We describe a hybrid photocatalytic system for hydrogen production consisting of nanocrystalline CdSe/CdS dot-in-rod (DIR) structures coupled to NiFe soluble hydrogenase I (SHI) from Pyrococcus ...furiosus . Electrons are shuttled to the catalyst by a redox mediator, either methyl viologen (MV 2+ , E 0 = −446 mV vs. NHE) or propyl-bridged 2-2′-bipyridinium (PDQ 2+ , E 0 = −550 mV vs. NHE). We demonstrate nearly equal photoreduction efficiencies for the two mediators, despite extracting ∼100 mV of additional driving force for proton reduction by PDQ 2+ . Femtosecond to microsecond transient absorption reveals that while electron transfer (ET) from the DIR to PDQ 2+ is slower than for MV 2+ , in both cases the ET process is complete by 1 ns and thus it efficiently outcompetes radiative decay. Long-lived charge separation is observed for both mediators, resulting in similar net efficiencies of photoreduction. Whereas both mediators are readily photoreduced, only PDQ 2+ yields measurable H 2 production, demonstrating the importance of optimizing the electron shuttling pathway to take advantage of the available reducing power of the DIR excited state. H 2 production in the PDQ 2+ system is highly efficient, with an internal quantum efficiency (IQE) as high as 77% and a TON SHI of 1.1 × 10 6 under mild (RT, pH = 7.35) conditions.
Highly sensitive: The azido analogue of methionine, azidohomoalanine (see picture), is shown to be a sensitive IR probe of protein structure, folding, and electrostatics, as demonstrated for ...ribosomal protein NTL9. It can be readily incorporated in to proteins, and the azido frequency is significantly blue‐shifted in the thermally unfolded state.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
We present a simple, yet flexible microfluidic mixer with a demonstrated mixing time as short as 80 μs that is widely accessible because it is made of commercially available parts. To simplify the ...study of fast protein dynamics, we have developed an inexpensive continuous-flow microfluidic mixer, requiring no specialized equipment or techniques. The mixer uses three-dimensional, hydrodynamic focusing of a protein sample stream by a surrounding sheath solution to achieve rapid diffusional mixing between the sample and sheath. Mixing initiates the reaction of interest. Reactions can be spatially observed by fluorescence or absorbance spectroscopy. We characterized the pixel-to-time calibration and diffusional mixing experimentally. We achieved a mixing time as short as 80 μs. We studied the kinetics of horse apomyoglobin (apoMb) unfolding from the intermediate (I) state to its completely unfolded (U) state, induced by a pH jump from the initial pH of 4.5 in the sample stream to a final pH of 2.0 in the sheath solution. The reaction time was probed using the fluorescence of 1-anilinonaphthalene-8-sulfonate (1,8-ANS) bound to the folded protein. We observed unfolding of apoMb within 760 μs, without populating additional intermediate states under these conditions. We also studied the reaction kinetics of the conversion of pyruvate to lactate catalyzed by lactate dehydrogenase using the intrinsic tryptophan emission of the enzyme. We observe sub-millisecond kinetics that we attribute to Michaelis complex formation and loop domain closure. These results demonstrate the utility of the three-dimensional focusing mixer for biophysical studies of protein dynamics.
