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Caspase-7 Loop Conformations as a Means of Allosteric ControlWitkowski, Witold Andrej 13 May 2011 (has links)
The caspase family of proteins is critical to biological understanding, because they serve as the final arbiters of life and death, being the initiators and executioners of cell death. Specifically, caspase-7 plays a key role in apoptosis, however its full complement of targets within the cell has not yet been elucidated, nor has its function been targeted by drug design efforts. These factors stem from the lack of fundamental understanding of the structural dynamics of the protein, including the mobile loops that constitute the active site binding groove of caspase-7, and their ability to modulate the function of the protein. In this work, we describe the importance of the entire loop bundle for catalysis, demonstrate a novel approach for allosteric control using loop movement, develop computational methods to engineer a new binding site for an allosteric effector and discover a hereunto unseen native disulfide within caspase-7 that may contribute to specificity and catalysis. The information obtained within this study is applicable for not only the study of caspase-7, but also the greater field of apoptosis research.
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Computational Modeling of Drug Resistance: Structural and Evolutionary ModelsSafi, Maryah 25 August 2014 (has links)
Active site mutations that disrupt drug binding are an important mechanism of drug resistance. Such resistance causing mutations impair drug binding, thus reducing drug efficacy. Knowledge of potential resistance mutations, before they are clinically observed, would be useful in a number of ways. During the lead prioritization phase of drug development, this knowledge may direct the research team away from candidate drugs that are most likely to experience resistance. In the clinical setting, knowledge of potential resistance mutations could allow the development of treatment regimens, with drug cocktails likely to maximize efficacy.
In this thesis I present a structure-based approach to predict resistance and its evolution. This method utilizes a two-pass search, which is based on a novel protein design algorithm, to identify mutations that impair drug binding while maintaining affinity for the native substrate. The approach is general and can be applied to any drug-target system where a structure of the target protein, its native substrate and the drug is available. Furthermore, it requires no training data for predictions and instead predicts resistance using structural principles.
Finally, I use approximate force-field calculations from MMPBSA and simple assumptions about the relationship between binding energy and fitness to build fitness landscapes for a target protein under selective pressure from either a single drug or a drug cocktail. I use a Markov-chain based model to simulate evolution on this fitness landscape and to predict the likely evolutionary trajectories for resistance starting from a wild-type. The structure-based method was used to probe resistance in four drug-target systems: isoniazid-enoyl-ACP reductase (tuberculosis), ritonavir-HIV protease (HIV), methotrexate-dihydrofolate reductase (breast cancer and leukemia), and gleevec-ABL kinase (leukemia). This method was validated using clinically known resistance mutations for all four test systems. In all cases, it correctly predicts the majority of known resistance mutations. Furthermore, exploiting the relationship between binding energy, drug resistance and fitness of a mutant, evolution was simulated on the HIV-protease fitness landscape. This hybrid evolutionary model further improves the resistance prediction. Finally, good agreement between these evolutionary simulations and observed evolution of drug resistance in patients was found.
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Exploring amino-acid radicals and quinone redox chemistry in model proteinsWesterlund, Kristina January 2008 (has links)
<p>Amino-acid radical enzymes have been studied extensively for 30 years but the experimental barriers to determine the thermodynamic properties of their key radical cofactors are so challenging that only a handful of reports exist in the literature. This is a major drawback when trying to understand the long-range radical transfer and/or catalytic mechanisms of this important family of enzymes. Here this issue is addressed by developing a library of well-structured model proteins specifically designed to study tyrosine and tryptophan radicals. The library is based on a 67-residue three-helix bundle (α<sub>3</sub>W) and a 117-residue four-helix bundle (α<sub>4</sub>W). α<sub>3</sub>W and α<sub>4</sub>W are single-chain and uniquely structured proteins. They are redox inert except for a single radical site (position 32 in α<sub>3</sub>W and 106 in α<sub>4</sub>W). Papers I and II describe the design process and the protein characteristics of α<sub>4</sub>W as well as a voltammetry study of its unique tryptophan. Paper III and V describe two projects based on α<sub>3</sub>C, which is a Trp-32 to Cys-32 variant of α<sub>3</sub>W. In Paper III we use α<sub>3</sub>C to investigate what effect the degree of solvent exposure of the phenolic OH group has on the redox characteristics of tyrosine analogs. We show that the potential of the PhO•/PhOH redox pair is dominated by interactions with the OH group and that the environment around the hydrophobic part of the phenol has no significant impact. In addition, we observe that interactions between the phenolic OH group and the protein matrix can raise the phenol potential by 0.11-0.12 V relative to solution values. The α<sub>3</sub>C system is extended in Paper V to study quinone redox chemistry. Papers III and V contain protocols to generate the cofactor-containing α<sub>3</sub>C systems and descriptions of their protein properties. Paper IV describes efforts to redesign α<sub>3</sub>Y (a Trp-32 to Tyr-32 variant of α<sub>3</sub>W) to contain an interacting Tyr-32/histidine pair. The aim is to engineer and study the effects of a redox-induced proton acceptor in the Tyr-32 site.</p>
