Structural Basis of Prospero-DNA Interaction Implications for Transcription Regulationin Developing Cells

SummaryThe crystal structure of a complex between the novel homeodomain of the neural transcription factor Prospero and DNA shows that the invariant residues Lys1290, Asn1294, and Asp1297 make specific contacts with the noncanonical DNA binding site. The overall structure includes the homeodomain and the adjacent Prospero domain and confirms that they act as a single structural unit, a Homeo-Prospero domain. The Prospero domain facilitates the proper alignment of the protein on the DNA. Knowledge of the structure reconciles two different DNA sequences that have been proposed as transcriptional targets for Prospero. As in the apo structure, the C terminus of the Prospero domain shields a short helix within the homeodomain that includes a nuclear export signal (NES). The structural results suggest that exposure of the NES is not coupled directly to DNA binding. We propose a DNA recognition mechanism specific to Prospero-type homeodomains in developing cells

Probing the Reaction Mechanism of the D-ala-D-ala Dipeptidase, VanX, by Using Stopped-Flow Kinetic and Rapid-Freeze Quench EPR Studies on the Co(II)-Substituted Enzyme

In an effort to probe the reaction mechanism of VanX, the D-ala-D-ala dipeptidase required for high-level vancomycin resistance in bacteria, stopped-flow kinetic and rapid-freeze quench EPR studies were conducted on the Co(II)-substituted enzyme when reacted with d-ala-d-ala. The intensity of the Co(II) ligand field band at 550 nm decreased (ε550 = 140 to 18 M-1 cm-1) when VanX was reacted with substrate, suggesting that the coordination number of the metal increases from 5 to 6 upon substrate binding. The stopped-flow trace was fitted to a kinetic mechanism that suggests the presence of an intermediate whose breakdown is rate-limiting. Rapid-freeze quench EPR studies verified the presence of a reaction intermediate that exhibits an unusually low hyperfine constant (33 G), which suggests a bidentate coordination of the intermediate to the metal center. The EPR studies also identified a distinct enzyme product complex. The results were used to offer a detailed reaction mechanism for VanX that can be used to guide future inhibitor design efforts

Kinetic and Spectroscopic Characterization of the H178A Methionyl Aminopeptidase from \u3cem\u3eEscherichia coli\u3c/em\u3e

To gain insight into the role of the strictly conserved histidine residue, H178, in the reaction mechanism of the methionyl aminopeptidase from Escherichia coli (EcMetAP-I), the H178A mutant enzyme was prepared. Metal-reconstituted H178A binds only one equivalent of Co(II) or Fe(II) tightly with affinities that are identical to the WT enzyme based on kinetic and isothermal titration calorimetry (ITC) data. Electronic absorption spectra of Co(II)-loaded H178A EcMetAP-I indicate that the active site divalent metal ion is pentacoordinate, identical to the WT enzyme. These data indicate that the metal binding site has not been affected by altering H178. The effect of altering H178 on activity is, in general, due to a decrease in kcat. The kcat value for Co(II)-loaded H178A decreased 70-fold toward MGMM and 290-fold toward MP-p-NA compared to the WT enzyme, while kcat decreased 50-fold toward MGMM for the Fe(II)-loaded H178A enzyme and 140-fold toward MP-p-NA. The Km values for MGMM remained unaffected, while those for MP-p-NA increased approximately 2-fold for Co(II)- and Fe(II)-loaded H178A. The kcat/Km values for both Co(II)- and Fe(II)-loaded H178A toward both substrates ranged from ∼50- to 580-fold reduction. The pH dependence of log Km, log kcat, and log(kcat/Km) of both WT and H178A EcMetAP-I were also obtained and are identical, within error, for H178A and WT EcMetAP-I. Therefore, H178A is catalytically important but is not required for catalysis. Assignment of one of the observed pKa values at 8.1 for WT EcMetAP-I was obtained from plots of molar absorptivity at λmax(640) vs pH for both WT and H178A EcMetAP-I. Apparent pKa values of 8.1 and 7.6 were obtained for WT and H178A EcMetAP-I, respectively, and were assigned to the deprotonation of a metal-bound water molecule. The data reported herein provide support for the key elements of the previously proposed mechanism and suggest that a similar mechanism can apply to the enzyme with a single metal in the active site

