate optimization toward low working temperatures remains unclear. In order to computationally address this problem, it is clear that reaction simulations rather than standard molecular dynamics calculations are needed. We have thus carried out extensive computer simulations of the keto-enol(ate) isomerization steps in differently adapted citrate synthases to explore the structure-function relationships behind catalytic rate adaptation to different temperatures. The calculations reproduce the absolute rates of the psychrophilic and mesophilic enzymes at 300 K, as well as the lower enthalpy and more negative entropy of activation of the cold-adapted enzyme, where the latter simulation result is obtained from high-precision Arrhenius plots. The overall catalytic effect originates from electrostatic stabilization of the transition state and enolate and the reduction of reorganization free energy. The simulations, however, show psychrophilic, mesophilic, and hyperthermophilic citrate synthases to have increasingly stronger electrostatic stabilization of the transition state, while the energetic penalty in terms of internal protein interactions follows the reverse order with the cold-adapted enzyme having the most favorable energy term. The lower activation enthalpy and more negative activation entropy observed for cold-adapted enzymes are found to be associated with a decreased protein stiffness. The origin of this effect is, however, not localized to the active site but to other regions of the protein structure.

  • 5.
    Bjelic, Sinisa
    et al.
    University of Washington, USA.
    Kipnis, Yakov
    University of Washington, USA.
    Wang, Ling
    University of Washington, USA.
    Pianowski, Zbigniew
    ETH Zurich, Switzerland.
    Vorobiev, Sergey
    Columbia University, USA.
    Su, Min
    Columbia University, USA.
    Seetharaman, Jayaraman
    Columbia University, USA.
    Xiao, Rong
    The State University of New Jersey, USA.
    Kornhaber, Gregory
    The State University of New Jersey, USA ; University of Medicine and Dentistry of New Jersey, USA ; Northeast Structural Genomics Consortium, USA.
    Hunt, John F
    Columbia University, USA.
    Tong, Liang
    Columbia University, USA.
    Hilvert, Donald
    ETH Zurich, Switzerland.
    Baker, David
    University of Washington, USA.
    Exploration of Alternate Catalytic Mechanisms and Optimization Strategies for Retroaldolase Design2014In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 426, no 1, p. 256-271Article in journal (Refereed)
    Abstract [en]

    Designed retroaldolases have utilized a nucleophilic lysine to promote carbon-carbon bond cleavage of β-hydroxy-ketones via a covalent Schiff base intermediate. Previous computational designs have incorporated a water molecule to facilitate formation and breakdown of the carbinolamine intermediate to give the Schiff base and to function as a general acid/base. Here we investigate an alternative active-site design in which the catalytic water molecule was replaced by the side chain of a glutamic acid. Five out of seven designs expressed solubly and exhibited catalytic efficiencies similar to previously designed retroaldolases for the conversion of 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone to 6-methoxy-2-naphthaldehyde and acetone. After one round of site-directed saturation mutagenesis, improved variants of the two best designs, RA114 and RA117, exhibited among the highest kcat (>10(-3)s(-1)) and kcat/KM (11-25M(-1)s(-1)) values observed for retroaldolase designs prior to comprehensive directed evolution. In both cases, the >10(5)-fold rate accelerations that were achieved are within 1-3 orders of magnitude of the rate enhancements reported for the best catalysts for related reactions, including catalytic antibodies (kcat/kuncat=10(6) to 10(8)) and an extensively evolved computational design (kcat/kuncat>10(7)). The catalytic sites, revealed by X-ray structures of optimized versions of the two active designs, are in close agreement with the design models except for the catalytic lysine in RA114. We further improved the variants by computational remodeling of the loops and yeast display selection for reactivity of the catalytic lysine with a diketone probe, obtaining an additional order of magnitude enhancement in activity with both approaches.