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Exploring amino-acid radicals and quinone redox chemistry in model proteinsWesterlund, Kristina January 2008 (has links)
Amino-acid radical enzymes have been studied extensively for 30 years but the experimental barriers to determine the thermodynamic properties of their key radical cofactors are so challenging that only a handful of reports exist in the literature. This is a major drawback when trying to understand the long-range radical transfer and/or catalytic mechanisms of this important family of enzymes. Here this issue is addressed by developing a library of well-structured model proteins specifically designed to study tyrosine and tryptophan radicals. The library is based on a 67-residue three-helix bundle (α3W) and a 117-residue four-helix bundle (α4W). α3W and α4W are single-chain and uniquely structured proteins. They are redox inert except for a single radical site (position 32 in α3W and 106 in α4W). Papers I and II describe the design process and the protein characteristics of α4W as well as a voltammetry study of its unique tryptophan. Paper III and V describe two projects based on α3C, which is a Trp-32 to Cys-32 variant of α3W. In Paper III we use α3C to investigate what effect the degree of solvent exposure of the phenolic OH group has on the redox characteristics of tyrosine analogs. We show that the potential of the PhO•/PhOH redox pair is dominated by interactions with the OH group and that the environment around the hydrophobic part of the phenol has no significant impact. In addition, we observe that interactions between the phenolic OH group and the protein matrix can raise the phenol potential by 0.11-0.12 V relative to solution values. The α3C system is extended in Paper V to study quinone redox chemistry. Papers III and V contain protocols to generate the cofactor-containing α3C systems and descriptions of their protein properties. Paper IV describes efforts to redesign α3Y (a Trp-32 to Tyr-32 variant of α3W) to contain an interacting Tyr-32/histidine pair. The aim is to engineer and study the effects of a redox-induced proton acceptor in the Tyr-32 site.
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Redesign of Alpha Class Glutathione Transferases to Study Their Catalytic PropertiesNilsson, Lisa O January 2001 (has links)
<p>A number of active site mutants of human Alpha class glutathione transferase A1-1 (hGST A1-1) were made and characterized to determine the structural determinants for alkenal activity. The choice of mutations was based on primary structure alignments of hGST A1-1 and the Alpha class enzyme with the highest alkenal activity, hGST A4-4, from three different species and crystal structure comparisons between the human enzymes. The result was an enzyme with a 3000-fold change in substrate specificity for nonenal over 1-chloro-2,4-dinitrobenzene (CDNB).</p><p>The C-terminus of the Alpha class enzymes is an α-helix that folds over the active site upon substrate binding. The rate-determining step is product release, which is influenced by the movements of the C-terminus, thereby opening the active site. Phenylalanine 220, near the end of the C-terminus, forms an aromatic cluster with tyrosine 9 and phenylalanine 10, positioning the β-carbon of the cysteinyl moiety of glutathione. The effects of phenylalanine 220 mutations on the mobility of the C-terminus were studied by the viscosity dependence of k<sub>cat</sub> and k<sub>cat</sub>/K<sub>m</sub> with glutathione and CDNB as the varied substrates. </p><p>The compatibility of slightly different subunit interfaces within the Alpha class has been studied by heterodimerization between monomers from hGST A1-1 and hGST A4-4. The heterodimer was temperature sensitive, and rehybridized into homodimers at 40 ˚C. The heterodimers did not show strictly additive activities with alkenals and CDNB. This result combined with further studies indicates that there are factors at the subunit interface influencing the catalytic properties of hGST A1-1.</p>
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Redesign of Alpha Class Glutathione Transferases to Study Their Catalytic PropertiesNilsson, Lisa O January 2001 (has links)
A number of active site mutants of human Alpha class glutathione transferase A1-1 (hGST A1-1) were made and characterized to determine the structural determinants for alkenal activity. The choice of mutations was based on primary structure alignments of hGST A1-1 and the Alpha class enzyme with the highest alkenal activity, hGST A4-4, from three different species and crystal structure comparisons between the human enzymes. The result was an enzyme with a 3000-fold change in substrate specificity for nonenal over 1-chloro-2,4-dinitrobenzene (CDNB). The C-terminus of the Alpha class enzymes is an α-helix that folds over the active site upon substrate binding. The rate-determining step is product release, which is influenced by the movements of the C-terminus, thereby opening the active site. Phenylalanine 220, near the end of the C-terminus, forms an aromatic cluster with tyrosine 9 and phenylalanine 10, positioning the β-carbon of the cysteinyl moiety of glutathione. The effects of phenylalanine 220 mutations on the mobility of the C-terminus were studied by the viscosity dependence of kcat and kcat/Km with glutathione and CDNB as the varied substrates. The compatibility of slightly different subunit interfaces within the Alpha class has been studied by heterodimerization between monomers from hGST A1-1 and hGST A4-4. The heterodimer was temperature sensitive, and rehybridized into homodimers at 40 ˚C. The heterodimers did not show strictly additive activities with alkenals and CDNB. This result combined with further studies indicates that there are factors at the subunit interface influencing the catalytic properties of hGST A1-1.