Nickel-chelating lipids offer a convenient platform for reversible immobilization of histidine-tagged proteins to liposome surfaces. This interaction recently found utility as a model system for ...studying membrane remodeling triggered by protein crowding. Despite its wide array of utility, the molecular details of transient protein association to the lipid surfaces decorated with such chelator lipids remains poorly understood. In this study, we explore the kinetics of protein–liposome association across a wide concentration range using stopped-flow fluorescence. The fluorescence of histidine-tagged protein containing an intrinsic fluorophore (superfolder green fluorescent protein, SfGFP) was quenched upon binding to Ni–NTA-modified liposomes containing the quencher Dabsyl-PE lipids. Stopped-flow fluorescence reveals a complex, multiexponential binding behavior with a fast (k obs ∼ 10–20 s–1) phase and slower (k obs < 4 s–1) phase. Interestingly, the observed rates for the slower phase increase initially under low concentrations but start decreasing once a critical concentration is reached. Despite differences in the binding time scales, we observe that the trend of decreasing rates is reproducible irrespective of the chelator lipid doping level, protein surface charge, or lipid composition. Consideration of the protein footprint and membrane surface area occupancy leads us to conclude that the multiphasic binding behavior is reflective of protein binding via two distinct binding conformations. We propose that preliminary steps in protein association involve binding of a sterically occlusive side-on conformation followed by reorganization that leads to an end-on conformation with increased packing density. These results are important for the improvement of histidine-tag-based immobilization strategies and offer mechanistic insight into intermediates preceding membrane bending driven by protein crowding.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM
The World Health Organization (WHO) recommends balanced energy and protein (BEP) supplementation be provided to all pregnant women living in undernourished populations, usually defined as having a ...prevalence > 20% of underweight women, to reduce the risk of stillbirths and small-for-gestational-age neonates. Few geographies meet this threshold, however, and a large proportion of undernourished women and those with inadequate gestational weight gain could miss benefiting from BEP. This study compares the effectiveness of individual targeting approaches for supplementation with micronutrient-fortified BEP vs. multiple micronutrient supplements (MMS) alone as control in pregnancy in improving birth outcomes.
The TARGET-BEP study is a four-arm, cluster-randomized controlled trial conducted in rural northwestern Bangladesh. Eligible participants are married women aged 15-35 years old identified early in pregnancy using a community-wide, monthly, urine-test-based pregnancy detection system. Beginning at 12-14 weeks of gestation, women in the study area comprising 240 predefined sectors are randomly assigned to one of four intervention arms, with sector serving as the unit of randomization. The interventions involving daily supplementation through end of pregnancy are as follows: (1) MMS (control); (2) BEP; (3) targeted BEP for those with pre-pregnancy body mass index (BMI) < 18.5 kg/m
and MMS for others; (4) targeted BEP for those with pre-pregnancy BMI < 18.5 kg/m
, MMS for others, and women with inadequate gestational weight gain switched from MMS to BEP until the end of pregnancy. Primary outcomes include birth weight, low birth weight (< 2500 g), and small for gestational age, defined using the 10
percentile of the INTERGROWTH-21st reference, for live-born infants measured within 72 h of birth. Project-hired local female staff visit pregnant women monthly to deliver the assigned supplements, monitor adherence biweekly, and assess weight regularly during pregnancy. Trained data collectors conduct pregnancy outcome assessment and measure newborn anthropometry in the facility or home depending on the place of birth.
This study will assess the effectiveness of targeted balanced energy and protein supplementation to improve birth outcomes among pregnant women in rural Bangladesh and similar settings.
ClinicalTrials.gov NCT05576207. Registered on October 5th, 2022.
Full text
Available for:
IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The folding mechanism of the β-sheet protein CspA, the major cold shock protein of Escherichia coli, was previously reported to be a concerted, two-state process. We have reexamined the folding of ...CspA using multiple spectroscopic probes of the equilibrium transition and laser-induced temperature jump (T-jump) to achieve better time resolution of the kinetics. Equilibrium temperature-dependent Fourier transform infrared (1634 cm–1) and tryptophan fluorescence measurements reveal probe-dependent thermal transitions with midpoints (T m) of 66 ± 1 and 61 ± 1 °C, respectively. Singular-value decomposition analysis with global fitting of the temperature-dependent infrared (IR) difference spectra reveals two spectral components with distinct melting transitions with different midpoints. T-jump relaxation measurements of CspA probed by IR and fluorescence spectroscopy show probe-dependent multiexponential kinetics characteristic of non-two-state folding. The frequency-dependent IR transients all show biphasic relaxation with average time constants of 50 ± 7 and 225 ± 25 μs at a T f of 77 °C and almost equal amplitudes. Similar biphasic kinetics are observed using Trp fluorescence of the wild-type protein and the Y42W and T68W mutants, with comparable lifetimes. All of these observations support a model for the folding of CspA through a compact intermediate state. The transient IR and fluorescence spectra are consistent with a diffuse intermediate having β-turns and substantial β-sheet structure. The loop β3−β4 structure is likely not folded in the intermediate state, allowing substantial solvent penetration into the barrel structure.