A Five-coordinate Metal Center in Co(II)-substituted VanX

In an effort to structurally probe the metal binding site in VanX, electronic absorption, EPR, and extended x-ray absorption fine structure (EXAFS) spectroscopic studies were conducted on Co(II)-substituted VanX. Electronic spectroscopy revealed the presence of Co(II) ligand field transitions that had molar absorptivities of ∼100 m–1 cm–1, which suggests that Co(II) is five-coordinate in Co(II)-substituted VanX. Low temperature EPR spectra of Co(II)-substituted VanX were simulated using spin Hamiltonian parameters of M = |±½〉, E/D = 0.14, greal(x,y) = 2.37, and grealS(z) = 2.03. These parameters lead to the prediction that Co(II) in the enzyme is five-coordinate and that there may be at least one solvent-derived ligand. Single scattering fits of EXAFS data indicate that the metal ions in both native Zn(II)-containing and Co(II)-substituted VanX have the same coordination number and that the metal ions are coordinated by 5 nitrogen/oxygen ligands at ∼ 2.0 Å. These data demonstrate that Co(II) (and Zn(II) from EXAFS studies) is five-coordinate in VanX in contrast to previous crystallographic studies (Bussiere, D. E., Pratt, S. D., Katz, L., Severin, J. M., Holzman, T., and Park, C. H. (1998) Mol. Cell 2, 75–84). These spectroscopic studies also demonstrate that the metal ion in Co(II)-substituted VanX when complexed with a phosphinate analog of substrate d-Ala-d-Ala is also five-coordinate

Operational Definitions of Sexual Orientation and Estimates of Adolescent Health Risk Behaviors

Increasing attention to the health of lesbian, gay, and bisexual (LGB) populations comes with requisite circumspection about measuring sexual orientation in surveys. However, operationalizing these variables also requires considerable thought. This research sought to document the consequences of different operational definitions of sexual orientation by examining variation in health risk behaviors

First test results from a high-resolution CdZnTe pixel detector with VLSI readout

We are developing a CdZnTe pixel detector with a custom low- noise analog VLSI readout for use in the High-Energy Focusing Telescope balloon experiment, as well as for future space astronomy applications. The goal of the program is to achieve good energy resolution (< 1 keV FWHM at 60 keV) and low threshold in a sensor with approximately 500 micrometers pixels. We have fabricated several prototype detector assemblies with 2 mm thick, 680 by 650 micrometers pitch CdZnTe pixel sensors indium bump bonded a VLSI readout chip developed at Caltech. Each readout circuit in the 8 X 8 prototype is matched to the detector pixel size, and contains a preamplifier, shaping amplifiers, and a peak stretcher/discriminator. In the first 8 X 8 prototype, we have demonstrated the low-noise preamplifier by routing the output signals off-chip for shaping and pulse-height analysis. Pulse height spectra obtained using a ^(241)Am source, collimated to illuminate a single pixel, show excellent energy resolution of 1.1 keV FWHM for the 60 keV line at room temperature. Line profiles are approximately Gaussian and dominated by electronic noise, however a small low energy tail is evident for the 60 keV line. We obtained slightly improved resolution of 0.9 keV FWHM at 60 keV by cooling the detector to 5 degree(s)C, near the expected balloon- flight operating temperature. Pulse height spectra obtained with the collimated source positioned between pixels show the effect of signal sharing for events occurring near the boundary. We are able to model the observed spectra using a Monte-Carlo simulation that includes the effects of photon interaction, charge transport and diffusion, pixel and collimator geometry, and electronic noise. By using the model to simulate the detector response to uncollimated radiation (including the effect of finite trigger threshold for reconstruction of the total energy of multi-pixel events), we find the energy resolution to be degraded by only 10% for full-face illumination, compared to the collimated case. The small value of the degradation is due directly to the low readout noise and amplifier threshold

Black Phosphorus with Near-Superhydrophic Properties and Long-Term Stability in Aqueous Media

