Tag Placement Research Articles

Soluble expression, purification, and secondary structure determination of human MESP1 transcription factor.

Transcription factor MESP1 is a crucial factor regulating cardiac, hematopoietic, and skeletal myogenic development. Besides, it also contributes to the generation of functional cardiomyocytes. Here, we report the soluble expression and purification of the full-length human MESP1 protein from the heterologous system, which can be delivered into the target mammalian cells. To generate this biological macromolecule, we cloned its codon-optimized gene sequence fused to a nuclear localization sequence, a cell-penetrating peptide, and a His-tag into the protein expression vector and expressed in the bacterial system (E. coli strain BL21(DE3)). Subsequently, we have screened and identified the optimal expression parameters to obtain this recombinant fusion protein in soluble form from E. coli and examined its expression concerning the placement of fusion tags at either terminal. Further, we have purified this recombinant fusion protein to homogeneity under native conditions. Notably, this purified fusion protein has maintained its secondary structure after purification, primarily comprising α-helices and random coils. This molecular tool can potentially replace its genetic and viral forms in the cardiac reprogramming of fibroblasts to induce a cardiac transcriptional profile in an integration-free manner and elucidating its role in various biological processes and diseases. KEY POINTS: • Screening of the suitable gene construct was performed and identified. • Screening of optimal expression conditions was performed and identified. • Native purification of recombinant human MESP1 protein from E. coli was performed. • Recombinant MESP1 protein has retained its secondary structure after purification.

Passive Wireless Dual-Tag UHF RFID Sensor System for Surface Crack Monitoring.

The generation and propagation of cracks are critical factors that affect the performance and life of large structures. Therefore, in order to minimize maintenance costs and ensure personal safety, it is necessary to monitor key structures. The sensor based on ultra-high frequency radio frequency identification (UHF RFID) antenna has the advantages of passive wireless, low cost, and great potential in the field of metallic structure health monitoring. In this paper, aimed at the key parts of a metallic structure, a dual-tag system is used for crack monitoring. In conjunction with mode analysis, the principles of the sensing tag and the coupling principles of the dual-tag are analyzed. Considering that the dual-tag is placed in different methods, the effect of mutual coupling on the sensing performance of the tag is studied. The results show that the frequency of the sensing tag can be tuned by adding the interference tag, and the dual-tag sensor system has reasonable sensitivity. The results also provide potential guidance for the optimal placement of multiple tags in the near-field region.

Remoras pick where they stick on blue whales.

Animal-borne video recordings from blue whales in the open ocean show that remoras preferentially adhere to specific regions on the surface of the whale. Using empirical and computational fluid dynamics analyses, we show that remora attachment was specific to regions of separating flow and wakes caused by surface features on the whale. Adhesion at these locations offers remoras drag reduction of up to 71-84% compared with the freestream. Remoras were observed to move freely along the surface of the whale using skimming and sliding behaviors. Skimming provided drag reduction as high as 50-72% at some locations for some remora sizes, but little to none was available in regions where few to no remoras were observed. Experimental work suggests that the Venturi effect may help remoras stay near the whale while skimming. Understanding the flow environment around a swimming blue whale will inform the placement of biosensor tags to increase attachment time for extended ecological monitoring.

Short-term responses of sperm whales Physeter macrocephalus to the attachment of suction cup tags

