Structure to function : case studies of hotdog-fold superfamily thioesterases from Escherichia coli.

My doctoral research primarily focuses on the Escherichia coli hotdog-fold thioesterases ydiI and ybgC. The ydiI gene is colocalized in the operon ydiHIJ. A substrate screen provided evidence that YdiI prefers aryl-CoA substrates and discriminated against the analogous aryl-ACP, unlike the paralog YbdB which works on 2,4-DHB-EntB in the enterobactin synthesis pathway. A bioinformatic approach showed that in some bacteria, ydiI is colocalized with genes from the menaquinone pathway. YdiI was shown to catalyze the hydrolysis of DHNA-CoA with physiological relevance (kcat/KM ~ 105). Furthermore, the E. coli YdiI- strain was shown to have perturbed growth, in good agreement with other menaquinone enzyme knockout experiments. Taking into account the evidence provided, YdiI is likely the DHNA-CoA thioesterase in the menaquinone pathway. Within the substrate binding pocket, changes to the YdiI Val68 to the YbdB equivalent of Met resulted in perturbed catalytic efficiency towards lauroyl-CoA, however not towards its physiological substrate DHNA-CoA. The YbdB M68V mutant resulted in increased efficiency towards lauroyl-CoA and DHNA-CoA, seemingly making it a gatekeeper. Secondly, YdiI catalyzes the hydrolysis of acyl-CoA\u27s using a Glu63, His54, and Gln48 triad determined by mutagenesis experiments. The utilization of 18-O incorporation and rapid-quench techniques provided insight into YdiI\u27s catalytic mechanism. A single phase multiple turnover reaction and the incorporation of 18-O in single turnover reactions suggests that ydiI uses a general base catalytic mechanism where Glu63 is the activating residue. The second E. coli hotdog-fold thioesterase protein discussed within is YbgC. YbgC is encoded by the first ORF of the tol-pal gene cluster. Previous work on the H. influenzae homologue demonstrated affinity for short chain acyl-CoA\u27s and tandem affintity purificiation experiments showed E. coli YbgC co-purifies with ACP. No previous attempts to screen for acyl-ACP have been made. To screen YbgC for ACP activity, a method was developed to stoichiometrically construct acyl-ACP\u27s, using apoACP, BF1558 acyl-transferase and acyl-CoA. The E. coli YbgC demonstrated preference for long chain acyl-CoA\u27s and their analogous acyl-ACPs. To understand the nature of ACP binding to YbgC, SAXS analysis was carried out on the complex demonstrating that 2 ACP molecules bind to 1 dimer of YbgC

Regulation of Set1-mediated methylation of Dam1

Eukaryotic genomes exist within a dynamic structure named chromatin in which DNA is wrapped around an octamer of histones forming the nucleosome. Histones are modified by a range of posttranslational modifications including methylation, phosphorylation, and ubiquitination, which are integral to a range of DNA-templated processes including transcriptional regulation. A hallmark for transcriptional activity is methylation of histone H3 on lysine (K) 4 within active gene promoters. In S. cerevisiae, H3K4 methylation is mediated by Set1 within the COMPASS complex. Methylation requires prior ubiquitination of histone H2BK123 by the E2-E3 ligases Rad6 and Bre1, as well as the Paf1 transcriptional elongation complex. This regulatory pathway exemplifies cross-talk in trans between posttranslational modifications on distinct histone molecules. Set1 has an additional substrate in the kinetochore protein Dam1, which is methylated on K233. This methylation antagonizes phosphorylation of adjacent serines by the Ipl1 Aurora kinase. The discovery of a second Set1 substrate raised the question of how Set1 function is regulated at the kinetochore. I hypothesized that transcriptional regulatory factors essential for H3K4 methylation at gene promoters might also regulate Set1-mediated methylation of Dam1K233. Here I show that the regulatory factors essential for COMPASS activity at gene promoters is also indispensable for the methylation of Dam1K233. Deletion of members of the COMPASS complex leads to loss of Dam1K233 methylation. In addition, deletion of Rad6, Bre1, or members of the Paf1 complex abolishes Dam1 methylation. The role of Rad6 and Bre1 in Dam1 methylation is dependent on H2BK123 ubiquitination, as mutation of K123 within H2B results in complete loss of Dam1 methylation. Importantly, methylation of Dam1K233 is independent of transcription and occurs at the kinetochore. My results demonstrate that Set1-mediated methylation is regulated by a general pathway regardless of substrate that is composed of transcriptional regulatory factors functioning independently of transcription at the kinetochore. My data provide the first example of cross-talk in trans between modifications on a histone and a non-histone protein. Additionally, my results indicate that several factors previously thought to be required for Set1 function at gene promoters are more generally required for the catalytic activity of the COMPASS complex regardless of substrate or cellular process