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Modulating Enzyme Functions by Semi-Rational Redesign and Chemical Modifications : A Study on Mu-class Glutathione TransferasesNorrgård, Malena A January 2011 (has links)
Today, enzymes are extensively used for many industrial applications, this includes bulk and fine-chemical synthesis, pharmaceuticals and consumer products. Though Nature has perfected enzymes for many millions of years, they seldom reach industrial performance targets. Natural enzymes could benefit from protein redesign experiments to gain novel functions or optimize existing functions. Glutathione transferases (GSTs) are detoxification enzymes, they also display disparate functions. Two Mu-class GSTs, M1-1 and M2-2, are closely related but display dissimilar substrate selectivity profiles. Saturation mutagenesis of a previously recognized hypervariable amino acid in GST M2-2, generated twenty enzyme variants with altered substrate selectivity profiles, as well as modified thermostabilities and expressivities. This indicates an evolutionary significance; GST Mu-class enzymes could easily alter functions in a duplicate gene by a single-point mutation. To further identify residues responsible for substrate selectivity in the GST M2-2 active site, three residues were chosen for iterative saturation mutagenesis. Mutations in position10, identified as highly conserved, rendered enzyme variants with substrate selectivity profiles resembling that of specialist enzymes. Ile10 could be conserved to sustain the broad substrate acceptance displayed by GST Mu-class enzymes. Enzymes are constructed from primarily twenty amino acids, it is a reasonable assumption that expansion of the amino acid repertoire could result in functional properties that cannot be accomplished with the natural set of building blocks. A combination approach of site-directed mutagenesis and chemical modifications in GST M2-2 and GST M1-1 resulted in novel enzyme variants that displayed altered substrate selectivity patterns as well as improved enantioselectivities. The results presented in this thesis demonstrate the use of different protein redesign techniques to modulate various functions in Mu-class GSTs. These techniques could be useful in search of optimized enzyme variants for industrial targets. / biokemi och organisk kemi
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Mutational Analysis and Redesign of Alpha-class Glutathione Transferases for Enhanced Azathioprine ActivityModén, Olof January 2013 (has links)
Glutathione transferase (GST) A2-2 is the human enzyme most efficient in catalyzing azathioprine activation. Structure-function relationships were sought explaining the higher catalytic efficiency compared to other alpha class GSTs. By screening a DNA shuffling library, five recombined segments were identified that were conserved among the most active mutants. Mutational analysis confirmed the importance of these short segments as their insertion into low-active GSTs introduced higher azathioprine activity. Besides, H-site mutagenesis led to decreased azathioprine activity when the targeted positions belonged to these conserved segments and mainly enhanced activity when other positions were targeted. Hydrophobic residues were preferred in positions 208 and 213. The prodrug azathioprine is today primarily used for maintaining remission in inflammatory bowel disease. Therapy leads to adverse effects for 30 % of the patients and genotyping of the metabolic genes involved can explain some of these incidences. Five genotypes of human A2-2 were characterized and variant A2*E had 3–4-fold higher catalytic efficiency with azathioprine, due to a proline mutated close to the H-site. Faster activation might lead to different metabolite distributions and possibly more adverse effects. Genotyping of GSTs is recommended for further studies. Molecular docking of azathioprine into a modeled structure of A2*E suggested three positions for mutagenesis. The most active mutants had small or polar residues in the mutated positions. Mutant L107G/L108D/F222H displayed a 70-fold improved catalytic efficiency with azathioprine. Determination of its structure by X-ray crystallography showed a widened H-site, suggesting that the transition state could be accommodated in a mode better suited for catalysis. The mutational analysis increased our understanding of the azathioprine activation in alpha class GSTs and highlighted A2*E as one factor possibly behind the adverse drug-effects. A successfully redesigned GST, with 200-fold enhanced catalytic efficiency towards azathioprine compared to the starting point A2*C, might find use in targeted enzyme-prodrug therapies.
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