Identifying the Catalytic and Ligand Binding Roles of Active Site Residues in Homotetrameric R67 Dihydrofolate Reductase

R67 dihydrofolate reductase (DHFR) is a novel protein that confers clinical resistance to trimethoprim (TMP). Surprisingly, this R-plasmid encoded enzyme does not share homology with chromosomal DHFR. Recently a high resolution crystal structure of R67 DHFR has been solved. From this structure, R67 DHFR is a homotetramer that possesses exact 222 symmetry and a single active site pore that traverses the length of the protein (Narayana et al., 1995). Although this symmetry implies that four symmetry related binding sites must exist for each substrate, isothermal titration calorimetry studies indicate only two molecules bind. Three possible combinations of bound ligands have been observed. These include two dihydrofolate molecules or two NADPH molecules or one substrate + one cofactor (Bradrick et al., 1996). The latter is the productive ternary complex. To date a crystal structure of this ternary complex has been solved. Computational docking studies, however have been used to develop a model of the productive ternary complex (Howell et al., 2001). This model has implicated several active site residues to be involved in ligand binding. Because of the unusual 222 symmetry of this enzyme and the fact it shares no structural similarities with the chromosomal enzyme, R67 DHFR must utilize a different strategy for ligand binding and catalysis. The research in this dissertation has been focused on utilizing site directed mutagenesis as a means to probe the function of active residues implicated by the computational studies to be important in ligand binding and catalysis. Another important goal of this work has been to probe the role interligand cooperativity v may play in the catalytic function of R67 DHFR. The results of the research presented in this support a model where R67 DHFR utilizes a an unusual “hot spot” binding surface capable of binding both ligands and facilitates catalysis simply by binding ligands in the appropriate orientation to stabilize the transition state. Thus R67 DHFR has adopted a novel yet simple strategy to reach the transition state compared with other more highly evolved DHFRs

Constructing a hybrid of R67 dihydrofolate reductase to study asymmetric mutations in the active site

R67 dihydrofolate reductase (DHFR) is a novel protein that confers clinical resistance to trimethoprim (TMP). Surprisingly, this R-plasmid encoded enzyme does not share any homology with chromosomal DHFR. The crystal structure of active homotetrameric R67 DHFR indicates that a single active site pore transverses the length of the molecule (Narayana et al., 1995). Since the center of the pore possesses exact 222 symmetry, site directed mutagenesis of the gene sequence encoding R67 DHFR will introduce four symmetrically related mutations at the single active site. To break this symmetry, a gene oligomerization strategy has been designed to construct an R67 DHFR hybrid possessing the essential tertiary structure of native tetrameric R67 DHFR. The procedures used in creation of the R67 DHFR hybrid require triple R67 DHFR (a gene triplication product) and a single gene copy containing an N-terrninal Histag sequence (Histag R67 DHFR). The Histag sequence is necessary for purification of the hybrid species utilizing nickel chelating affinity chromatography. Triple R67 DHFR is a covalently linked three domain monomer created by linking in frame three copies of the R67 DHFR gene. The results in this thesis show that both triple and Histag R67 DHFRs are kinetically similar to native R67 DHFR and are accordingly suitable for constructing the hybrid. Two important requirements for creating a functional hybrid from the above gene oligomerization strategy are that both triple and Histag R67 DHFR associate to form a stable structure that kinetically mimics the wildtype homotetramer, and that the N-terrninal Histag sequence does not block access to the active site. The pH titration, gel filtration, and CD data, in this thesis, show substantial evidence that both proteins associate to produce a homogenous hybrid species corresponding to a 1:1 molar ratio of triple and Histag R67 DHFRs. Furthermore, this species is also stable and kinetically similar to the wildtype enzyme. The data shown in this thesis are significant in that they show that the R67 DHFR hybrid is very similar to the native homotetramer. Thus, the next step would be to use this procedure to create a mutant R67 DHFR hybrid to study asymmetric mutations in the active site of R67 DHFR

An open source, FPGA-based LeKID readout for BLAST-TNG: Pre-flight results

We present a highly frequency multiplexed readout for large-format superconducting detector arrays intended for use in the next generation of balloon-borne and space-based sub-millimeter and far-infrared missions. We will demonstrate this technology on the upcoming NASA Next Generation Balloon-borne Large Aperture Sub-millimeter Telescope (BLAST-TNG) to measure the polarized emission of Galactic dust at wavelengths of 250, 350 and 500 microns. The BLAST-TNG receiver incorporates the first arrays of Lumped Element Kinetic Inductance Detectors (LeKID) along with the first microwave multiplexing readout electronics to fly in a space-like environment and will significantly advance the TRL for these technologies. After the flight of BLAST-TNG, we will continue to improve the performance of the detectors and readout electronics for the next generation of balloon-borne instruments and for use in a future FIR Surveyor. Read More: http://www.worldscientific.com/doi/abs/10.1142/S225117171641003

The Providence Mutation (βK82D) in Human Hemoglobin Substantially Reduces βCysteine 93 Oxidation and Oxidative Stress in Endothelial Cells

