A miniaturized spectrophotometric in situ pH sensor for seawater

Since the Industrial Revolution, the world\u27s oceans have absorbed increasing amounts of CO2 and the resultant changes to the marine carbonate chemical system have reduced the pH by \u3e 0.1 units (~ 30%) in surface waters. This acidification of the oceans has many far reaching impacts on marine life and there is great need of quality instrumentation to assess and follow the changing carbonate system. This MIS project aims to develop a low cost pH sensor with high precision and accuracy for open sea measurements with special emphasis on reduced size and cost. Design effort is based on the commercially available in situ ocean pH sensor, the SAMI-pH. Emphasis on small size and low cost will allow deployment of the sensors on a much wider variety of platforms than are currently viable thus greatly extending the spatial and temporal resolution of ocean acidification measurements. One such platform is NOAAs Global Drifter Program, a network of non-recovered drifting buoys that has potential for ocean carbon cycle research. A prototype instrument was designed, the inexpensive SAMI-pH or iSAMI-pH. This instrument was entered into the Wendy Schmidt Ocean Health (WSOH) XPRIZE. This was an incentivized global competition to spur innovation in pH sensor technology with both accuracy and affordability prize purses totaling $2 million dollars. The affordability purse consisted of three phases of testing that explored accuracy, precision and stability using a variety of tests that spanned 6 months. It progressed from bench testing in a temperature controlled chamber and a 60 day tank test at the Monterey Bay Aquarium Research Institute (MBARI), to a month long deployment in a specially designed tank at the Seattle Aquarium that used the highly variable waters of Puget Sound. In lab testing, the iSAMI showed ± 0.01 accuracy. In the MBARI test tank, the iSAMI showed precision of ± 0.004 pH units and stability of 0.008 pH units per month with validation uncertainty of ± 0.009 pH units. In the coastal trials, the iSAMI again showed a precision of ± 0.004 pH units and a stability of 0.011 pH units per month with a validation uncertainty of ± 0.012 pH units. Stability or drift was statistically indistinguishable from that of the validation measurements. The iSAMI was in excellent agreement with the commercially available SAMI-pH which won the accuracy prize purse of the WSOH XPRIZE. The iSAMI won the affordability prize purse exceeding the performance metrics by several fold

Loop Dynamics of the Extracellular Domain of Human Tissue Factor and Activation of Factor VIIa

In the crystal structure of the complex between the soluble extracellular domain of tissue factor (sTF) and activesite- inhibited VIIa, residues 91 and 92 in the Pro79-Pro92 loop of sTF interact with the catalytic domain of VIIa. It is not known, however, whether this loop has a role in allosteric activation of VIIa. Time-resolved fluorescence anisotropy measurements of probes covalently bound to sTF mutants E84C and T121C show that binding uninhibited Factor VIIa affects segmental motions in sTF. Glu84 resides in the Pro79-Pro92 loop, and Thr121 resides in the turn between the first and second antiparallel b-strands of the sTF subdomain that interacts with the Gla and EGF1 domains of VIIa; neither Glu84 nor Thr121 makes direct contact with VIIa. Probes bound to T121C report limited segmental flexibility in free sTF, which is lost after VIIa binding. Probes bound to E84C report substantial segmental flexibility in the Pro79-Pro92 loop in free sTF, which is greatly reduced after VIIa binding. Thus, VIIa binding reduces dynamic motions in sTF. In particular, the decrease in the Pro79-Pro92 loop motions indicates that loop entropy has a role in the thermodynamics of the protein-protein interactions involved in allosteric control of VIIa activation

Pressure dissociation of integration host factor–DNA complexes reveals flexibility-dependent structural variation at the protein–DNA interface

E. coli Integration host factor (IHF) condenses the bacterial nucleoid by wrapping DNA. Previously, we showed that DNA flexibility compensates for structural characteristics of the four consensus recognition elements associated with specific binding (Aeling et al., J. Biol. Chem. 281, 39236–39248, 2006). If elements are missing, high-affinity binding occurs only if DNA deformation energy is low. In contrast, if all elements are present, net binding energy is unaffected by deformation energy. We tested two hypotheses for this observation: in complexes containing all elements, (1) stiff DNA sequences are less bent upon binding IHF than flexible ones; or (2) DNA sequences with differing flexibility have interactions with IHF that compensate for unfavorable deformation energy. Time-resolved Förster resonance energy transfer (FRET) shows that global topologies are indistinguishable for three complexes with oligonucleotides of different flexibility. However, pressure perturbation shows that the volume change upon binding is smaller with increasing flexibility. We interpret these results in the context of Record and coworker's model for IHF binding (J. Mol. Biol. 310, 379–401, 2001). We propose that the volume changes reflect differences in hydration that arise from structural variation at IHF–DNA interfaces while the resulting energetic compensation maintains the same net binding energy

Autonomous Optofluidic Chemical Analyzers for Marine Applications: Insights from the Submersible Autonomous Moored Instruments (SAMI) for pH and pCO2

