Iterative approach to computational enzyme design

A general approach for the computational design of enzymes to catalyze arbitrary reactions is a goal at the forefront of the field of protein design. Recently, computationally designed enzymes have been produced for three chemical reactions through the synthesis and screening of a large number of variants. Here, we present an iterative approach that has led to the development of the most catalytically efficient computationally designed enzyme for the Kemp elimination to date. Previously established computational techniques were used to generate an initial design, HG-1, which was catalytically inactive. Analysis of HG-1 with molecular dynamics simulations (MD) and X-ray crystallography indicated that the inactivity might be due to bound waters and high flexibility of residues within the active site. This analysis guided changes to our design procedure, moved the design deeper into the interior of the protein, and resulted in an active Kemp eliminase, HG-2. The cocrystal structure of this enzyme with a transition state analog (TSA) revealed that the TSA was bound in the active site, interacted with the intended catalytic base in a catalytically relevant manner, but was flipped relative to the design model. MD analysis of HG-2 led to an additional point mutation, HG-3, that produced a further threefold improvement in activity. This iterative approach to computational enzyme design, including detailed MD and structural analysis of both active and inactive designs, promises a more complete understanding of the underlying principles of enzymatic catalysis and furthers progress toward reliably producing active enzymes

Cloning, sequencing and expression of glucose dehydrogenase from Thermoplasma acidophilum

SIGLEAvailable from British Library Document Supply Centre- DSC:DX95643 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Synthetic and Computational Studies on Polycyclic Aromatic Hydrocarbon Derivatives, Nucleoside Analogs and Peptides

In recent years, the understanding of the structure and functions of biological macromolecules has advanced rapidly, the result of which is a better mechanistic understanding of many biological processes. As an outgrowth of this understanding, organic molecules that react with biological macromolecules (DNA) or adopt conformations responsible for specific functions in biological macromolecules (peptides and proteins) have been synthesized and computational modeling studies performed. Polycyclic aromatic hydrocarbons (PAHs) and β-peptides are among synthetic organic compounds known to interact with natural biological macromolecules. This interaction may affect the specific biological functions of the biomacromolecules. A variety of synthetic methodologies have been employed in the synthesis of benzo[c]phenanthrene derivatives, single electron oxidation nucleoside adducts and deoxynucleoside derivatives (Part 1). In Part 2 heterogeneous backbone oligomers containing the β-amino acid, trans-2-aminocyclohexanecarboxylic acid (ACHC), and α-amino acids Ala, Phe, Val, Lys, and Tyr in an alternating sequence have been synthesized. Computational modeling studies have been applied in studying the diastereoselectivity of reaction intermediates in the PAH syntheses (Part 1), the interaction between the organic compounds and biomacromolecules (β-peptides with proteins Fos and Jun, Part 2), and the conformational preference (conformations of α/β-peptides, Part 2). Computational modeling based on molecular and quantum mechanical techniques were applied to complement the syntheses in Parts 1 and 2

Mechanistic studies of the Class I ribonucleotide reductase from Escherichia coli

Thesis (Sc. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2006.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Vita.Includes bibliographical references.Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides, providing the monomeric precursors required for DNA replication and repair. The class I RNRs are found in many bacteria, DNA viruses, and all eukaryotes including humans, and are composed of two homodimeric subunits: R1 and R2. RNR from Escherichia coli (E. coli ) serves as the prototype of this class. R1 has the active site where nucleotide reduction occurs, and R2 contains the diferric-tyrosyl radical (Y · ) cofactor essential for radical initiation on R1. The rate-determining step in E. coli RNR has recently been shown to be a physical step prior to generation of the putative thiyl radical (S · ) on C439. Thus, the chemistry of nucleotide reduction is kinetically invisible, which has precluded detection of intermediates in the reduction process with the normal substrate. Perturbation of the system using mechanism-based inhibitors and site-directed mutants of R1 and R2 has provided the bulk of the insight into the reduction mechanism by inference.(cont.) The work described in this thesis makes use of two mechanism-based inhibitors, 20 - azido - 20 - deoxyuridine - 50 - diphosphate (N3UDP) and 20 - deoxy - 20,20 - difluorocytidine - 50 - diphosphate (dFdCDP), and one active site mutant, E441Q R1, to further our understanding of the catalytic capabilities of RNR. The results provide strong support for a 30 - ketodeoxynucleotide intermediate postulated to lie on the normal reduction pathway, as well as for the elimination of nitrogenous base in the active site of R1 during inhibition. The studies further show that under physiologically relevant reducing conditions, inhibition of RNR by the clinically important nucleotide analog dFdCDP is a result of covalent modification. An essential part of these studies was the development of a robust, high-yielding enzymatic method for the selective 50 - phosphorylation of cytidine, 20 - deoxycytidine, 20 - deoxyuridine and their analogs that are not amenable to standard chemical phosphorylation methods.by Erin Jelena Artin.Sc.D

Development and optimization of a workflow to enable mass spectrometry-based quantitative membrane proteomics of mature and tolerogenic dendritic cells