The highly toxic oxidative transformation of hemoglobin (Hb) to the ferryl state (HbFe4+) is known to occur in both in vitro and in vivo settings. We recently constructed oxidatively stable human Hbs, based on the Hb Providence (βK82D) mutation in sickle cell Hb (βE6V/βK82D) and in a recombinant crosslinked Hb (rHb0.1/βK82D). Using High Resolution Accurate Mass (HRAM) mass spectrometry, we first quantified the degree of irreversible oxidation of βCys93 in these proteins, induced by hydrogen peroxide (H2O2), and compared it to their respective controls (HbA and HbS). Both Hbs containing the βK82D mutation showed considerably less cysteic acid formation, a byproduct of cysteine irreversible oxidation. Next, we performed a novel study aimed at exploring the impact of introducing βK82D containing Hbs on vascular endothelial redox homeostasis and energy metabolism. Incubation of the mutants carrying βK82D with endothelial cells resulted in altered bioenergetic function, by improving basal cellular glycolysis and glycolytic capacity. Treatment of cells with Hb variants containing βK82D resulted in lower heme oxygenase-1 and ferritin expressions, compared to native Hbs. We conclude that the presence of βK82D confers oxidative stability to Hb and adds significant resistance to oxidative toxicity. Therefore, we propose that βK82D is a potential gene-editing target in the treatment of sickle cell disease and in the design of safe and effective oxygen therapeutics

The Providence Mutation (βK82D) in Human Hemoglobin Substantially Reduces βCysteine 93 Oxidation and Oxidative Stress in Endothelial Cells

The highly toxic oxidative transformation of hemoglobin (Hb) to the ferryl state (HbFe4+) is known to occur in both in vitro and in vivo settings. We recently constructed oxidatively stable human Hbs, based on the Hb Providence (βK82D) mutation in sickle cell Hb (βE6V/βK82D) and in a recombinant crosslinked Hb (rHb0.1/βK82D). Using High Resolution Accurate Mass (HRAM) mass spectrometry, we first quantified the degree of irreversible oxidation of βCys93 in these proteins, induced by hydrogen peroxide (H2O2), and compared it to their respective controls (HbA and HbS). Both Hbs containing the βK82D mutation showed considerably less cysteic acid formation, a byproduct of cysteine irreversible oxidation. Next, we performed a novel study aimed at exploring the impact of introducing βK82D containing Hbs on vascular endothelial redox homeostasis and energy metabolism. Incubation of the mutants carrying βK82D with endothelial cells resulted in altered bioenergetic function, by improving basal cellular glycolysis and glycolytic capacity. Treatment of cells with Hb variants containing βK82D resulted in lower heme oxygenase-1 and ferritin expressions, compared to native Hbs. We conclude that the presence of βK82D confers oxidative stability to Hb and adds significant resistance to oxidative toxicity. Therefore, we propose that βK82D is a potential gene-editing target in the treatment of sickle cell disease and in the design of safe and effective oxygen therapeutics.</jats:p

Comparison of Digestion Protocols for Microgram Quantities of Enriched Protein Samples

Standard biochemical techniques that are used for protein enrichments, such as affinity isolation and density gradient centrifugation, frequently yield high-nanogram to low-microgram quantities at a significant expenditure of resources and time. The characterization of selected protein enrichments by the “shotgun” mass spectrometry approach is often compromised by the lack of effective and efficient in-solution proteolysis protocols specifically tailored for these small quantities of proteins. This study compares the results of five different digestion protocols that were applied to 2.5 μg portions of protein isolates from two disparate sources:  Rhodopseudomonas palustris 70S ribosomal proteins, and Bos taurus microtubule-associated proteins (MAPs). Proteolytic peptides produced according to each protocol in each type of protein isolate were analyzed by one-dimensional liquid chromatography−tandem mass spectrometry (LC−MS/MS). The effectiveness of each digestion protocol was assessed on the basis of three parameters:  number of peptide identifications, number of protein identifications, and sequence coverage. The two protocols using a solvent containing 80% acetonitrile (CH3CN) for trypsin digestions performed as well as, and in some instances better than, protocols employing other solvents and chaotropes in both types of protein isolates. A primary advantage of the 80% CH3CN protocol is that it requires fewer sample manipulation steps. Keywords: trypsin • digestion • proteolysis • acetonitrile • mass spectrometry • liquid chromatography • ribosome • microtubule-associated protei

Comparison of Digestion Protocols for Microgram Quantities of Enriched Protein Samples

Standard biochemical techniques that are used for protein enrichments, such as affinity isolation and density gradient centrifugation, frequently yield high-nanogram to low-microgram quantities at a significant expenditure of resources and time. The characterization of selected protein enrichments by the “shotgun” mass spectrometry approach is often compromised by the lack of effective and efficient in-solution proteolysis protocols specifically tailored for these small quantities of proteins. This study compares the results of five different digestion protocols that were applied to 2.5 μg portions of protein isolates from two disparate sources:  Rhodopseudomonas palustris 70S ribosomal proteins, and Bos taurus microtubule-associated proteins (MAPs). Proteolytic peptides produced according to each protocol in each type of protein isolate were analyzed by one-dimensional liquid chromatography−tandem mass spectrometry (LC−MS/MS). The effectiveness of each digestion protocol was assessed on the basis of three parameters:  number of peptide identifications, number of protein identifications, and sequence coverage. The two protocols using a solvent containing 80% acetonitrile (CH3CN) for trypsin digestions performed as well as, and in some instances better than, protocols employing other solvents and chaotropes in both types of protein isolates. A primary advantage of the 80% CH3CN protocol is that it requires fewer sample manipulation steps. Keywords: trypsin • digestion • proteolysis • acetonitrile • mass spectrometry • liquid chromatography • ribosome • microtubule-associated protei