Line Broadening in Field Metal-poor Red Giant and Red Horizontal Branch Stars

We report 349 radial velocities for 45 metal-poor field red giant and red horizontal branch stars. We have have identified one new spectroscopic binary, HD 4306, and one possible such system, HD 184711. We also report 57 radial velocities for 11 of the 91 stars reported on previously by Carney et al. (2003). As was found in the previous study, radial velocity "jitter" is present in many of the most luminous stars. Excluding stars showing spectroscopic binary orbital motion, all 7 of the red giants with M(V) <= -2.0 display jitter, as well as 3 of the 14 stars with -2.0 <= M(V) <= -1.4. We have also measured line broadening in all of the new spectra, using synthetic spectra as templates. The most luminous red giants show significant line broadening, as do many of the red horizontal branch stars, and we discuss briefly possible causes.Comment: To appear in the Astronomical Journa

Multiwavelength transit observations of the candidate disintegrating planetesimals orbiting WD 1145+017

We present multiwavelength, ground-based follow-up photometry of the white dwarf WD 1145+017, which has recently been suggested to be orbited by up to six or more short-period, low-mass, disintegrating planetesimals. We detect nine significant dips in flux of between 10% and 30% of the stellar flux in our ~32 hr of photometry, suggesting that WD 1145+017 is indeed being orbited by multiple, short-period objects. Through fits to the asymmetric transits that we observe, we confirm that the transit egress is usually longer than the ingress, and that the transit duration is longer than expected for a solid body at these short periods, all suggesting that these objects have cometary tails streaming behind them. The precise orbital periods of the planetesimals are unclear, but at least one object, and likely more, have orbital periods of ~4.5 hr. We are otherwise unable to confirm the specific periods that have been reported, bringing into question the long-term stability of these periods. Our high-precision photometry also displays low-amplitude variations, suggesting that dusty material is consistently passing in front of the white dwarf, either from discarded material from these disintegrating planetesimals or from the detected dusty debris disk. We compare the transit depths in the V- and R-bands of our multiwavelength photometry, and find no significant difference; therefore, for likely compositions, the radius of single-size particles in the cometary tails streaming behind the planetesimals must be ~0.15 μm or larger, or ~0.06 μm or smaller, with 2σ confidence

Novel use Of Hydroxyurea in an African Region with Malaria (NOHARM): a trial for children with sickle cell anemia

