Photolithography based patterning of bacteriorhodopsin films

The patterning of photoactive purple membrane (PM) films onto electronic substrates to create a biologically based light detection device was investigated. This research is part of a larger collaborative effort to develop a miniaturized toxin detection platform. This platform will utilize PM films containing the photoactive protein bacteriorhodopsin to convert light energy to electrical energy. Following an effort to pattern PM films using focused ion beam machining, the photolithography based bacteriorhodopsin patterning technique (PBBPT) was developed. This technique utilizes conventional photolithography techniques to pattern oriented PM films onto flat substrates. After the basic patterning process was developed, studies were conducted that confirmed the photoelectric functionality of the PM films after patterning. Several process variables were studied and optimized in order to increase the pattern quality of the PM films. Optical microscopy, scanning electron microscopy, and interferometric microscopy were used to evaluate the PM films produced by the patterning technique. Patterned PM films with lateral dimensions of 15 μm have been demonstrated using this technique. Unlike other patterning techniques, the PBBPT uses standard photolithographic processes that make its integration with conventional semiconductor fabrication feasible. The final effort of this research involved integrating PM films patterned using the PBBPT with PMOS transistors. An indirect integration of PM films with PMOS transistors was successfully demonstrated. This indirect integration used the voltage produced by a patterned PM film under light exposure to modulate the gate of a PMOS transistor, activating the transistor. Following this success, a study investigating how this PM based light detection system responded to variations in light intensity supplied to the PM film. This work provides a successful proof of concept for a portion of the toxin detection platform currently under development.

The effect of long-term water table manipulations on vegetation, pore water, substrate quality, and carbon cycling in a Northern poor fen peatland

Northern peatlands are large reservoirs of soil organic carbon (C). Historically peatlands have served as a sink for C since decomposition is slowed primarily because of a raised water table (WT) that creates anoxic conditions. Climate models are predicting dramatic changes in temperature and precipitation patterns for the northern hemisphere that contain more than 90% of the world’s peatlands. It is uncertain whether climate change will shift northern peatlands from C sequestering systems to a major global C source within the next century because of alterations to peatland hydrology. This research investigated the effects of 80 years of hydrological manipulations on peatland C cycling in a poor fen peatland in northern Michigan. The construction of an earthen levee within the Seney National Wildlife Refuge in the 1930’s resulted in areas of raised and lowered WT position relative to an intermediate WT site that was unaltered by the levee. We established sites across the gradient of long-term WT manipulations to examine how decadal changes in WT position alter peatland C cycling. We quantified vegetation dynamics, peat substrate quality, and pore water chemistry in relation to trace gas C cycling in these manipulated areas as well as the intermediate site. Vegetation in both the raised and lowered WT treatments has different community structure, biomass, and productivity dynamics compared to the intermediate site. Peat substrate quality exhibited differences in chemical composition and lability across the WT treatments. Pore water dissolved organic carbon (DOC) concentrations increased with impoundment and WT drawdown. The raised WT treatment DOC has a low aromaticity and is a highly labile C source, whereas WT drawdown has increased DOC aromaticity. This study has demonstrated a subtle change of the long-term WT position in a northern peatland will induce a significant influence on ecosystem C cycling with implications for the fate of peatland C stocks.

Serine residues 1177/78/82 of the insulin receptor are required for substrate phosphorylation but not autophosphorylation

Serine residues of the human insulin receptor (HIR) may be phosphorylated and negatively regulate the insulin signal. We studied the impact of 16 serine residues in HIR by mutation to alanine and co-overexpression in human embryonic kidney (HEK) 293 cells together with the docking proteins insulin receptor substrate (IRS)-1, IRS-2, or (SHC) Src homologous and collagen-like. As a control, IRS-1 was also cotransfected with an HIR with a juxtamembrane deletion (HIR delta JM) and therefore not containing the domain required for interaction with IRS-1. Coexpression of HIR with IRS-1, IRS-2, and SHC strongly enhanced tyrosine phosphorylation of these proteins. A similar increase in tyrosine phosphorylation was observed in cells overexpressing IRS-1, IRS-2, or SHC together with all HIR mutants except HIR delta JM and a mutant carrying exchanges of serines 1177, 1178, and 1182 to alanine (HIR1177/78/82), although this mutant showed normal autophosphorylation. Analysis of total cell lysates with anti-phosphotyrosine antibodies showed that in addition to the overexpressed substrates, other cellular proteins displayed reduced levels of tyrosine phosphorylation in these cells. To study consequences for phosphatidylinositol 3-kinase (PI 3-kinase) activation, we established stable NIH3T3 fibroblast cell lines overexpressing wild-type HIR, HIR1177/78/82, and other HIR mutants as the control. Again, HIR1177/78/82 showed normal autophosphorylation but showed a clear decrease in tyrosine phosphorylation of endogenous IRS-1 and activation of PI 3-kinase. This decrease in kinase activity also occurred in an in vitro kinase assay towards recombinant IRS-1. Finally, we performed a separation of the phosphopeptides by high-performance liquid chromatography and could not detect any differences in the profiles of HIR and HIR1177/78/82. In conclusion, we have defined a region in HIR that is important for substrate phosphorylation but not autophosphorylation. Therefore, this mutant may provide new insights into the mechanism of kinase activation and substrate phosphorylation.