PhD ThesisTolerogenic dendritic cells are monocyte-derived dendritic cells (DC) cultured such that they adopt an immunoregulatory phenotype. In vitro, these cells are able to induce and maintain T cell tolerance through deviation of naive T cells to an anti-inflammatory phenotype and induction of anergy in memory T cells. Equivalent cells suppress established arthritis in murine models and tolerogenic DC are presently the subject of a phase I safety and efficacy trial at Newcastle University as part of the AutoDeCRA study. However, in spite of these promising data, we are yet to rigorously explore the basis of the phenotype of tolerogenic DC and lack markers to unequivocally distinguish them from other types of DC. This body of work is concerned with the development of a workflow to enable these questions to be addressed using mass spectrometry-based quantitative proteomics. Specifically, methods have been optimized and validated to enable a) enrichment and proteolytic digestion of membrane proteins, favouring their detection over more abundant cytoplasmic and nuclear proteins in LC/MS; b) differential stable isotope labelling of peptide N- and C-termini, enabling ‘isobaric peptide termini labelling’-based relative quantitation at the MS2 level; c) pipette-tip based anion exchange fractionation of IPTL-labelled peptides prior to LC/MS analysis, broadening depth of proteome coverage. Efforts to apply aspects of the workflow to perform quantitative comparisons of the whole cell proteomes and qualitative profiling of the membrane proteomes of mature and tolerogenic DC are also documented. It is envisaged that future application of this optimized workflow as a whole will enable the identification and relative quantitation of significant numbers of mature and tolerogenic DC plasma membrane proteins. Differentially expressed proteins of interest identified through this approach may then be further investigated for putative roles in tolerance induction

Structural Characterisation of Proteins from the Peroxiredoxin Family

The oligomerisation of protein subunits is an area of much research interest, in particular the relationship to protein function. In the last decade, the potential to control the interactions involved in order to design constructs with tuneable oligomeric properties in vitro has been pursued. The subject of this thesis is the quaternary structure of members of the peroxiredoxin family, which have been seen to assume an intriguing array of organisations. Human Peroxiredoxin 3 (HsPrx3) and Mycobacterium tuberculosis alkyl hydroperoxide reductase (MtAhpE) catalyse the detoxification of reactive species, preferentially hydrogen peroxide and peroxynitrite respectively, and form an essential part of the antioxidant defence system. As well as their biomedical interest, the ability of these proteins to form organised supramolecular assemblies makes them of interest in protein nanotechnology. The work described focusses on the elucidation of the quaternary structure of both proteins, resolving previous debates about their oligomeric state. The factors influencing oligomerisation were examined through biophysical characterisation in different conditions, using solution techniques including chromatography, light and X-ray scattering, and electron microscopy. The insight gained, along with analysis of the protein-protein interfaces, was used to alter the quaternary structure through site-directed mutagenesis. This resulted in a level of control over the protein’s oligomeric state to be achieved, and novel structures with potential applications in nanotechnology to be generated. The activity of the non-native structures was also assessed, to begin to unravel the relationship between peroxiredoxin quaternary structure to enzyme activity. The formation and structure of very high molecular weight complexes of HsPrx3 were explored using electron microscopy. The first high resolution structural data for such a complex is presented, analysis of which allowed the theory of an assembly mechanism to be proposed

Design and synthesis of modified deoxynucleosides, site specifically damaged oligonucleotides, and repair inhibiting chemotherapeutic agents

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1994.Includes bibliographical references (leaves 276-314).by Marshall Lee Morningstar.Ph.D

Untersuchung des Beitrags der Substratsammelantenne Sammelantenne zum Protonen/Laktat-Cotransport (in PfFNT und MCT1)

Some cells rely intensively on the coupled activity of glycolysis and lactate dehydrogenase to constantly generate ATP. This process releases lactate and protons, the accumulation of which would become detrimental to the cell´s survival. Therefore, the removal of these metabolites is critical to maintain the cells energy generation. This is ensured by monocarboxylate transporters (MCT), such as the human MCT and the Plasmodium falciparum formate nitrite transporter (PfFNT). In the case of human MCT, it has been established that their transport activity was modulated by partner proteins: its chaperone Basigin and carbonic anhydrases enzymes. The surface of such proteins act as proton and substrate collecting antennas, generating microenvironments of greater substrate concentration close to the transport sites. This work set out to investigate how such antennas were involved in the modulation of the monocarboxylate transport functionality of MCT1 and PfFNT. Initial attempts of expressing fusion constructs of carbonic anhydrase, Basigin chaperone and MCT1 transporter proved unsuccessful. Then, alternative methods of protein production were explored to observe interaction between the transporter and the proton antenna. Moreover, this work identified that C-terminal poly-Histidine tag initially intended for protein purification and identification would affect the transport capacity of such MCT1 transporter. This work also hypothesized that the PfFNT C-terminal helix, highly conserved among all human-infecting Plasmodia, plays the role of an endogenous proton collecting antenna facilitating the proton/lactate cotransport. Experimental results suggested that this terminus does modulate the substrate transport (radiolabeled lactate influx capacity was increased in acidic extracellular pH upon its deletion), but it remains to be determined whenever this collecting antenna increases the local concentration of protons or lactate