Black phosphorus is a two-dimensional material that has potential applications in energy storage, high frequency electronics and sensing, yet it suffers from instability in oxygenated and/or aqueous systems. Here we present the use of a polymeric stabilizer which prevents the degradation of nearly 68% of the material in aqueous media over the course of ca. 1 month

SARS-CoV-2 antibody trajectories after a single COVID-19 vaccination with and without prior infection

Given high SARS-CoV-2 incidence, coupled with slow and inequitable vaccine roll-out in many settings, there is a need for evidence to underpin optimum vaccine deployment, aiming to maximise global population immunity. We evaluate whether a single vaccination in individuals who have already been infected with SARS-CoV-2 generates similar initial and subsequent antibody responses to two vaccinations in those without prior infection. We compared anti-spike IgG antibody responses after a single vaccination with ChAdOx1, BNT162b2, or mRNA-1273 SARS-CoV-2 vaccines in the COVID-19 Infection Survey in the UK general population. In 100,849 adults median (50 (IQR: 37–63) years) receiving at least one vaccination, 13,404 (13.3%) had serological/PCR evidence of prior infection. Prior infection significantly boosted antibody responses, producing higher peak levels and/or longer half-lives after one dose of all three vaccines than those without prior infection receiving one or two vaccinations. In those with prior infection, the median time above the positivity threshold was >1 year after the first vaccination. Single-dose vaccination targeted to those previously infected may provide at least as good protection to two-dose vaccination among those without previous infection

First test results from a high-resolution CdZnTe pixel detector with VLSI readout

We are developing a CdZnTe pixel detector with a custom low- noise analog VLSI readout for use in the High-Energy Focusing Telescope balloon experiment, as well as for future space astronomy applications. The goal of the program is to achieve good energy resolution (< 1 keV FWHM at 60 keV) and low threshold in a sensor with approximately 500 micrometers pixels. We have fabricated several prototype detector assemblies with 2 mm thick, 680 by 650 micrometers pitch CdZnTe pixel sensors indium bump bonded a VLSI readout chip developed at Caltech. Each readout circuit in the 8 X 8 prototype is matched to the detector pixel size, and contains a preamplifier, shaping amplifiers, and a peak stretcher/discriminator. In the first 8 X 8 prototype, we have demonstrated the low-noise preamplifier by routing the output signals off-chip for shaping and pulse-height analysis. Pulse height spectra obtained using a ^(241)Am source, collimated to illuminate a single pixel, show excellent energy resolution of 1.1 keV FWHM for the 60 keV line at room temperature. Line profiles are approximately Gaussian and dominated by electronic noise, however a small low energy tail is evident for the 60 keV line. We obtained slightly improved resolution of 0.9 keV FWHM at 60 keV by cooling the detector to 5 degree(s)C, near the expected balloon- flight operating temperature. Pulse height spectra obtained with the collimated source positioned between pixels show the effect of signal sharing for events occurring near the boundary. We are able to model the observed spectra using a Monte-Carlo simulation that includes the effects of photon interaction, charge transport and diffusion, pixel and collimator geometry, and electronic noise. By using the model to simulate the detector response to uncollimated radiation (including the effect of finite trigger threshold for reconstruction of the total energy of multi-pixel events), we find the energy resolution to be degraded by only 10% for full-face illumination, compared to the collimated case. The small value of the degradation is due directly to the low readout noise and amplifier threshold

Cryogenic Optical Performance of a Lightweighted Mirror Assembly for Future Space Astronomical Telescopes: Correlating Optical Test Results and Thermal Optical Model

A 43cm diameter stacked core mirror demonstrator was interferometrically tested at room temperature down to 250 degrees Kelvin for thermal deformation. The 2.5m radius of curvature spherical mirror assembly was constructed by low temperature fusing three abrasive waterjet core sections between two CNC pocket milled face sheets. The 93% lightweighted Corning ULE mirror assembly represents the current state of the art for future UV, optical, near IR space telescopes. During the multiple thermal test cycles, test results of interferometric test, thermal IR images of the front face were recorded in order to validate thermal optical model