  • 6.
    Bjelic, Sinisa
    et al.
    Uppsala University.
    Nervall, M
    Uppsala University.
    Gutiérrez-de-Terán, H
    Uppsala University.
    Ersmark, K
    Uppsala University.
    Hallberg, A
    Uppsala University.
    Aqvist, J
    Uppsala University.
    Computational inhibitor design against malaria plasmepsins2007In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 64, no 17, p. 2285-2305Article in journal (Refereed)
    Abstract [en]

    Plasmepsins are aspartic proteases involved in the degradation of the host cell hemoglobin that is used as a food source by the malaria parasite. Plasmepsins are highly promising as drug targets, especially when combined with the inhibition of falcipains that are also involved in hemoglobin catabolism. In this review, we discuss the mechanism of plasmepsins I-IV in view of the interest in transition state mimetics as potential compounds for lead development. Inhibitor development against plasmepsin II as well as relevant crystal structures are summarized in order to give an overview of the field. Application of computational techniques, especially binding affinity prediction by the linear interaction energy method, in the development of malarial plasmepsin inhibitors has been highly successful and is discussed in detail. Homology modeling and molecular docking have been useful in the current inhibitor design project, and the combination of such methods with binding free energy calculations is analyzed.

  • 7.
    Bjelic, Sinisa
    et al.
    University of Washington, USA.
    Nivón, Lucas G
    University of Washington, USA.
    Çelebi-Ölçüm, Nihan
    University of California, USA ; Yeditepe University, Turkey.
    Kiss, Gert
    University of California, USA.
    Rosewall, Carolyn F
    Lovick, Helena M
    Ingalls, Erica L
    Gallaher, Jasmine Lynn
    University of Washington, USA.
    Seetharaman, Jayaraman
    Columbia University, USA.
    Lew, Scott
    Columbia University, USA.
    Montelione, Gaetano Thomas
    Columbia University, USA.
    Hunt, John Francis
    Columbia University, USA.
    Michael, Forrest Edwin
    Houk, K N
    University of California, USA.
    Baker, David
    University of Washington, USA.
    Computational design of enone-binding proteins with catalytic activity for the Morita-Baylis-Hillman reaction2013In: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 8, no 4, p. 749-757Article in journal (Refereed)
    Abstract [en]

    The Morita-Baylis-Hillman reaction forms a carbon-carbon bond between the α-carbon of a conjugated carbonyl compound and a carbon electrophile. The reaction mechanism involves Michael addition of a nucleophile catalyst at the carbonyl β-carbon, followed by bond formation with the electrophile and catalyst disassociation to release the product. We used Rosetta to design 48 proteins containing active sites predicted to carry out this mechanism, of which two show catalytic activity by mass spectrometry (MS). Substrate labeling measured by MS and site-directed mutagenesis experiments show that the designed active-site residues are responsible for activity, although rate acceleration over background is modest. To characterize the designed proteins, we developed a fluorescence-based screen for intermediate formation in cell lysates, carried out microsecond molecular dynamics simulations, and solved X-ray crystal structures. These data indicate a partially formed active site and suggest several clear avenues for designing more active catalysts.

  • 8.
    Bjelic, Sinisa
    et al.
    Uppsala University.
    Åqvist, Johan
    Uppsala University.
    Catalysis and linear free energy relationships in aspartic proteases2006In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 45, no 25, p. 7709-7723Article in journal (Refereed)
    Abstract [en]

    Aspartic proteases are receiving considerable attention as potential drug targets in several serious diseases, such as AIDS, malaria, and Alzheimer's disease. These enzymes cleave polypeptide chains, often between specific amino acid residues, but despite the common reaction mechanism, they exhibit large structural differences. Here, the catalytic mechanism of aspartic proteases plasmepsin II, cathepsin D, and HIV-1 protease is examined by computer simulations utilizing the empirical valence bond approach in combination with molecular dynamics and free energy perturbation calculations. Free energy profiles are established for four different substrates, each six amino acids long and containing hydrophobic side chains in the P1 and P1' positions. Our simulations reproduce the catalytic effect of these enzymes, which accelerate the reaction rate by a factor of approximately 10(10) compared to that of the corresponding uncatalyzed reaction in water. The calculations elucidate the origin of the catalytic effect and allow a rationalization of the fact that, despite large structural differences between plasmepsin II/cathepsin D and HIV-1 protease, the magnitude of their rate enhancement is very similar. Amino acid residues surrounding the active site together with structurally conserved water molecules are found to play an important role in catalysis, mainly through dipolar (electrostatic) stabilization. A linear free energy relationship for the reactions in the different enzymes is established that also demonstrates the reduced reorganization energy in the enzymes compared to that in the uncatalyzed water reaction.