Animal-mounted data logging devices are used to study the behaviour, physiology, and ecology of free-ranging marine mammals, as well as their reactions to controlled exposures. It is important to consider whether collected data are representative of natural behaviour or biased by responses to tagging. In species with stereotypical diving behaviour, tagging responses can be quantified by identifying anomalous dives. Data from 36 suction cup tag deployments on sperm whalesPhyseter macrocephalusfrom 4 locations were analysed to consider whether tagging effects were evident within 5 dive parameters: maximum dive depth, dive duration, descent speed, depth difference between start of clicking and first prey capture attempt, and buzz rate. Linear mixed models were generated for each response parameter and covariates for dive index were added to assess whether model fit improved when the order of dives was taken into account. Time-decaying tagging effects were noted in maximum dive depth (first dives were 25% shallower than average) and buzz rate (first dives contained 34% fewer buzzes per minute than average). In the Azores, the first 3 dives subsequent to tag attachment featured faster descent speeds than average. The whales were likely responding to the cumulative ‘dose’ of research activity at the surface: multiple boat approaches, tag placement, and general disturbance. Disturbance should be minimised during tagging, and the extent and duration of responses should be quantified. Modelling of quantified tagging responses could enable correction of these responses in tag data.

Variations in received levels on a sound and movement tag on a singing humpback whale: Implications for caller identification.

Bio-logging devices are advancing the understanding of marine animal behavior, but linking sound production and behavior of individual baleen whales is still unreliable. Tag placement potentially within the near field of the sound source creates uncertainty about how tagged animal sounds will register on recorders. This study used data from a tagged singing humpback whale to evaluate this question of how sound levels present on a tag when calls are produced by a tagged animal. Root-mean-square (rms) received levels (RLs) of song units ranged from 112 to 164 dB re 1 μPa rms, with some, but not all, of the lower frequency units registering on the tag's 800 Hz accelerometer sensor. Fifty-nine percent of recorded units measured lower acoustic RLs than previously reported source levels for humpback song, but signal-to-noise ratios (SNRs) were 30-45 dB during periods of the dive with low noise. This research highlights that tag RL does not alone predict caller identity, argues for higher SNR thresholds if using SNR to inform decisions about the source of a call, and provides a baseline for future research identifying diagnostic properties of tagged animal calls in cetacean bioacoustic tag datasets.

Impact of satellite linked radio transmitting (SLRT) tags on the dorsal fin of subadult and adult white sharks (Carcharodon carcharias)

Satellite linked radio transmitting (SLRT) tags provide long-term, high accuracy, near real-time tracking data for marine wildlife. Adult white sharks (Carcharodon carcharias Linnaeus, 1758) in the northeastern Pacific at both Guadalupe Island, Mexico and Southeast Farallon Island, USA were tagged with SLRT oval and inline finmout tags. These tags provided up to 7.4 yrs of tracking data. A previous study showed structural dorsal fin damage for SLRT tagged juvenile and subadult white sharks off South Africa. Sharks tagged in the northeastern Pacific were resighted between 1 and 11 yrs post tagging and included 10 adults and 2 subadults. Sharks that were resighted did not exhibit significant fin deformation, although in one case the oval finmount tag did cause bending of the dorsal fin due to tag shape, weight, and placement of the tag on the dorsal fin. Four inline tags came off after deployments of at least 2.2 to 3.7 yrs (based on date of last message received), and two of these caused a tear to the trailing edge of the dorsal fin upon release. Overall, SLRT tags provided long-term tracking data and there was no apparent impact on the behavior or migration cycles of the sharks. Serious dorsal fin deformation seems to occur in the case of juvenile and subadult white sharks where the fin is rapidly growing and thus for tracking juvenile white sharks, alternate methods should be considered.

Implications of tag positioning and performance on the analysis of cetacean movement

