Energy Transfer Measurements Research Articles

Systematic mapping of chemoreceptor specificities for Pseudomonas aeruginosa.

Chemotaxis of motile bacteria has multiple physiological functions. It enables bacteria to locate optimal ecological niches, mediates collective behaviors, and can play an important role in infection. These multiple functions largely depend on ligand specificities of chemoreceptors, and the number and identities of chemoreceptors show high diversity between organisms. Similar diversity is observed for the spectra of chemoeffectors, which include not only chemicals of high metabolic value but also bacterial, plant, and animal signaling molecules. However, the systematic identification of chemoeffectors and their mapping to specific chemoreceptors remains a challenge. Here, we combined several in vivo and in vitro approaches to establish a systematic screening strategy for the identification of receptor ligands and we applied it to identify a number of new physiologically relevant chemoeffectors for the important opportunistic human pathogen P. aeruginosa. This strategy can be equally applicable to map specificities of sensory domains from a wide variety of receptor types and bacteria.

Lipopolysaccharide of Legionella pneumophila Serogroup 1 Facilitates Interaction with Host Cells.

Legionella pneumophila is the primary causative agent of Legionnaires' disease. The mutant-type strain interrupted in the ORF7 gene region responsible for the lipopolysaccharide biosynthesis of the L. pneumophila strain Heysham-1, lacking the O-acetyl groups attached to the rhamnose of the core part, showed a higher surface polarity compared with the wild-type strain. The measurement of excitation energy transfer between fluorophores located on the surface of bacteria and eukaryotic cells showed that, at an early stage of interaction with host cells, the mutant exhibited weaker interactions with Acanthamoeba castellanii cells and THP-1-derived macrophages. The mutant displayed reduced adherence to macrophages but enhanced adherence to A. castellanii, suggesting that the O-acetyl group of the LPS core region plays a crucial role in facilitating interaction with macrophages. The lack of core rhamnose O-acetyl groups made it easier for the bacteria to multiply in amoebae and macrophages. The mutant induced TNF-α production more strongly compared with the wild-type strain. The mutant synthesized twice as many ceramides Cer(t34:0) and Cer(t38:0) than the wild-type strain. The study showed that the internal sugars of the LPS core region of L. pneumophila sg 1 can interact with eukaryotic cell surface receptors and mediate in contacting and attaching bacteria to host cells as well as modulating the immune response to infection.

Utility of FRET in studies of membrane protein oligomerization: The concept of the effective dissociation constant

The activity of many membrane receptors is controlled through their lateral association into dimers or higher-order oligomers. Although Förster resonance energy transfer (FRET) measurements have been used extensively to characterize the stability of receptor dimers, the utility of FRET in studies of larger oligomers has been limited. Here we introduce an effective equilibrium dissociation constant that can be extracted from FRET measurements for EphA2, a receptor tyrosine kinase (RTK) known to form active oligomers of heterogeneous distributions in response to its ligand ephrinA1-Fc. The newly introduced effective equilibrium dissociation constant has a well-defined physical meaning and biological significance. It denotes the receptor concentration for which half of the receptors are monomeric and inactive, and the other half are associated into oligomers and are active, irrespective of the exact oligomer size. This work introduces a new dimension to the utility of FRET in studies of membrane receptor association and signaling in the plasma membrane.

Multiscale computational framework to investigate integrin mechanosensing and cell adhesion

Integrin mechanosensing plays an instrumental role in cell behavior, phenotype, and fate by transmitting mechanical signals that trigger downstream molecular and cellular changes. For instance, force transfer along key amino acid residues can mediate cell adhesion. Disrupting key binding sites within α5β1 integrin’s binding partner, fibronectin (FN) diminishes adhesive strength. While past studies have shown the importance of these residues in cell adhesion, the relationship between the dynamics of these residues and how integrin distributes force across the cell surface remains less explored. Here, we present a multiscale mechanical model to investigate the mechanical coupling between integrin nanoscale dynamics and whole-cell adhesion mechanics. Our framework leverages molecular dynamics simulations to investigate residues within α5β1-FN during stretching and the finite element method to visualize the whole-cell adhesion mechanics. The forces per integrin across the cell surface of the whole-cell model were consistent with past atomic force microscopy and Förster resonance energy transfer measurements from the literature. The molecular dynamics simulations also confirmed past studies that implicate two key sites within FN that maintain cell adhesion: the synergy site and arginine-glycine-aspartic acid (RGD) motif. Our study contributed to our understanding of molecular mechanisms by which these sites collaborate to mediate whole-cell integrin adhesion dynamics. Specifically, we showed how FN unfolding, residue binding/unbinding, and molecular structure contribute to α5β1-FN’s nonlinear force–extension behavior during stretching. Our computational framework could be used to explain how the dynamics of key residues influence cell differentiation or how uniquely designed protein structures could dynamically limit the spread of metastatic cells.