  • 9.
    Ersmark, Karolina
    et al.
    Uppsala University.
    Feierberg, Isabella
    Uppsala University.
    Bjelic, Sinisa
    Uppsala University.
    Hamelink, Elizabeth
    National Institute for Medical Research, UK.
    Hackett, Fiona
    Medivir AB.
    Blackman, Michael J
    Medivir AB.
    Hultén, Johan
    Uppsala University.
    Samuelsson, Bertil
    Medivir AB.
    Aqvist, Johan
    Uppsala University.
    Hallberg, Anders
    Uppsala University.
    Potent inhibitors of the Plasmodium falciparum enzymes plasmepsin I and II devoid of cathepsin D inhibitory activity2004In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 47, no 1, p. 110-122Article in journal (Refereed)
    Abstract [en]

    The hemoglobin-degrading aspartic proteases plasmepsin I (Plm I) and plasmepsin II (Plm II) of the malaria parasite Plasmodium falciparum have lately emerged as putative drug targets. A series of C(2)-symmetric compounds encompassing the 1,2-dihydroxyethylene scaffold and a variety of elongated P1/P1' side chains were synthesized via microwave-assisted palladium-catalyzed coupling reactions. Binding affinity calculations with the linear interaction energy method and molecular dynamics simulations reproduced the experimental binding data obtained in a Plm II assay with very good accuracy. Bioactive conformations of the elongated P1/P1' chains were predicted and agreed essentially with a recent X-ray structure. The compounds exhibited picomolar to nanomolar inhibition constants for the plasmepsins and no measurable affinity to the human enzyme cathepsin D. Some of the compounds also demonstrated significant inhibition of parasite growth in cell culture. To the best of our knowledge, these plasmepsin inhibitors represent the most selective reported to date and constitute promising lead compounds for further optimization.

  • 10.
    Ersmark, Karolina
    et al.
    Uppsala University.
    Feierberg, Isabella
    Uppsala University.
    Bjelic, Sinisa
    Uppsala University.
    Hultén, Johan
    Uppsala University.
    Samuelsson, Bertil
    Medivir AB.
    Åqvist, Johan
    Uppsala University.
    Hallberg, Anders
    Uppsala University.
    C2-symmetric inhibitors of Plasmodium falciparum plasmepsin II: synthesis and theoretical predictions2003In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 11, no 17, p. 3723-3733Article in journal (Refereed)
    Abstract [en]

    A series of C(2)-symmetric compounds with a mannitol-based scaffold has been investigated, both theoretically and experimentally, as Plm II inhibitors. Four different stereoisomers with either benzyloxy or allyloxy P1/P1' side chains were studied. Computational ranking of the binding affinities of the eight compounds was carried out using the linear interaction energy (LIE) method relying on a complex previously determined by crystallography. Within both series of isomers the theoretical binding energies were in agreement with the enzymatic measurements, illustrating the power of the LIE method for the prediction of ligand affinities prior to synthesis. The structural models of the enzyme-inhibitor complexes obtained from the MD simulations provided a basis for interpretation of further structure-activity relationships. Hence, the affinity of a structurally similar ligand, but with a different P2/P2' substituent was examined using the same procedure. The predicted improvement in binding constant agreed well with experimental results.

  • 11.
    Georgoulia, Panagiota S.
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Todde, Guido
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Bjelic, Sinisa
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Friedman, Ran
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    The catalytic activity of Abl1 single and compound mutations: Implications for the mechanism of drug resistance mutations in chronic myeloid leukaemia2019In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1863, no 4, p. 732-741Article in journal (Refereed)
    Abstract [en]

    Background

    Abl1 is a protein tyrosine kinase whose aberrant activation due to mutations is the culprit of several cancers, most notably chronic myeloid leukaemia. Several Abl1 inhibitors are used as anti-cancer drugs. Unfortunately, drug resistance limits their effectiveness. The main cause for drug resistance is mutations in the kinase domain (KD) of Abl1 that evolve in patients. The T315I mutation confers resistance against all clinically-available inhibitors except ponatinib. Resistance to ponatinib can develop by compound (double) mutations.

    Methods

    Kinetic measurements of the KD of Abl1 and its mutants were carried out to examine their catalytic activity. Specifically, mutants that lead to drug resistance against ponatinib were considered. Molecular dynamics simulations and multiple sequence analysis were used for explanation of the experimental findings.

    Results

    The catalytic efficiency of the T315I pan-resistance mutant is more than two times lower than that of the native KD. All ponatinib resistant mutations restore the catalytic efficiency of the enzyme. Two of them (G250E/T315I and Y253H/E255V) have a catalytic efficiency that is more than five times that of the native KD.