BackgroundSatellite-linked animal-borne tags enable the study of free-ranging marine mammals. These tags can only transmit data while their antenna is above the surface for a sufficient amount of time. Thus, the position of the tag on the animal’s body will likely influence the quality and the quantity of location estimates. We explored the effects of tag placement and tag performance on the analysis of cetacean movement, by deploying two identical Argos tags 33 cm apart on the dorsal fin of a male killer whale in Norway in January 2017.ResultsThe highest placed (top tag) generated 540 location estimates, while the lowest placed tag (bottom tag) generated 245 locations. In addition, the top tag generated locations of higher quality, with less than 50% of the location estimates in Argos class B (the class with the highest estimated uncertainty), compared to the bottom tag (90% Argos class B locations). The distance between two reconstructed paths ranged from 81 m to 31 km. The path based on the top tag was 1.5 times longer, yielding a higher average speed and more extreme turning angles. The estimated uncertainty around the top track was smaller than that of the bottom track. Switches between searching and travelling behaviour, based on data from the top and the bottom tags, occurred at different positions and times. A significant relationship between core utilization areas and a simulated environmental variable was detectable at a finer spatial scale using data collected by the top tag compared to the bottom tag. A literature search yielded no evidence that tag performance or tag placement is commonly discussed in killer whale telemetry articles.ConclusionsThe differences in quality and quantity of location estimates from our two tags had a substantial effect on derived movement metrics, behavioural inferences and significance of a simulated environmental variable. These differences in tag performance are likely linked to the height difference in tag placement of 33 cm. We suggest that tag positioning on free-ranging marine mammals and tag performance should be considered as a covariate in telemetry studies, especially at a fine scale.

TIN-based Tag Map Layout

ABSTRACTWith the widespread use of tag clouds, multiple map-based variations have been proposed. Like standard tag clouds (also called word clouds), these ‘tag maps’ all share the basic strategy of displaying words within a ‘geographic space’ and scaling the word size to depict frequency (or importance) of those words within some dataset. While some tag maps simply plot a standard tag cloud on top of a map, the subset of tag maps we focus on here are those in which the collection of words are displayed within bounded geographic regions (often of irregular shape) that the words are relevant for. For this form of tag map, map scale and polygon shape add constraints to word size and position that have not been considered in most prior approaches to tag map word layout. In this paper, we present a layout strategy for tag map generation that includes consideration of the shape and size of the geographical regions acting as containers for the tags. The method introduced here uses a triangulated irregular network (TIN) to subdivide the geographical region into many triangle subareas, with the centroid of each triangle being a potential location to centre a tag on. All the triangles are sorted by their area and all the tags are sorted by their weight value (e.g. frequency, importance or popularity). Positioning of tags is undertaken sequentially from most important (or frequent or popular) with potential locations being the TIN triangle centroids (tried from largest to smallest triangle). After each tag placement, the TIN is recalculated to integrate the tag centroid and bounding corners into the TIN creation. The limited whitespace in the geographical region, at any specific scale, is used fully by dynamically adjusting the font size along with the number and the direction of tags. The method can be applied to add tags within geographic polygons that are convex, concave and other more complex regions containing holes or islands.

Experimental Evaluation of UWB Indoor Positioning for Sport Postures.

Radio frequency (RF)-based indoor positioning systems (IPSs) use wireless technologies (including Wi-Fi, Zigbee, Bluetooth, and ultra-wide band (UWB)) to estimate the location of persons in areas where no Global Positioning System (GPS) reception is available, for example in indoor stadiums or sports halls. Of the above-mentioned forms of radio frequency (RF) technology, UWB is considered one of the most accurate approaches because it can provide positioning estimates with centimeter-level accuracy. However, it is not yet known whether UWB can also offer such accurate position estimates during strenuous dynamic activities in which moves are characterized by fast changes in direction and velocity. To answer this question, this paper investigates the capabilities of UWB indoor localization systems for tracking athletes during their complex (and most of the time unpredictable) movements. To this end, we analyze the impact of on-body tag placement locations and human movement patterns on localization accuracy and communication reliability. Moreover, two localization algorithms (particle filter and Kalman filter) with different optimizations (bias removal, non-line-of-sight (NLoS) detection, and path determination) are implemented. It is shown that although the optimal choice of optimization depends on the type of movement patterns, some of the improvements can reduce the localization error by up to 31%. Overall, depending on the selected optimization and on-body tag placement, our algorithms show good results in terms of positioning accuracy, with average errors in position estimates of 20 cm. This makes UWB a suitable approach for tracking dynamic athletic activities.