Hydroxyurea treatment is recommended for children with sickle cell anemia (SCA) living in high-resource malaria-free regions, but its safety and efficacy in malaria-endemic sub-Saharan Africa, where the greatest sickle-cell burden exists, remain unknown. In vitro studies suggest hydroxyurea could increase malaria severity, and hydroxyurea-associated neutropenia could worsen infections. NOHARM (Novel use Of Hydroxyurea in an African Region with Malaria) was a randomized, double-blinded, placebo-controlled trial conducted in malaria-endemic Uganda, comparing hydroxyurea to placebo at 20 ± 2.5 mg/kg per day for 12 months. The primary outcome was incidence of clinical malaria. Secondary outcomes included SCA-related adverse events (AEs), clinical and laboratory effects, and hematological toxicities. Children received either hydroxyurea (N = 104) or placebo (N = 103). Malaria incidence did not differ between children on hydroxyurea (0.05 episodes per child per year; 95% confidence interval [0.02, 0.13]) vs placebo (0.07 episodes per child per year [0.03, 0.16]); the hydroxyurea/placebo malaria incidence rate ratio was 0.7 ([0.2, 2.7]; P = .61). Time to infection also did not differ significantly between treatment arms. A composite SCA-related clinical outcome (vaso-occlusive painful crisis, dactylitis, acute chest syndrome, splenic sequestration, or blood transfusion) was less frequent with hydroxyurea (45%) than placebo (69%; P = .001). Children receiving hydroxyurea had significantly increased hemoglobin concentration and fetal hemoglobin, with decreased leukocytes and reticulocytes. Serious AEs, sepsis episodes, and dose-limiting toxicities were similar between treatment arms. Three deaths occurred (2 hydroxyurea, 1 placebo, and none from malaria). Hydroxyurea treatment appears safe for children with SCA living in malaria-endemic sub-Saharan Africa, without increased severe malaria, infections, or AEs. Hydroxyurea provides SCA-related laboratory and clinical efficacy, but optimal dosing and monitoring regimens for Africa remain undefined. This trial was registered at www.clinicaltrials.gov as #NCT01976416

Multiwavelength Transit Observations of the Candidate Disintegrating Planetesimals Orbiting WD 1145+017

We present multiwavelength, multi-telescope, ground-based follow-up photometry of the white dwarf WD 1145+017, that has recently been suggested to be orbited by up to six or more, short-period, low-mass, disintegrating planetesimals. We detect 9 significant dips in flux of between 10% and 30% of the stellar flux from our ground-based photometry. We observe transits deeper than 10% on average every ~3.6 hr in our photometry. This suggests that WD 1145+017 is indeed being orbited by multiple, short-period objects. Through fits to the multiple asymmetric transits that we observe, we confirm that the transit egress timescale is usually longer than the ingress timescale, and that the transit duration is longer than expected for a solid body at these short periods, all suggesting that these objects have cometary tails streaming behind them. The precise orbital periods of the planetesimals in this system are unclear from the transit-times, but at least one object, and likely more, have orbital periods of ~4.5 hours. We are otherwise unable to confirm the specific periods that have been reported, bringing into question the long-term stability of these periods. Our high precision photometry also displays low amplitude variations suggesting that dusty material is consistently passing in front of the white dwarf, either from discarded material from these disintegrating planetesimals or from the detected dusty debris disk. For the significant transits we observe, we compare the transit depths in the V- and R-bands of our multiwavelength photometry, and find no significant difference; therefore, for likely compositions the radius of single-size particles in the cometary tails streaming behind the planetesimals in this system must be ~0.15 microns or larger, or ~0.06 microns or smaller, with 2-sigma confidence.Comment: 16 pages, 12 figures, submitted to ApJ on October 8th, 201

The Mass of the White Dwarf Companion in the Self-Lensing Binary KOI-3278: Einstein vs. Newton

KOI-3278 is a self-lensing stellar binary consisting of a white-dwarf secondary orbiting a Sun-like primary star. Kruse and Agol (2014) noticed small periodic brightenings every 88.18 days in the Kepler photometry and interpreted these as the result of microlensing by a white dwarf with about 63$\%$ of the mass of the Sun. We obtained two sets of spectra for the primary that allowed us to derive three sets of spectroscopic estimates for its effective temperature, surface gravity, and metallicity for the first time. We used these values to update the Kruse and Agol (2014) Einsteinian microlensing model, resulting in a revised mass for the white dwarf of $0.539^{+0.022}_{-0.020} \, M_{\odot}$. The spectra also allowed us to determine radial velocities and derive orbital solutions, with good agreement between the two independent data sets. An independent Newtonian dynamical MCMC model of the combined velocities yielded a mass for the white dwarf of $0.5122^{+0.0057}_{-0.0058} \, M_{\odot}$. The nominal uncertainty for the Newtonian mass is about four times better than for the Einsteinian, $\pm 1.1\%$ vs. $\pm 4.1\%$ and the difference between the two mass determinations is $5.2 \%$. We then present a joint Einsteinian microlensing and Newtonian radial velocity model for KOI-3278, which yielded a mass for the white dwarf of $0.5250^{+0.0082}_{-0.0089} \, M_{\odot}$. This joint model does not rely on any white dwarf evolutionary models or assumptions on the white dwarf mass-radius relation. We discuss the benefits of a joint model of self-lensing binaries, and how future studies of these systems can provide insight into the mass-radius relation of white dwarfs.Comment: ApJ Accepted; 22 Pages, 8 Figures, 6 Tables and 4 Supplementary Table