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM
Dihydrofolate reductase (DHFR) is a well-studied, clinically relevant enzyme known for being highly dynamic over the course of its catalytic cycle. However, the role dynamic motions play in the ...explicit hydride transfer from the nicotinamide cofactor to the dihydrofolate substrate remains unclear because reaction initiation and direct spectroscopic examination on the appropriate time scale for such femtosecond to picosecond motions is challenging. Here, we employ pre-steady-state kinetics to observe the hydride transfer as directly as possible in two different species of DHFR: Escherichia coli and Homo sapiens. While the hydride transfer has been well-characterized in DHFR from E. coli, improvements in time resolution now allow for sub-millisecond dead times for stopped-flow spectroscopy, which reveals that the maximum rate is indeed faster than previously recorded. The rate in the human enzyme, previously only estimated, is also able to be directly observed using cutting-edge stopped-flow instrumentation. In addition to the pH dependence of the hydride transfer rates for both enzymes, we examine the primary H/D kinetic isotope effect to reveal a temperature dependence in the human enzyme that is absent from the E. coli counterpart. This dependence, which appears above a temperature of 15 °C is a shared feature among other hydride transfer enzymes and is also consistent with computational work suggesting the presence of a fast promoting-vibration that provides donor–acceptor compression on the time scale of catalysis to facilitate the chemistry step.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM
Triosephosphate isomerase (TIM) catalyzes the interconversion between dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (GAP) via an enediol(ate) intermediate. The active-site ...residue Glu165 serves as the catalytic base during catalysis. It abstracts a proton from C1 carbon of DHAP to form the reaction intermediate and donates a proton to C2 carbon of the intermediate to form product GAP. Our difference Fourier transform infrared spectroscopy studies on the yeast TIM (YeTIM)/phosphate complex revealed a CO stretch band at 1706 cm–1 from the protonated Glu165 carboxyl group at pH 7.5, indicating that the pK a of the catalytic base is increased by >3.0 pH units upon phosphate binding, and that the Glu165 carboxyl environment in the complex is still hydrophilic in spite of the increased pK a. Hence, the results show that the binding of the phosphodianion group is part of the activation mechanism which involves the pK a elevation of the catalytic base Glu165. The deprotonation kinetics of Glu165 in the μs to ms time range were determined via infrared (IR) T-jump studies on the YeTIM/phosphate and (“heavy enzyme”) U-13C,-15NYeTIM/phosphate complexes. The slower deprotonation kinetics in the ms time scale is due to phosphate dissociation modulated by the loop motion, which slows down by enzyme mass increase to show a normal heavy enzyme kinetic isotope effect (KIE) ∼1.2 (i.e., slower rate in the heavy enzyme). The faster deprotonation kinetics in the tens of μs time scale is assigned to temperature-induced pK a decrease, while phosphate is still bound, and it shows an inverse heavy enzyme KIE ∼0.89 (faster rate in the heavy enzyme). The IR static and T-jump spectroscopy provides atomic-level resolution of the catalytic mechanism because of its ability to directly observe the bond breaking/forming process.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM
A series of viologen related redox mediators of varying reduction potential has been characterized and their utility as electron shuttles between CdSe quantum dots and hydrogenase enzyme has been ...demonstrated. Tuning the mediator LUMO energy optimizes the performance of this hybrid photocatalytic system by balancing electron transfer rates of the shuttle.
The abundance of early stages of the surf silverside Notocheirus hubbsi in nearshore waters of central Chile, collected in samplings set up to assess the lunar cycle during austral spring and summer ...is reported. A total of 19 specimens were collected with light traps, 16 larvae (7.89-16.20 mm SL) in austral spring and 3 juveniles (30.70-34.60 mm SL) in summer. Capture per unit effort (CPUE) varied from 0.33 to 2.00 ind. light trap-1 night-1 during the entire study period (September 2015-February 2016, and September 2016-February 2017). N. hubbsi catches recorded maximum abundance during the new moon and no catches during full moon.
PDF