Computational generation and characterization of IsdA-binding aptamers with single-molecule FRET analysis.

Staphylococcus aureus is a major foodborne bacterial pathogen. Early detection of S. aureus is crucial to prevent infections and ensure food quality. The iron-regulated surface determinant protein A (IsdA) of S. aureus is a unique surface protein necessary for sourcing vital iron from host cells for the survival and colonization of the bacteria. The function, structure, and location of the IsdA protein make it an important protein for biosensing applications relating to the pathogen. Here, we report an in-silico approach to develop and validate high-affinity binding aptamers for the IsdA protein detection using custom-designed in-silico tools and small-molecule Fluorescence Resonance Energy Transfer (smFRET) measurements. We utilized in-silico oligonucleotide screening methods and metadynamics-based methods to generate 10 aptamer candidates and characterized them based on the Dissociation Free Energy (DFE) of the IsdA-aptamer complexes. Three of the aptamer candidates were shortlisted for smFRET experimental analysis of binding properties. Limits of detection in the low picomolar range were observed for the aptamers, and the results correlated well with the DFE calculations, indicating the potential of the in-silico approach to support aptamer discovery. This study showcasesa computational SELEXmethod in combination with single-molecule binding studies decipheringeffectiveaptamersagainst S. aureusIsdA, protein. Theestablished approach demonstrates the abilityto expediteaptamerdiscovery that has the potential to cutcosts and predict binding efficacy.The application can be extendedto designingaptamersfor various protein targets, enhancing molecular recognition, and facilitating the development of high-affinityaptamersfor multiple uses. This article is protected by copyright. All rights reserved.

From Microstates to Macrostates in the Conformational Dynamics of GroEL: A Single-Molecule Förster Resonance Energy Transfer Study.

The chaperonin GroEL is a multisubunit molecular machine that assists in protein folding in the Escherichia coli cytosol. Past studies have shown that GroEL undergoes large allosteric conformational changes during its reaction cycle. Here, we report single-molecule Förster resonance energy transfer measurements that directly probe the conformational transitions of one subunit within GroEL and its single-ring variant under equilibrium conditions. We find that four microstates span the conformational manifold of the protein and interconvert on the submillisecond time scale. A unique set of relative populations of these microstates, termed a macrostate, is obtained by varying solution conditions, e.g., adding different nucleotides or the cochaperone GroES. Strikingly, ATP titration studies demonstrate that the partition between the apo and ATP-ligated conformational macrostates traces a sigmoidal response with a Hill coefficient similar to that obtained in bulk experiments of ATP hydrolysis. These coinciding results from bulk measurements for an entire ring and single-molecule measurements for a single subunit provide new evidence for the concerted allosteric transition of all seven subunits.

Establishing Fluorine-Containing Amino Acids as an Orthogonal Tool in Coiled Coil Assembly.

The α-helical coiled coil (CC) is one of the best-characterized folding motifs in the protein world. In this context, fluorinated amino acids have been shown to be capable of tuning the properties of CC assemblies, and especially fluorinated derivatives of aliphatic amino acids can significantly increase the stability of this folding motif when placed in the hydrophobic a and d positions. However, it has not been shown yet whether fluorinated amino acids, by means of rational design, can be used as an orthogonal tool to control CC assembly processes. In the current work, we approached this question by creating a combinatorial peptide library based on a VPE/VPK heteromeric CC system previously established and characterized in our group. This CC model allowed us to screen fluorinated amino acids for interaction with different potential binding partners in position a of the VPE/VPK model with a particular emphasis on studying the impact of stereochemistry within the side chain of α-branched aliphatic fluorinated amino acids on CC properties such as oligomerization state, thermodynamic stability, and orientation. 28 combinations of library members were characterized regarding structure, oligomerization, and thermal stability utilizing circular dichroism, size exclusion chromatography, and Förster resonance energy transfer measurements. This detailed approach showed that the stability and oligomerization state of the motif were not only dependent on the steric demand and the fluorination of corresponding amino acids but also on the stereochemistry within the side chain. The results were applied for a rational design of the fluorine-driven orthogonal assembly, and we could show that CC dimer formation occurred based on specific interactions between fluorinated amino acids. These results demonstrate the potential of fluorinated amino acids as an orthogonal tool besides classical electrostatic and hydrophobic interactions for the fine-tuning and direction of peptide-peptide interactions. Furthermore, within the space of fluorinated amino acids, we could demonstrate the specificity of interactions between differently fluorinated side chains.

