• Experimental Neurobiology
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• v.26 no.5
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• pp.241-251
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• 2017

Saturation mutagenesis was performed on a single position in the voltage-sensing domain (VSD) of a genetically encoded voltage indicator (GEVI). The VSD consists of four transmembrane helixes designated S1-S4. The V220 position located near the plasma membrane/extracellular interface had previously been shown to affect the voltage range of the optical signal. Introduction of polar amino acids at this position reduced the voltage-dependent optical signal of the GEVI. Negatively charged amino acids slightly reduced the optical signal by 33 percent while positively charge amino acids at this position reduced the optical signal by 80%. Surprisingly, the range of V220D was similar to that of V220K with shifted optical responses towards negative potentials. In contrast, the V220E mutant mirrored the responses of the V220R mutation suggesting that the length of the side chain plays in role in determining the voltage range of the GEVI. Charged mutations at the 219 position all behaved similarly slightly shifting the optical response to more negative potentials. Charged mutations to the 221 position behaved erratically suggesting interactions with the plasma membrane and/or other amino acids in the VSD. Introduction of bulky amino acids at the V220 position increased the range of the optical response to include hyperpolarizing signals. Combining The V220W mutant with the R217Q mutation resulted in a probe that reduced the depolarizing signal and enhanced the hyperpolarizing signal which may lead to GEVIs that only report neuronal inhibition.

• Journal of Microbiology and Biotechnology
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• v.25 no.4
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• pp.503-510
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• 2015

The iLOV protein belongs to a family of blue-light photoreceptor proteins containing a light-oxygen-voltage sensing domain with a noncovalently bound flavin mononucleotide (FMN) as its chromophore. Owing to advantages such as its small size, oxygen-independent nature, and pH stability, iLOV is an ideal candidate over other reporter fluorescent proteins such as GFP and DsRed. Here, for the first time, we describe the feasibility of applying LOV domain-based fluorescent iLOV as a metal sensor by measuring the fluorescence quenching of a protein with respect to the concentration of metal ions. In the present study, we demonstrated the inherent copper sensing property of the iLOV protein and identified the possible amino acids responsible for metal binding. The fluorescence quenching upon exposure to Cu²⁺ was highly sensitive and exhibited reversibility upon the addition of the metal chelator EDTA. The copper binding constant was found to be 4.72 ± 0.84 µM. In addition, Cu²⁺-bound iLOV showed high fluorescence quenching at near physiological pH. Further computational analysis yielded a better insight into understanding the possible amino acids responsible for Cu²⁺ binding with the iLOV protein.

• Proceedings of the KIPE Conference
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• 2004.07b
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• pp.587-591
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• 2004

This paper proposes a novel current compensation technique that can eliminate the harmonic currents included in line currents without computation of harmonic current components. A current controller with fast dynamics for an active filter is described. Harmonic currents are directly controlled without the need for sensing and computing the harmonic current of the load current, thus simplifying the control system. Current compensation is done in the time domain, allowing a fast time response. The DC voltage control loop keeps the voltage across the DC capacitor constant. High power factor control by an active filter is described. All control functions are implemented in software using a single-chip microcontroller, thus simplifying the control circuit. Any current-controlled synchronous rectifier can be used as a shunt active filter through only the simple modification of the software and the addition of current sensors. It is shown through experimental results that the proposed controller gives good performance for the shunt active filter.

• Structural Monitoring and Maintenance
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• v.1 no.2
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• pp.183-195
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• 2014