Evaluating the placement of PIT tags in tropical river fishes: a case study involving two Mekong River species

Fish communities are becoming increasingly threatened in many tropical river-floodplain systems due to the construction of dams and other physical barriers. Efficient tagging techniques are urgently needed to better understand the movement ecology of tropical river-floodplain species — both at a fundamental level and in response to the effects of barriers. Passive Integrated Transponder (PIT) tagging has been successfully used to quantify fish movements in many temperate riverine species, but its effectiveness on tropical riverine species remains largely untested. We investigated the potential use of PIT tags in two tropical species from the Mekong River — Pangasianodon hypophthalmus (Striped catfish) and Hypsibarbus malcolmi (Goldfin tinfoil barbs). Two separate, but concurrent, 50-day experiments were conducted on the two species to determine whether (1) the PIT tags can be retained within the fish, without affecting their mortality or growth, and (2) the outcomes for tag retention, fish mortality, and/or fish growth are influenced by the location of the tags in the fish. Results indicated that, for both species, PIT tags can be retained in the chest, gut or shoulder without affecting mortality or growth. This suggests that PIT tags could be successfully used in a range of body locations in Striped catfish and Goldfin tinfoil barbs in the Mekong River. However, the Mekong fishery is a highly important food source for the people of its neighbouring countries — thus, the most suitable tag location in large-bodied species would be the gut region, as the gut, and tag, are most likely to be removed prior to human consumption.

Development of cell-penetrating bispecific antibodies targeting the N-terminal domain of androgen receptor for prostate cancer therapy.

Castration-resistant prostate cancer cells exhibit continued androgen receptor signaling in spite of low levels of ligand. Current therapies to block androgen receptor signaling act by inhibiting ligand production or binding. We developed bispecific antibodies capable of penetrating cells and binding androgen receptor outside of the ligand-binding domain. Half of the bispecific antibody molecule consists of a single-chain variable fragment of 3E10, an anti-DNA antibody that enters cells. The other half is a single-chain variable fragment version of AR441, an anti-AR antibody. The resulting 3E10-AR441 bispecific antibody enters human LNCaP prostate cells and accumulates in the nucleus. The antibody binds to wild-type, mutant and splice variant androgen receptor. Binding affinity of 3E10-AR441 to androgen receptor (284 nM) was lower than that of the parental AR441 mAb (4.6 nM), but could be improved (45 nM) through alternative placement of the affinity tags, and ordering of the VH and VK domains. The 3E10-AR441 bispecific antibody blocked genomic signaling by wild-type or splice variant androgen receptor in LNCaP cells. It also blocked non-genomic signaling by the wild-type receptor. Furthermore, bispecific antibody inhibited the growth of C4-2 prostate cancer cells under androgen-stimulated conditions. The 3E10-AR441 biAb can enter prostate cancer cells and inhibits androgen receptor function in a ligand-independent manner. It may be an attractive prototype agent for prostate cancer therapy.

Controlling Protein Surface Orientation by Strategic Placement of Oligo-Histidine Tags

We report oriented immobilization of proteins using the standard hexahistidine (His6)-Ni2+:NTA (nitrilotriacetic acid) methodology, which we systematically tuned to give control of surface coverage. Fluorescence microscopy and surface plasmon resonance measurements of self-assembled monolayers (SAMs) of red fluorescent proteins (TagRFP) showed that binding strength increased by 1 order of magnitude for each additional His6-tag on the TagRFP proteins. All TagRFP variants with His6-tags located on only one side of the barrel-shaped protein yielded a 1.5 times higher surface coverage compared to variants with His6-tags on opposite sides of the so-called β-barrel. Time-resolved fluorescence anisotropy measurements supported by polarized infrared spectroscopy verified that the orientation (and thus coverage and functionality) of proteins on surfaces can be controlled by strategic placement of a His6-tag on the protein. Molecular dynamics simulations show how the differently tagged proteins reside at the surface in “end-on” and “side-on” orientations with each His6-tag contributing to binding. Also, not every dihistidine subunit in a given His6-tag forms a full coordination bond with the Ni2+:NTA SAMs, which varied with the position of the His6-tag on the protein. At equal valency but different tag positions on the protein, differences in binding were caused by probing for Ni2+:NTA moieties and by additional electrostatic interactions between different fractions of the β-barrel structure and charged NTA moieties. Potential of mean force calculations indicate there is no specific single-protein interaction mode that provides a clear preferential surface orientation, suggesting that the experimentally measured preference for the end-on orientation is a supra-protein, not a single-protein, effect.