    Conclusions

    The measurements and analysis suggest that resistance is at least partially due to the development of a highly efficient kinase through subsequent mutations. The simulations highlight modifications in two structurally important regions of Abl1, the activation and phosphate binding loops, upon mutations.

    General significance

    Experimental and computational methods were used together to explain how mutations in the kinase domain of Abl1 lead to resistance against the most advanced drug currently in use to treat chronic myeloid leukaemia.

  • 12.
    Liu, Yu
    et al.
    The Scripps Research Institute, USA.
    Tan, Yun Lei
    The Scripps Research Institute, USA.
    Zhang, Xin
    The Scripps Research Institute, USA.
    Bhabha, Gira
    University of California, USA.
    Ekiert, Damian C
    University of California, USA.
    Genereux, Joseph C
    The Scripps Research Institute, USA.
    Cho, Younhee
    The Scripps Research Institute, USA.
    Kipnis, Yakov
    University of Washington, USA.
    Bjelic, Sinisa
    University of Washington, USA.
    Baker, David
    University of Washington, USA.
    Kelly, Jeffery W
    The Scripps Research Institute, USA.
    Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts.2014In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, no 12, p. 4449-4454Article in journal (Refereed)
    Abstract [en]

    Although much is known about protein folding in buffers, it remains unclear how the cellular protein homeostasis network functions as a system to partition client proteins between folded and functional, soluble and misfolded, and aggregated conformations. Herein, we develop small molecule folding probes that specifically react with the folded and functional fraction of the protein of interest, enabling fluorescence-based quantification of this fraction in cell lysate at a time point of interest. Importantly, these probes minimally perturb a protein's folding equilibria within cells during and after cell lysis, because sufficient cellular chaperone/chaperonin holdase activity is created by rapid ATP depletion during cell lysis. The folding probe strategy and the faithful quantification of a particular protein's functional fraction are exemplified with retroaldolase, a de novo designed enzyme, and transthyretin, a nonenzyme protein. Our findings challenge the often invoked assumption that the soluble fraction of a client protein is fully folded in the cell. Moreover, our results reveal that the partitioning of destabilized retroaldolase and transthyretin mutants between the aforementioned conformational states is strongly influenced by cytosolic proteostasis network perturbations. Overall, our results suggest that applying a chemical folding probe strategy to other client proteins offers opportunities to reveal how the proteostasis network functions as a system to regulate the folding and function of individual client proteins in vivo.

  • 13.
    Liu, Yu
    et al.
    The Scripps Research Institute, USA.
    Zhang, Xin
    The Scripps Research Institute, USA.
    Tan, Yun Lei
    The Scripps Research Institute, USA.
    Bhabha, Gira
    University of California, USA.
    Ekiert, Damian C
    University of California, USA.
    Kipnis, Yakov
    University of Washington, USA.
    Bjelic, Sinisa
    University of Washington, USA.
    Baker, David
    University of Washington, USA.
    Kelly, Jeffery W
    The Scripps Research Institute, USA.
    De Novo-Designed Enzymes as Small-Molecule-Regulated Fluorescence Imaging Tags and Fluorescent Reporters2014In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 136, no 38, p. 13102-13105Article in journal (Refereed)
    Abstract [en]

    Enzyme-based tags attached to a protein-of-interest (POI) that react with a small molecule, rendering the conjugate fluorescent, are very useful for studying the POI in living cells. These tags are typically based on endogenous enzymes, so protein engineering is required to ensure that the small-molecule probe does not react with the endogenous enzyme in the cell of interest. Here we demonstrate that de novo-designed enzymes can be used as tags to attach to POIs. The inherent bioorthogonality of the de novo-designed enzyme-small-molecule probe reaction circumvents the need for protein engineering, since these enzyme activities are not present in living organisms. Herein, we transform a family of de novo-designed retroaldolases into variable-molecular-weight tags exhibiting fluorescence imaging, reporter, and electrophoresis applications that are regulated by tailored, reactive small-molecule fluorophores.