PqqD is a novel peptide chaperone that forms a ternary complex with the radical S-adenosylmethionine protein PqqE in the pyrroloquinoline quinone biosynthetic pathway

Pyrroloquinoline quinone (PQQ) is a product of a ribosomally synthesized and post-translationally modified pathway consisting of five conserved genes, pqqA-E. PqqE is a radical S-adenosylmethionine (RS) protein with a C-terminal SPASM domain, and is proposed to catalyze the formation of a carbon-carbon bond between the glutamate and tyrosine side chains of the peptide substrate PqqA. PqqD is a 10-kDa protein with an unknown function, but is essential for PQQ production. Recently, in Klebsiella pneumoniae (Kp), PqqD and PqqE were shown to interact; however, the stoichiometry and KD were not obtained. Here, we show that the PqqE and PqqD interaction transcends species, also occurring in Methylobacterium extorquens AM1 (Me). The stoichiometry of the MePqqD and MePqqE interaction is 1:1 and the KD, determined by surface plasmon resonance spectroscopy (SPR), was found to be ∼12 μm. Moreover, using SPR and isothermal calorimetry techniques, we establish for the first time that MePqqD binds MePqqA tightly (KD ∼200 nm). The formation of a ternary MePqqA-D-E complex was captured by native mass spectrometry and the KD for the MePqqAD-MePqqE interaction was found to be ∼5 μm. Finally, using a bioinformatic analysis, we found that PqqD orthologues are associated with the RS-SPASM family of proteins (subtilosin, pyrroloquinoline quinone, anaerobic sulfatase maturating enzyme, and mycofactocin), all of which modify either peptides or proteins. In conclusion, we propose that PqqD is a novel peptide chaperone and that PqqD orthologues may play a similar role in peptide modification pathways that use an RS-SPASM protein

Detecting the Companions and Ellipsoidal Variations of RS CVn Primaries: I. sigma Geminorum

To measure the properties of both components of the RS CVn binary sigma Geminorum (sigma Gem), we directly detect the faint companion, measure the orbit, obtain model-independent masses and evolutionary histories, detect ellipsoidal variations of the primary caused by the gravity of the companion, and measure gravity darkening. We detect the companion with interferometric observations obtained with the Michigan InfraRed Combiner (MIRC) at Georgia State University's Center for High Angular Resolution Astronomy (CHARA) Array with a primary-to-secondary H-band flux ratio of 270+/-70. A radial velocity curve of the companion was obtained with spectra from the Tillinghast Reflector Echelle Spectrograph (TRES) on the 1.5-m Tillinghast Reflector at Fred Lawrence Whipple Observatory (FLWO). We additionally use new observations from the Tennessee State University Automated Spectroscopic and Photometric Telescopes (AST and APT, respectively). From our orbit, we determine model-independent masses of the components (M_1 = 1.28 +/- 0.07 M_Sun, M_2 = 0.73 +/- 0.03 M_Sun), and estimate a system age of 5 -/+ 1 Gyr. An average of the 27-year APT light curve of sigma Gem folded over the orbital period (P = 19.6027 +/- 0.0005 days) reveals a quasi-sinusoidal signature, which has previously been attributed to active longitudes 180 deg apart on the surface of sigma Gem. With the component masses, diameters, and orbit, we find that the predicted light curve for ellipsoidal variations due to the primary star partially filling its Roche lobe potential matches well with the observed average light curve, offering a compelling alternative explanation to the active longitudes hypothesis. Measuring gravity darkening from the light curve gives beta < 0.1, a value slightly lower than that expected from recent theory.Comment: Accepted to ApJ, 11 pages, 6 figures, 8 table