Assessment of wound healing of tagged gray (Eschrichtius robustus) and blue (Balaenoptera musculus) whales in the eastern North Pacific using long‐term series of photographs

AbstractTags have been used to examine migration routes and habitat use of large whales for >40 yr, however, evaluation of tag wound healing has largely been short‐term, anecdotal or generalized. This study developed methods for systematic photographic assessment of long‐term external consequences of tag placement, to determine potential differences in wound healing between species and tag types and thus advise future tagging efforts to possibly minimize undesirable side effects. Tag site appearance and healing characteristics were evaluated by two reviewers and a time series evaluated by five veterinarians from photographs during 995 postdeployment encounters with 34 gray and 63 blue whales tagged in the North Pacific. Blue whale resightings were less frequent, but spanned a longer time period due to earlier tag deployments than the more frequent gray whale follow‐up observations. Swelling occurred in 74% of reencountered gray whales, with the highest frequency 6 mo postdeployment. Swellings were common in blue whales with early tag designs but rare with current models. Depressions occurred in 82% of gray and 71% of blue whales. This study demonstrates the value of follow‐up studies of tagged animals and systematic scoring of photographs to quantitatively compare tag response.

An investigation of factors influencing performance of Radio Frequency Identification (RFID): applications in transportation

This study investigates the effect of speed, tag and reader location, vehicle speed, and over-shadowing on RFID performance. The study tested two reader/antenna heights and three tag locations. Three vehicle speeds were tested for each tag location including the simultaneous placement of RFID tags on the vehicle. The study results show, with tags individually placed on the vehicle, the appropriate position for a tag is on the vehicle windshield, and the reader/antenna set at 7ft above the ground on a signpost. Placing multiple tags on the vehicle simultaneously improved the signal strength and detection rates. Vehicle speed had a negative effect on tag detection rate; that is, at higher vehicle speeds the detection rate decreased. With respect to horizontal distance between the reader and the tag, the results show that, the closer the tag and reader are to each other the higher the signal strength and so is the detection rate.

Post-tagging behaviour and habitat use in shortnose sturgeon measured with high-frequency accelerometer and PSATs

External tagging of fish using pop-up satellite tags (PSATs) can cause trauma and stress associated with capture, handling, tagging injury and tag placement that impedes body function and mobility, and these can affect the behaviour of the fish. We measured short-term (days) post-release behavioural response of estuarine shortnose sturgeon to tagging with PSATs and high-frequency accelerometers. We developed a secure, low-noise attachment method for high-resolution (50 Hz) accelerometer PSATs in shortnose sturgeon. The archived time series of acceleration was used to determine sturgeon post-tagging activity, estimate swimming speed, erratic behaviour and behavioural clusters in relation to ambient light level and temperature, depth and tidal flow. Short-term (hours) post-release response was characterized by resting periods on the river bottom and decreased swimming activity that was associated with individual-specific modulations in the swimming gait and high-energy burst acceleration movements. Locomotion routines suggested a relation to tidal flow, yet the short-term time series did not allow for routine movements to be classified. The approach used provides a useful method of revealing behavioural modifications during the post-release recovery period of PSAT-tagged fish. We discuss how short-term (acute) effects on behaviour and potential longer-term (chronic) effects on survival are especially relevant in tagging studies.