Bioaffinity Nanoprobes for Foodborne Pathogen Sensing.

Bioaffinity nanoprobes are a type of biosensor that utilize the specific binding properties of biological molecules, such as antibodies, enzymes, and nucleic acids, for the detection of foodborne pathogens. These probes serve as nanosensors and can provide highly specific and sensitive detection of pathogens in food samples, making them an attractive option for food safety testing. The advantages of bioaffinity nanoprobes include their ability to detect low levels of pathogens, rapid analysis time, and cost-effectiveness. However, limitations include the need for specialized equipment and the potential for cross-reactivity with other biological molecules. Current research efforts focus on optimizing the performance of bioaffinity probes and expanding their application in the food industry. This article discusses relevant analytical methods, such as surface plasmon resonance (SPR) analysis, Fluorescence Resonance Energy Transfer (FRET) measurements, circular dichroism, and flow cytometry, that are used to evaluate the efficacy of bioaffinity nanoprobes. Additionally, it discusses advances in the development and application of biosensors in monitoring foodborne pathogens.

Quantitative fluorescence emission anisotropy microscopy for implementing homo-fluorescence resonance energy transfer measurements in living cells.

Quantitative fluorescence emission anisotropy microscopy reveals the organization of fluorescently labeled cellular components and allows their characterization in terms of changes in either rotational diffusion or homo-Förster's energy transfer characteristics in living cells. These properties provide insights into molecular organization, such as orientation, confinement, and oligomerization in situ. Here we elucidate how quantitative measurements of anisotropy using multiple microscope systems may be made by bringing out the main parameters that influence the quantification of fluorescence emission anisotropy. We focus on a variety of parameters that contribute to errors associated with the measurement of emission anisotropy in a microscope. These include the requirement for adequate photon counts for the necessary discrimination of anisotropy values, the influence of extinction ratios of the illumination source, the detector system, the role of numerical aperture, and excitation wavelength. All these parameters also affect the ability to capture the dynamic range of emission anisotropy necessary for quantifying its reduction due to homo-FRET and other processes. Finally, we provide easily implementable tests to assess whether homo-FRET is a cause for the observed emission depolarization.

Transthyretin binds soluble endoglin and increases its uptake by hepatocytes: A possible role for transthyretin in preeclampsia?

BackgroundPreeclampsia is a common but life-threatening condition of pregnancy. It is caused by poor placentation resulting in release of trophoblast material (including soluble endoglin (sEng)) into the maternal circulation leading to maternal vascular dysfunction and to the life-threatening condition of eclampsia. The only cure is early delivery, which can have lifelong consequences for the premature child. The thyroid hormone binding protein transthyretin is dysregulated in preeclampsia, however it is not known if this plays a role in disease pathology. We hypothesised that transthyretin may bind sEng and abrogate its negative effects by removing it from the maternal serum. MethodsThe effect of transthyretin on hepatocyte uptake of Alexa-labelled sEng was measured using live cell imaging. Interactions between transthyretin, and sEng were investigated using molecular modelling, direct binding on CnBr Sepharose columns, confocal imaging, and measurement of fluorescence resonance energy transfer. ResultsTransthyretin directly bound to sEng and increased its uptake by hepatocytes. This uptake was altered in the presence of transforming growth factor-β1 (TGF-β1). Molecular modelling predicted that transthyretin and TGF-β1 bind at the same site in sEng and may compete for binding. Endocytosed transthyretin and endoglin entered cells together and co-localised inside hepatocyte cells. ConclusionTransthyretin can bind sEng and increase its uptake from the extracellular medium. This suggests that increasing transthyretin levels or developing drugs that normalise or mimic transthyretin, may provide treatment options to reduce sEng induced vascular dysfunction.