  • 14.
    Nivón, Lucas G
    et al.
    University of Washington, USA.
    Bjelic, Sinisa
    University of Washington, USA.
    King, Chris
    University of Washington, USA.
    Baker, David
    University of Washington, USA.
    Automating human intuition for protein design2014In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 82, no 5, p. 858-866Article in journal (Refereed)
    Abstract [en]

    In the design of new enzymes and binding proteins, human intuition is often used to modify computationally designed amino acid sequences prior to experimental characterization. The manual sequence changes involve both reversions of amino acid mutations back to the identity present in the parent scaffold and the introduction of residues making additional interactions with the binding partner or backing up first shell interactions. Automation of this manual sequence refinement process would allow more systematic evaluation and considerably reduce the amount of human designer effort involved. Here we introduce a benchmark for evaluating the ability of automated methods to recapitulate the sequence changes made to computer-generated models by human designers, and use it to assess alternative computational methods. We find the best performance for a greedy one-position-at-a-time optimization protocol that utilizes metrics (such as shape complementarity) and local refinement methods too computationally expensive for global Monte Carlo (MC) sequence optimization. This protocol should be broadly useful for improving the stability and function of designed binding proteins.

  • 15.
    Richter, Florian
    et al.
    University of Washington, USA.
    Leaver-Fay, Andrew
    University of North Carolina, USA.
    Khare, Sagar D
    University of Washington, USA.
    Bjelic, Sinisa
    University of Washington, USA.
    Baker, David
    University of Washington, USA.
    De novo enzyme design using Rosetta32011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 5Article in journal (Refereed)
    Abstract [en]

    The Rosetta de novo enzyme design protocol has been used to design enzyme catalysts for a variety of chemical reactions, and in principle can be applied to any arbitrary chemical reaction of interest. The process has four stages: 1) choice of a catalytic mechanism and corresponding minimal model active site, 2) identification of sites in a set of scaffold proteins where this minimal active site can be realized, 3) optimization of the identities of the surrounding residues for stabilizing interactions with the transition state and primary catalytic residues, and 4) evaluation and ranking the resulting designed sequences. Stages two through four of this process can be carried out with the Rosetta package, while stage one needs to be done externally. Here, we demonstrate how to carry out the Rosetta enzyme design protocol from start to end in detail using for illustration the triosephosphate isomerase reaction.

  • 16.
    Wijma, Hein J
    et al.
    University of Groningen, The Netherlands.
    Floor, Robert J
    University of Groningen, The Netherlands.
    Bjelic, Sinisa
    University of Washington, USA.
    Marrink, Siewert J
    University of Groningen, The Netherlands.
    Baker, David
    University of Washington, USA.
    Janssen, Dick B
    University of Groningen, The Netherlands.
    Enantioselective enzymes by computational design and in silico screening2015In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 54, no 12, p. 3726-3730Article in journal (Refereed)
    Abstract [en]

    Computational enzyme design holds great promise for providing new biocatalysts for synthetic chemistry. A strategy to design small mutant libraries of complementary enantioselective epoxide hydrolase variants for the production of highly enantioenriched (S,S)-diols and (R,R)-diols is developed. Key features of this strategy (CASCO, catalytic selectivity by computational design) are the design of mutations that favor binding of the substrate in a predefined orientation, the introduction of steric hindrance to prevent unwanted substrate binding modes, and ranking of designs by high-throughput molecular dynamics simulations. Using this strategy we obtained highly stereoselective mutants of limonene epoxide hydrolase after experimental screening of only 37 variants. The results indicate that computational methods can replace a substantial amount of laboratory work when developing enantioselective enzymes.

  • 17.
    Åqvist, Johan
    et al.
    Uppsala University.
    Wennerström, Petra
    Uppsala University.
    Nervall, Martin
    Uppsala University.
    Bjelic, Sinisa
    Uppsala University.
    Brandsdal, Bjørn O.
    Uppsala University.
    Molecular dynamics simulations of water and biomolecules wit a Monte Carlo constant pressure algorithm2004In: Chemical Physics Letters, ISSN 0009-2614, E-ISSN 1873-4448, Vol. 384, no 4-6, p. 288-294Article in journal (Refereed)
    Abstract [en]

    A mixed molecular dynamics/Monte Carlo (MD/MC) algorithm for constant pressure simulations of arbitrary molecular systems is examined. Calculations are reported at ambient and high pressures both for liquid water systems and for a chemical reaction step in a solvated enzyme utilizing empirical valence bond potentials. The present method reproduces earlier reported results well and is computationally efficient since it does not require the virial to be evaluated at each MD step. It is also found that the effects of introducing MC volume steps on the dynamics of the system are negligible provided that the volume step sizes and updating frequencies are appropriately chosen.

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