Quantitative Measures of Anthropogenic Noise on Harbor Porpoises: Testing the Reliability of Acoustic Tag Recordings.

In recent years, several sound and movement recording tags have been developed to sample the acoustic field experienced by cetaceans and their reactions to it. However, little is known about how tag placement and an animal's orientation in the sound field affect the reliability of on-animal recordings as proxies for actual exposure. Here, we quantify sound exposure levels recorded with a DTAG-3 tag on a captive harbor porpoise exposed to vessel noise in a controlled acoustic environment. Results show that flow noise is limiting onboard noise recordings, whereas no evidence of body shading has been found for frequencies of 2-20 kHz.

In the belly of the beast: resolving stomach tag data to link temperature, acceleration and feeding in white sharks (Carcharodon carcharias)

Motion detecting archival data loggers such as accelerometers have become increasingly important in animal biotelemetry and offer unique insights into animal behavior, energetics, and kinematics. However, challenges remain for successful deployment and interpretation of data from captive and wild animals. Accelerometer sensors require being packaged in an archival tag that has a firm attachment in a fixed (known) orientation to accurately measure the relevant motion of the animal. This requirement can lead to handling stress and attachment techniques that can affect the tagged animal’s natural behavior and welfare, and lead to behavioral artifacts in the data. Accelerometer data also require careful interpretation to correctly identify behavioral events of interest such as foraging. For endothermic species, changes in stomach temperature can produce temperature signatures indicative of foraging events. In this paper, we present a novel method for recording foraging events in free-swimming white sharks. We used a combination of accelerometer loggers and pop-up archival transmitting (PAT) tags (MK10, Wildlife Computers) to examine the feeding and kinematics of white sharks (Carcharodon carcharias) in the wild. We validated feeding results using a captive juvenile white shark where controlled feeding experiments could be conducted in an aquarium setting at the Monterey Bay Aquarium. We fed data logger instrument packages to eight free-swimming white sharks. Deployment durations ended naturally when the package was regurgitated and ranged from 2 to 12 days. While inside the stomach, the orientation of the data logger package was arbitrary and resulted in slow shifting over time, a challenge for normal analysis routines. We present one of these datasets to illustrate a novel methodology for calibrating accelerometer orientation, and evaluate the utility of resulting data. We obtained accurate accelerometer measurements including surge, heave, and sway from data loggers with shifting orientation through data post-processing. We measured consistent dips in stomach temperature followed by a steady prolonged heating during controlled feeding events in a captive white shark. Similar thermal signatures identified in wild white shark records confirmed feeding events while acceleration data characterized the associated prey capture behavior. We provided proof of concept for a novel and non-invasive technique for accelerometer data logger deployment extending the possibilities for their use in bio-logging. The placement of data-logging tags in the stomachs of endothermic white sharks produced high quality tri-axial acceleration data in addition to stomach temperature data capable of detecting feeding events. The technique has the potential for distinguishing between attempted and successful foraging events. This approach to accelerometer coordinate correction could be applied in other systems where logger orientation is unknown or changes after deployment.

Relation between tag position and degree of visualized cerebrospinal fluid reflux into the lateral ventricles in time-spatial labeling inversion pulse magnetic resonance imaging at the foramen of Monro.