Cell shape and tension alter focal adhesion structure

Cells are not only anchored to the extracellular matrix via the focal adhesion complex, the focal adhesion complex also serves as a sensor for force transduction. How tension influences the structure of focal adhesions is not well understood. Here, we analyse the effect of tension on the location of key focal adhesion proteins, namely vinculin, paxillin and actin. We use micropatterning on gold surfaces to manipulate the cell shape, to create focal adhesions at specific cell areas, and to perform metal-induced energy transfer (MIET) measurements on the patterned cells. MIET resolves the different protein locations with respect to the gold surface with nanometer accuracy. Further, we use drugs influencing the cellular motor protein myosin or mechanosensitive ion channels to get deeper insight into focal adhesions at different tension states. We show here that in particular actin is affected by the rationally tuned force balance. Blocking mechanosensitive ion channels has a particularly high influence on the actin and focal adhesion architecture, resulting in larger focal adhesions with elevated paxillin and vinculin and strongly lowered actin stress fibres. Our results can be explained by a balance of adhesion tension with cellular tension together with ion channel-controlled focal adhesion homeostasis, where high cellular tension leads to an elevation of vinculin and actin, while high adhesion tension lowers these proteins.

Measurement of Proton Beam Dose-Averaged Linear Energy Transfer Using a Radiochromic Film

Seohyeon An, Sang-il Pak, Seonghoon Jeong, Soonki Min, Tae Jeong Kim, Dongho Shin, Youngkyung Lim, Jong Hwi Jeong, Haksoo Kim, Se Byeong Lee. Progress in Medical Physics 2022;33:80-7. https://doi.org/10.14316/pmp.2022.33.4.80

Ubiquitin modulates 26S proteasome conformational dynamics and promotes substrate degradation.

The 26S proteasome recognizes thousands of appropriate protein substrates in eukaryotic cells through attached ubiquitin chains and uses its adenosine triphosphatase (ATPase) motor for mechanical unfolding and translocation into a proteolytic chamber. Here, we used single-molecule Förster resonance energy transfer measurements to monitor the conformational dynamics of the proteasome, observe individual substrates during their progression toward degradation, and elucidate how these processes are regulated by ubiquitin chains. Rapid transitions between engagement- and processing-competent proteasome conformations control substrate access to the ATPase motor. Ubiquitin chain binding functions as an allosteric regulator to slow these transitions, stabilize the engagement-competent state, and aid substrate capture to accelerate degradation initiation. Upon substrate engagement, the proteasome remains in processing-competent states for translocation and unfolding, except for apparent motor slips when encountering stably folded domains. Our studies revealed how ubiquitin chains allosterically regulate degradation initiation, which ensures substrate selectivity in a crowded cellular environment.

Multilayered allosteric modulation of coupled folding and binding by phosphorylation, peptidyl-prolyl cis/trans isomerization, and diversity of interaction partners

Intrinsically disordered proteins (IDPs) play key roles in cellular regulation, including signal transduction, transcription, and cell-cycle control. Accordingly, IDPs can commonly interact with numerous different target proteins, and their interaction networks are expected to be highly regulated. However, many of the underlying regulatory mechanisms have remained unclear. Here, we examine the representative case of the nuclear coactivator binding domain (NCBD) of the large multidomain protein CBP, a hub in transcriptional regulation, and the interaction with several of its binding partners. Single-molecule Förster resonance energy transfer measurements show that phosphorylation of NCBD reduces its binding affinity, with effects that vary depending on the binding partner and the site and number of modifications. The complexity of the interaction is further increased by the dependence of the affinities on peptidyl-prolyl cis/trans isomerization in NCBD. Overall, our results reveal the potential for allosteric regulation on at least three levels: the different affinities of NCBD for its different binding partners, the differential modulation of these affinities by phosphorylation, and the effect of peptidyl-prolyl cis/trans isomerization on binding.

Clinical dose assessment for scanned carbon-ion radiotherapy using linear energy transfer measurements and Monte Carlo simulations