Fabrication methods for creating flexible polymer substrate sensor tags

The authors describe the design, fabrication, and testing of a passive wireless sensor platform utilizing low-cost commercial surface acoustic wave filters and sensors. Polyimide and polyethylene terephthalate sheets are used as substrates to create a flexible sensor tag that can be applied to curved surfaces. A microfabricated antenna is integrated on the substrate in order to create a compact form factor. The sensor tags are fabricated using 315 MHz surface acoustic wave filters and photodiodes and tested with the aid of a fiber-coupled tungsten lamp. Microwave energy transmitted from a network analyzer is used to interrogate the sensor tag. Due to an electrical impedance mismatch at the SAW filter and sensor, energy is reflected at the sensor load and reradiated from the integrated antenna. By selecting sensors that change electrical impedance based on environmental conditions, the sensor state can be inferred through measurement of the reflected energy profile. Testing has shown that a calibrated system utilizing this type of sensor tag can detect distinct light levels wireless and passively. The authors also demonstrate simultaneous operation of two tags with different center passbands that detects light. Ranging tests show that the sensor tags can operate at a distance of at least 3.6 m.

Substrate binding tunes conformational flexibility and kinetic stability of an amino acid antiporter

We used single molecule dynamic force spectroscopy to unfold individual serine/threonine antiporters SteT from Bacillus subtilis. The unfolding force patterns revealed interactions and energy barriers that stabilized structural segments of SteT. Substrate binding did not establish strong localized interactions but appeared to be facilitated by the formation of weak interactions with several structural segments. Upon substrate binding, all energy barriers of the antiporter changed thereby describing the transition from brittle mechanical properties of SteT in the unbound state to structurally flexible conformations in the substrate-bound state. The lifetime of the unbound state was much shorter than that of the substrate-bound state. This leads to the conclusion that the unbound state of SteT shows a reduced conformational flexibility to facilitate specific substrate binding and a reduced kinetic stability to enable rapid switching to the bound state. In contrast, the bound state of SteT showed an increased conformational flexibility and kinetic stability such as required to enable transport of substrate across the cell membrane. This result supports the working model of antiporters in which alternate substrate access from one to the other membrane surface occurs in the substrate-bound state.

The platelet protein kinase C substrate pleckstrin binds directly to SDPR protein

Pleckstrin is a modular platelet protein consisting of N- and C-terminal pleckstrin homology (PH) domains, a central dishevelled egl10 and pleckstrin (DEP) domain and a phosphorylation region. Following agonist-induced platelet stimulation, dimeric pleckstrin translocates to the plasma membrane, is phosphorylated and then monomerizes. A recent study found that pleckstrin null platelets from a knockout mouse have a defect in granule secretion, actin polymerization and aggregation. However, the mechanism of pleckstrin signaling for this function is unknown. Our recent studies have led to the identification of a novel pleckstrin-binding protein, serum deprivation response protein (SDPR), by co-immunoprecipitation, GST-pulldowns and nanospray quadruple time of flight mass spectrometry. We show that this interaction occurs directly through N-terminal sequences of pleckstrin. Both pleckstrin and SDPR are phosphorylated by protein kinase C (PKC), but the interaction between pleckstrin and SDPR was shown to be independent of PKC inhibition or activation. These results suggest that SDPR may facilitate the translocation of nonphosphorylated pleckstrin to the plasma membrane in conjunction with phosphoinositides that bind to the C-terminal PH domain. After binding of pleckstrin to the plasma membrane, its phosphorylation by PKC exerts downstream effects on platelet aggregation/secretion.