BackgroundTime-spatial labeling inversion pulse (Time-SLIP) magnetic resonance imaging allows non-invasive visualization of cerebrospinal fluid (CSF) movement. Our study evaluated the sensitivity of the Time-SLIP tag placement on the measurement of CSF reflux from the third ventricle into the lateral ventricles via the foramen of Monro.FindingsMultiple Time-SLIP MRI scans were obtained in three healthy volunteers (23–55 years of age) evaluating the observed CSF pulsation and reflux from the third ventricle into the lateral ventricles while varying the placement of the tag. Linear regression was performed to evaluate the effects of tag position on the amount of visualized reflux and pulsation. Variation in the position of the tag relative to the plane of the free margin of the septum pellucidum produced a significant inverse variation in the observed reflux into the lateral ventricles (R2 = 0.74). The further the distance of the top (superior edge) of the tag from the plane of the free margin of the septum pellucidum, the less reflux into the lateral ventricles was observed (P = 0.006).ConclusionsThe amount of observed CSF reflux into the lateral ventricles in Time-SLIP MR imaging is dependent on the positioning of the CSF tag with decreasing amount of visualized reflux the further caudal the CSF tag is relative to the free margin of the septum pellucidum.Electronic supplementary materialThe online version of this article (doi:10.1186/s12987-015-0011-0) contains supplementary material, which is available to authorized users.

The use of thiafentanil oxalate and azaperone for reversible immobilisation of African buffalo (Syncerus caffer) within a nature reserve - Short communication.

Although thiafentanil oxalate has been widely used for wildlife immobilisation on different species, no report has been published about its usefulness on African buffalo (Syncerus caffer). Thirty-four African buffaloes were successfully immobilised at Loskop Dam Nature Reserve, South Africa in July 2013. The animals were kept in bomas of the nature reserve. The purpose of the immobilisation was to provide opportunity for microchip implantation, ear tag placement, intradermal tuberculin test and blood sampling. All animals were immobilised with thiafentanil oxalate 6 mg/animal (0.007-0.01 mg/kg) and azaperone 40 mg/animal (0.07-0.04 mg/kg) using Dan-inject darts and gun. The opioid reversal agent naltrexone hydrochloride 60 mg/animal (0.07-0.1 mg/kg) was given intravenously to the ear vein. The mean induction time was 3.9 ± 0.2 min, the recovery time was 1.65 ± 0.87 min. The results of the present study indicate that thiafentanil oxalate, this low-volume, high-potency, reversible drug combined with azaperone provides fast induction and smooth recovery. The authors recommend this drug combination as a reliable immobilising regimen for African buffalo.

FRET-Based Structural Analysis of the Skeletal Muscle DHPR using Biarsenical Labeling

The α1s skeletal muscle dihydropyridine receptor (DHPR) subunit triggers muscle contraction during excitation contraction (EC) coupling. The intracellular loops of this voltage-gated calcium channel have diverse functions, though the spatial organization of these loops relative to each other and to other DHPR subunits, is poorly understood. We employed a FRET-based assay to determine the proximity of these intracellular loops relative to the N-terminus of the α1s DHPR subunit and then determined how these proximities changed upon binding of the β1a DHPR subunit. Recombinant α1s DHPRs were expressed in HEK293T cells and then FRET was measured from YFP fused to the DHPR N-terminus to the biarsenical reagent ReAsH targeted to a tetracysteine (Tc) tag inserted into each of the DHPR intracellular loops. Energy transfer was highest to ReAsH bound to a Tc tag placed adjacent to the N-terminally fused YFP (E∼40-50%) whereas low FRET (E∼10%) was observed for a negative control construct lacking a Tc tag. Placement of Tc tags in each of the DHPR intracellular loops resulted in intermediate FRET efficiencies whereas insignificant energy transfer was observed between the DHPR N- and C-termini. Addition of purified recombinant DHPR β1a subunit resulted in significant enhancement of energy transfer between the N-terminal YFP and ReAsH targeted to the DHPR α1s II-III loop, a major EC coupling determinant. Our data demonstrate that FRET measurements using biarsenical labeling reagents offer an effective approach to study DHPR structure since disruption of native conformation is minimal. In addition, these studies should allow for subsequent measurements of changes in DHPR structure that occur during EC coupling. Supported by NIH grants R01AR059126 (to JDF) and R03AR066359 (to CFP).