Objective. Dosimetric commissioning of treatment planning systems (TPS) focuses on validating the agreement of the physical dose with experimental data. For carbon-ion radiotherapy, the commissioning of the relative biological effectiveness (RBE) is necessary to predict the clinical outcome based on the radiation quality of the mixed radiation field. In this study, we proposed a approach for RBE commissioning using Monte Carlo (MC) simulations, which was further strengthen by RBE validation based on linear energy transfer (LET) measurements. Approach. First, we tuned the MC simulation based on the results of dosimetric experiments including the beam ranges, beam sizes, and MU calibrations. Furthermore, we compared simulated results to measured depth- and radial-LET distributions of the 430 MeV u−1 carbon-ion spot beam with a 1.5 mm2, 36 μm thick silicon detector. The measured dose-averaged LET (LETd) and RBE were compared with the simulated results. The RBE was calculated based on the mixed beam model with linear-quadratic parameters depending on the LET. Finally, TPS-calculated clinical dose profiles were validated through the tuned MC-based calculations. Main results. A 10 keV μm−1 and 0.15 agreement for LETd and RBE, respectively, were found between simulation and measurement results obtained for a 2σ lateral size of 430 MeV u−1 carbon-ion spot beam in water. These results suggested that the tuned MC simulation can be used with acceptable precision for the RBE and LET calculations of carbon-ion spot beam within the clinical energy range. For physical and clinical doses, the TPS- and MC-based calculations showed good agreements within 1.0% at the centre of the spread-out Bragg peaks. Significance. The tuned MC simulation can accurately reproduce the actual carbon-ion beams, and it can be used to validate the physical and clinical dose distributions calculated by TPS. Moreover, the MC simulation can be used for dosimetric commissioning, including clinical doses, without LET measurements.

New N-aryl-N-alkyl-thiophene-2-carboxamide compound enhances intracellular Ca2+ dynamics by increasing SERCA2a Ca2+ pumping

The type 2a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) plays a central role in the intracellular Ca2+ homeostasis of cardiac myocytes, pumping Ca2+ from the cytoplasm into the sarcoplasmic reticulum (SR) lumen to maintain relaxation (diastole) and prepare for contraction (systole). Diminished SERCA2a function has been reported in several pathological conditions, including heart failure. Therefore, development of new drugs that improve SERCA2a Ca2+ transport is of great clinical significance. In this study, we characterized the effect of a recently identified N-aryl-N-alkyl-thiophene-2-carboxamide (or compound 1) on SERCA2a Ca2+-ATPase and Ca2+ transport activities in cardiac SR vesicles, and on Ca2+ regulation in a HEK293 cell expression system and in mouse ventricular myocytes. We found that compound 1 enhances SERCA2a Ca2+-ATPase and Ca2+ transport in SR vesicles. Fluorescence lifetime measurements of fluorescence resonance energy transfer between SERCA2a and phospholamban indicated that compound 1 interacts with the SERCA-phospholamban complex. Measurement of endoplasmic reticulum Ca2+ dynamics in HEK293 cells expressing human SERCA2a showed that compound 1 increases endoplasmic reticulum Ca2+ load by enhancing SERCA2a-mediated Ca2+ transport. Analysis of cytosolic Ca2+ dynamics in mouse ventricular myocytes revealed that compound 1 increases the action potential-induced Ca2+ transients and SR Ca2+ load, with negligible effects on L-type Ca2+ channels and Na+/Ca2+ exchanger. However, during adrenergic receptor activation, compound 1 did not further increase Ca2+ transients and SR Ca2+ load, but it decreased the propensity toward Ca2+ waves. Suggestive of concurrent desirable effects of compound 1 on RyR2, [3H]-ryanodine binding to cardiac SR vesicles shows a small decrease in nM Ca2+ and a small increase in μM Ca2+. Accordingly, compound 1 slightly decreased Ca2+ sparks in permeabilized myocytes. Thus, this novel compound shows promising characteristics to improve intracellular Ca2+ dynamics in cardiomyocytes that exhibit reduced SERCA2a Ca2+ uptake, as found in failing hearts.

Internalized Amyloid-β (1-42) Peptide Inhibits the Store-Operated Calcium Entry in HT-22 Cells.