Efforts towards the synthesis of fully N-differentiated heparin-like glycosaminoglycans; and, Investigations into the mechanism of inactivation of RTPR by gemcitabine triphosphate

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, February 2007.Vita.Includes bibliographical references.Efforts towards the Synthesis of Fully N-Differentiated Heparin-like Glycosaminoglycans. Heparin-like glycosaminoglycans (HLGAGs) are complex information-carrying biopolymers and are an important component of the coagulation cascade. They have also been implicated in interactions with growth factors, cytokines, virus entry, and other functions. Currently, no general synthesis of arbitrary HLGAG sequences has been demonstrated. The modular synthesis of glycosaminoglycans requires straightforward methods for the production of large quantities of protected uronic acid building blocks. An efficient route to methyl 3-0- benzyl-1,2-O-isopropylidene-a-L-idopyranosiduronate from diacetone glucose in nine steps and 36% overall yield is described. Additionally, a general method for the conversion of glycals to the corresponding 1,2-cis-isopropylidene-a-glycosides is reported. Epoxidation of glycals with dimethyldioxirane followed by ZnC12-catalyzed addition of acetone converted a variety of protected glycals into 1,2-cis-isopropylidene-a-glycosides in good yield. The reaction is compatible with a range of protecting groups, as well as free hydroxyl groups. This method has been applied to develop a synthesis of 3-O-benzyl-1,2-O-isopropylidene-P-D-glucopyranosiduronate in seven steps and 32% overall yield.(cont.) These compounds are useful as glycosyl acceptors and as intermediates that may be further elaborated into uronic acid trichloroacetimidate glycosyl donors for the assembly of glycosaminoglycan structures. The glucosamine residues in HLGAGs have been found to exist as amines, acetamides, and N-sulfonates. In order to develop a completely general, modular synthesis of heparin, three degrees of orthogonal nitrogen protection are required. Reported is a strategy for the synthesis of fully N-differentiated heparin oligosaccharides in the context of target octasaccharide 3-1, which contains an N-acetate, N-sulfonates, and a free amine. The protecting group scheme used in the synthesis blocked the N-acetate as a N-diacetate, the N-sulfonates as azido groups, and the amine as a N-CBz; free hydroxyls were masked as benzyl ethers and O-sulfonates as acetate esters. Disaccharide and tetrasaccharide modules were synthesized using this strategy; however, the union of tetrasaccharide trichloroacetimidate 3-4 with disaccharide acceptor 3-5 unexpectedly formed the undesired P-linked glycoside in addition to the a-linkage anticipated for iduronic acid nucleophiles, resulting in an inseparable 6:1 a: p mixture of products. Detailed studies into the basis for this unexpected result were conducted and are also reported.(cont.) Investigations into the Mechanism of Inactivation of RTPR by Gemcitabine Triphosphate. Ribonucleoside triphosphate reductase (RTPR) is an adenosylcobalamin (AdoCbl) dependant enzyme that catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates via controlled radical chemistry. The antitumor agent 2',2'-difluoro-2'- deoxycytidine (gemcitabine, F2C) has been shown to owe some of its in vivo activity to inhibition of human RNR by the 5'-diphosphate (F2CDP). Previous studies have shown that RTPR is rapidly inactivated by one equivalent of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F2CTP). This inactivation is associated with the release of two equivalents of fluoride and modification of RTPR by a Co-S bond between C419 and the cobalamin cofactor. In order to further characterize this inactivation, isotopically labeled derivatives of F2CTP were synthesized: radiolabeled 1'-[3H]-F2C and mass labeled 1'-[2H]-F2C and 3'-[2H]-F2C. These compounds were converted to F2CTP through a set of enzymatic phosphorylation steps which overcome difficulties found using traditional, chemical methods. Biochemical investigations were performed using these labeled derivatives to track the fate of the base and sugar during RTPR inactivation by F2CTP.(cont.) The release of cytosine base, previously overlooked in this system, was detected utilizing 5-[3H]-F2CTP: 0.7 equiv. of cytosine were released, with 0.15-0.2 equiv. of unreacted F2CTP remaining. Size exclusion chromatography (SEC) was used to quantify covalent labeling of RTPR by F2CTP: 0.15 equiv. were detected using 5-[3H]-F2CTP, 0.45 equiv. were detected using 1'-[3H]-F2CTP. A small molecule nucleotide product was identified in inactivation mixtures quenched with NaBH4 and identified as an isomer of cytidine, indicating the loss of both fluorides and the addition of an oxygen at the 2' carbon. RTPR inactivated with 1'-[3H]-F2CTP was digested with trypsin and peptides containing radioactivity purified. Identical peptides were prepared using partially deuterated F2CTP, allowing identification by MALDI-MS. Post source decay (PSD) MS/MS methods were used to further characterize these peptides, identifying the site of label as the C-terminal tryptic peptide of RTPR at C731 and C736. The cysteines were labeled through conjugate addition with a furanone-like precursor that had lost cytosine, triphosphate, and both fluorines. The results of these studies have allowed for the first time the proposal of a mechanistic hypothesis for RTPR inactivation by F2CTP.by Gregory J.S. Lohman.Ph.D