The commercial availability of inexpensive fiber optics and small volume pumps in the early 1990's provided the components necessary for the successful development of low power, low reagent consumption, autonomous optofluidic analyzers for marine applications. It was evident that to achieve calibration-free performance, reagent-based sensors would require frequent renewal of the reagent by pumping the reagent from an impermeable, inert reservoir to the sensing interface. Pumping also enabled measurement of a spectral blank further enhancing accuracy and stability. The first instrument that was developed based on this strategy, the Submersible Autonomous Moored Instrument for CO2 (SAMI-CO2), uses a pH indicator for measurement of the partial pressure of CO2 (pCO2). Because the pH indicator gives an optical response, the instrument requires an optofluidic design where the indicator is pumped into a gas permeable membrane and then to an optical cell for analysis. The pH indicator is periodically flushed from the optical cell by using a valve to switch from the pH indicator to a blank solution. Because of the small volume and low power light source, over 8,500 measurements can be obtained with a ~500 mL reagent bag and 8 alkaline D-cell battery pack. The primary drawback is that the design is more complex compared to the single-ended electrode or optode that is envisioned as the ideal sensor. The SAMI technology has subsequently been used for the successful development of autonomous pH and total alkalinity analyzers. In this manuscript, we will discuss the pros and cons of the SAMI pCO2 and pH optofluidic technology and highlight some past data sets and applications for studying the carbon cycle in aquatic ecosystems

Pressure dissociation of integration host factor-DNA complexes reveals flexibility-dependent structural variation at the protein-DNA interface

E. coli Integration host factor (IHF) condenses the bacterial nucleoid by wrapping DNA. Previously, we showed that DNA flexibility compensates for structural characteristics of the four consensus recognition elements associated with specific binding (Aeling et al., J. Biol. Chem. 281, 39236–39248, 2006). If elements are missing, high-affinity binding occurs only if DNA deformation energy is low. In contrast, if all elements are present, net binding energy is unaffected by deformation energy. We tested two hypotheses for this observation: in complexes containing all elements, (1) stiff DNA sequences are less bent upon binding IHF than flexible ones; or (2) DNA sequences with differing flexibility have interactions with IHF that compensate for unfavorable deformation energy. Time-resolved Förster resonance energy transfer (FRET) shows that global topologies are indistinguishable for three complexes with oligonucleotides of different flexibility. However, pressure perturbation shows that the volume change upon binding is smaller with increasing flexibility. We interpret these results in the context of Record and coworker's model for IHF binding (J. Mol. Biol. 310, 379–401, 2001). We propose that the volume changes reflect differences in hydration that arise from structural variation at IHF–DNA interfaces while the resulting energetic compensation maintains the same net binding energy

Electrophoretic mobility-shift assay of IHF binding to oligonucleotide A

<p><b>Copyright information:</b></p><p>Taken from "Pressure dissociation of integration host factor–DNA complexes reveals flexibility-dependent structural variation at the protein–DNA interface"</p><p></p><p>Nucleic Acids Research 2007;35(6):1761-1772.</p><p>Published online 25 Feb 2007</p><p>PMCID:PMC1874591.</p><p>© 2007 The Author(s)</p>2. IHF concentrations in Lanes 1–10 are 0, 20, 40, 60, 81, 99, 120, 165, 201 and 240 nM, respectively. This pseudo-color image was generated by coloring the emission collected through a 520-nm band pass filter green (FAM fluorescence) and coloring the emission collected through a 580-nm band pass filter red (TAMRA fluorescence). With excitation at 488 nm, the unliganded oligonucleotide is green, reflecting only FAM fluorescence. The yellow color of the mobility-shifted band results from a combination of green and red fluorescence, indicating efficient FRET due to the wrapped DNA in the bound complex

Panel A shows the pressure FRET ratio baseline data (open circle) and polynomial smoothing curve (solid line for oligonucleotide A

<p><b>Copyright information:</b></p><p>Taken from "Pressure dissociation of integration host factor–DNA complexes reveals flexibility-dependent structural variation at the protein–DNA interface"</p><p></p><p>Nucleic Acids Research 2007;35(6):1761-1772.</p><p>Published online 25 Feb 2007</p><p>PMCID:PMC1874591.</p><p>© 2007 The Author(s)</p>6 in the absence of IHF compared with unprocessed data for 10 nM DNA and 25 nM IHF (filled square) (10 mM Tris pH 8.0, 100 mM NaCl and 1 mM EDTA). Panel B compares fraction bound for oligonucleotides A.2 (filled diamond) and A.6 (filled square) at 10 nM DNA, 25 nM IHF, i.e. same A.6 data as panel A and same reaction conditions. Solid and dashed curves are the fits and 95% confidence intervals to these individual experiments, using equations () as described in the text

Loop Dynamics of the Extracellular Domain of Human Tissue Factor and Activation of Factor VIIa

In the crystal structure of the complex between the soluble extracellular domain of tissue factor (sTF) and active-site-inhibited VIIa, residues 91 and 92 in the Pro79-Pro92 loop of sTF interact with the catalytic domain of VIIa. It is not known, however, whether this loop has a role in allosteric activation of VIIa. Time-resolved fluorescence anisotropy measurements of probes covalently bound to sTF mutants E84C and T121C show that binding uninhibited Factor VIIa affects segmental motions in sTF. Glu84 resides in the Pro79-Pro92 loop, and Thr121 resides in the turn between the first and second antiparallel β-strands of the sTF subdomain that interacts with the Gla and EGF1 domains of VIIa; neither Glu84 nor Thr121 makes direct contact with VIIa. Probes bound to T121C report limited segmental flexibility in free sTF, which is lost after VIIa binding. Probes bound to E84C report substantial segmental flexibility in the Pro79-Pro92 loop in free sTF, which is greatly reduced after VIIa binding. Thus, VIIa binding reduces dynamic motions in sTF. In particular, the decrease in the Pro79-Pro92 loop motions indicates that loop entropy has a role in the thermodynamics of the protein-protein interactions involved in allosteric control of VIIa activation