Dysregulation in calcium signaling pathways plays a major role in the initiation of Alzheimer’s disease (AD) pathogenesis. Accumulative experimental evidence obtained with cellular and animal models, as well as with AD brain samples, points out the high cytotoxicity of soluble small oligomeric forms of amyloid-β peptides (Aβ) in AD. In recent works, we have proposed that Aβ-calmodulin (CaM) complexation may play a major role in neuronal Ca2+ signaling, mediated by CaM-binding proteins (CaMBPs). STIM1, a recognized CaMBP, plays a key role in store-operated calcium entry (SOCE), and it has been shown that the SOCE function is diminished in AD, resulting in the instability of dendric spines and enhanced amyloidogenesis. In this work, we show that 2 and 5 h of incubation with 2 μM Aβ(1-42) oligomers of the immortalized mouse hippocampal cell line HT-22 leads to the internalization of 62 ± 11 nM and 135 ± 15 nM of Aβ(1-42), respectively. Internalized Aβ(1-42) oligomers colocalize with the endoplasmic reticulum (ER) and co-immunoprecipitated with STIM1, unveiling that this protein is a novel target of Aβ. Fluorescence resonance energy transfer measurements between STIM1 tagged with a green fluorescent protein (GFP) and Aβ(1-42)-HiLyte™-Fluor555 show that STIM1 can bind nanomolar concentrations of Aβ(1-42) oligomers at a site located close to the CaM-binding site in STIM1. Internalized Aβ(1-42) produced dysregulation of the SOCE in the HT-22 cells before a sustained alteration of cytosolic Ca2+ homeostasis can be detected, and is elicited by only 2 h of incubation with 2 μM Aβ(1-42) oligomers. We conclude that Aβ(1-42)-induced SOCE dysregulation in HT-22 cells is caused by the inhibitory modulation of STIM1, and the partial activation of ER Ca2+-leak channels.

A fluorescent multi-domain protein reveals the unfolding mechanism of Hsp70

Detailed understanding of the mechanism by which Hsp70 chaperones protect cells against protein aggregation is hampered by the lack of a comprehensive characterization of the aggregates, which are typically heterogeneous. Here we designed a reporter chaperone substrate, MLucV, composed of a stress-labile luciferase flanked by stress-resistant fluorescent domains, which upon denaturation formed a discrete population of small aggregates. Combining Förster resonance energy transfer and enzymatic activity measurements provided unprecedented details on the aggregated, unfolded, Hsp70-bound and native MLucV conformations. The Hsp70 mechanism first involved ATP-fueled disaggregation and unfolding of the stable pre-aggregated substrate, which stretched MLucV beyond simply unfolded conformations, followed by native refolding. The ATP-fueled unfolding and refolding action of Hsp70 on MLucV aggregates could accumulate native MLucV species under elevated denaturing temperatures highly adverse to the native state. These results unambiguously exclude binding and preventing of aggregation from the non-equilibrium mechanism by which Hsp70 converts stable aggregates into metastable native proteins.

Linear energy transfer (LET) spectroscopy and relative biological effect estimation by SiC-based dosimeter at clinical carbon-beam cancer therapy field

A radiation-tolerant silicon carbide (SiC) based dosimeter was developed for the measurement of linear energy transfer (LET) distributions along the Bragg curve for pristine and spread-out Bragg peak (SOBP) clinical carbon beam. Since the conventional ionized chamber is not suitable and effective for LET measurement, several energy-dispersive dosimeters are being developed and tested in the clinical field. While most of the detector lacks radiation hardness against carbon beam, we have utilized wide-bandgap semiconductors of SiC with different epitaxial layers from 25, 69, and 170 μm as a dosimeter for the clinical carbon field at Gunma University Heavy Ion Medical Center (GHMC). LET spectra were obtained not only for pristine carbon beams with an energy of 290 MeV/n but also SOBP conditions. It was confirmed through the changes in LET distribution, primally carbons and fragments are identified, and those energy depositions are successfully obtained by the fabricated SiC dosimeter. The distribution of relative biological effectiveness (RBE) was estimated from LET through biological estimation models. Results suggested that SiC-based dosimeter is successful in being utilized in the detailed characterization of the clinical carbon therapy field.

Phased Array Beamforming Methods for Powering Biomedical Ultrasonic Implants.

Millimeter-scale implants using ultrasound (US) for power and communication have been proposed for a range of deep-tissue applications, including neural recording and stimulation. However, published implementations have shown high sensitivity to misalignment with the external US transducer. Ultrasonic beamforming using a phased array to these implants can improve tolerance to misalignment, reduce implant volume, and allow multiple implants to be operated simultaneously in different locations. This article details the design of a custom planar phased array US system, which is capable of steering and focusing US power within a 3-D volume. Analysis and simulation is performed to determine the choice of array element pitch, with special attention given to maximizing the power available at the implant while meeting FDA limits for diagnostic US. Time reversal (TR) is proposed as a computationally simple approach to beamforming that is robust despite scattering and inhomogeneity of the acoustic medium. This technique is demonstrated both in active drive and pulse-echo modes, and it is experimentally compared with other beamforming techniques by measuring energy transfer efficiency. Simultaneous power delivery to multiple implants is also demonstrated.