Scour is one of the leading causes of overwater bridge failures worldwide. While monitoring systems have already been implemented or are still being developed, they suffer from limitations such as high costs, inaccuracies, and low reliability, among others. Also, most sensors only measure scour depth at one location and near the pier. Thus, the objective is to design a simple, low cost, scour hole topography monitoring system that could better characterize the entire depth, shape, and size of bridge scour holes. The design is based on burying a robust, waterproofed, piezoelectric sensor strip in the streambed. When scour erodes sediments to expose the sensor, flowing water excites it to cause the generation of time-varying voltage signals. An algorithm then takes the time-domain data and maps it to the frequency-domain for identifying the sensor's resonant frequency, which is used for calculating the exposed sensor length or scour depth. Here, three different sets of tests were conducted to validate this new technique. First, a single sensor was tested in ambient air, and its exposed length was varied. Upon verifying the sensing concept, a waterproofed prototype was buried in soil and tested in a tank filled with water. Sensor performance was characterized as soil was manually eroded away, which simulated various scour depths. The results confirmed that sensor resonant frequencies decreased with increasing scour depths. Finally, a network of 11 sensors was configured to form a distributed monitoring system in the lab. Their exposed lengths were adjusted to simulate scour hole formation and evolution. Results showed promise that the proposed sensing system could be scaled up and used for bridge scour topography monitoring.

• Journal of Life Science
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• v.21 no.7
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• pp.1032-1038
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• 2011

Recently identified Ciona intestinalis voltage sensor-containing phosphatase (Ci-VSP) consists of an ion channel-like transmembrane domain (VSD) and a phosphatase-like domain. Ci-VSP senses the change of membrane potential by its VSD and works as a phosphoinositide phosphatase by its phosphatase domain. In this study, we present the construction of His-tagged phosphatase-like domain of Ci-VSP, its recombinant expression and purification, and its enzymatic activity behavior in order to examine the biochemical behavior of phosphatase domain of Ci-VSP without interference. We found that Ci-VSP(248-576)-His can be eluted with an elution buffer containing 25 mM NaCl and 100 mM imidazole during His-tag purification. In addition, we found the proper measurement condition for kinetics study of Ci-VSP(248-576)-His against p-nitrophenyl phosphate (pNPP). We measured the kinetic constant of Ci-VSP(248-576)-His at $37^{\circ}C$, pH 5.0 or 5.5, under 30 min of reaction time, and less than $2.0\;{\mu}g$ of protein amount. With these conditions, we acquired that Ci-VSP(248-576)-His has $K_m$ of $354{\pm}0.143\;{\mu}M$, $V_{max}$ of $0.0607{\pm}0.0137\;{\mu}mol$/min/mg and $k_{cat}$ of $0.359{\pm}0.009751\;min^{-1}$ for pNPP dephosphorylation. Therefore, we produced a pure form of Ci-VSP(248-576)-His, and this showed a higher activity against pNPP. This purified protein will provide the road to a structural investigation on an interesting protein, Ci-VSP.

• Smart Structures and Systems
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• v.29 no.2
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• pp.267-278
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• 2022

Pre-stressed concrete circular spun piles are widely used in various infrastructure projects around the world and offer an economical deep foundation system with consistent and superior quality compared to cast in-situ and other concrete piles. Conventional methods for measuring the lateral response of piles have been limited to conventional instrumentation, such as electrical based gauges and pressure transducers. The problem with existing technology is that the sensors are not able to assist in recording the lateral stiffness changes of the pile which varies along the length depending on the distribution of the flexural moments and appearance of tensile cracks. This paper describes a full-scale bending test of a 1-m diameter spun pile of 30 m long and instrumented using advanced fibre optic distributed sensor, known as Brillouin Optical Time Domain Analysis (BOTDA). Optical fibre sensors were embedded inside the concrete during the manufacturing stage and attached on the concrete surface in order to measure the pile's full-length flexural behaviour under the prescribed serviceability and ultimate limit state. The relationship between moments-deflections and bending moments-curvatures are examined with respect to the lateral forces. Tensile cracks were measured and compared with the peak strains observed from BOTDA data which corroborated very well. By analysing the moment-curvature response of the pile, the structure can be represented by two bending stiffness parameters, namely the pre-yield (EI) and post-yield (EI_cr), where the cracks reduce the stiffness property by 89%. The pile deflection profile can be attained from optical fibre data through closed-form solutions, which generally matched with the displacements recorded by Linear Voltage Displacement Transducers (LVDTs).