Anabaena Sensory Rhodopsin Research Articles

Modeling pH-Dependent Biomolecular Photochemistry.

The tuning mechanism of pH can be extremely challenging to model computationally in complex biological systems, especially with respect to the photochemical properties. This article reports a protocol aimed at modeling pH-dependent photodynamics using a combination of constant-pH molecular dynamics and semiclassical nonadiabatic molecular dynamics simulations. With retinal photoisomerization in Anabaena sensory rhodopsin (ASR) as a testbed, we show that our protocol produces pH-dependent photochemical properties, such as the isomerization quantum yield or decay rates. We decompose our results into single-titrated residue contributions, identifying some key tuning amino acids. Additionally, we assess the validity of the single protonation state picture to represent the system at a given pH and propose the most populated protein charge state as a compromise between cost and accuracy.

On the Role and Structural Outlook of Carboxy-Terminal Domain of Anabaena Sensory Rhodopsin

Anabaena Sensory Rhodopsin, ASR represents a small family of sensory rhodopsins among a vast family of microbial rhodopsins. In contrast to other microbial sensory rhodopsins, which are known to mediate photostimulated signal relay using cognate membrane transducer, ASR interacts to plausible soluble oligomeric transducer protein. Though the atomic resolution structure ASR is available, it only included only the transmembrane domain. Interestingly the available structure is remarkably different from other microbial sensory rhodopsins, with presence of water. Additionally, the sequence of missing cytoplasmic domain contains a series of positively charged Arginine residues in it. Authors have outlined the influence of this domain in addition to flexible structural outlook using Deep Mind’s Nova Fold-2. Authors propose that charged amino acids are vital in ASR-transducer interaction. Subsequent attempts to explore the molecular basis of ASR and cognate transducer binding would be vital to understand the unique photo-mediated signal cascade in Anabaena PCC 7120.

Protein structure–function exploration initiative in undergraduate biochemistry and independent research courses

Protein structure-function relationship serves as the primary learning outcome in any undergraduate biochemistry course. We expanded the protein structure-function exploration, PSFE initiative during COVID-19 to provide more effective and engaging experience to our undergraduates in biochemistry and independent research courses. Multiple alignments of protein sequences provided crucial insight into sequence conservation across many species and thus allow identification of those sections of the sequence most critical to protein function. We used Anabaena Sensory Rhodopsin, ASR its transducer, ASRT and downstream novel kinase gene products of Anabaena PCC 7120 to seek their alignment with homologs in available database. Pymol served an opportunity to achieve this goal (interactive learning during lab session and stimulation of course content discussion) in interesting ways. The PSFE initiative expansion continued during pandemic using online/hybrid modality. Initially model examples all helical ASR and beta-sheet ASRT were introduced to connect and integrate our ongoing research interest into classroom activities. Subsequently, undergraduates in biochemistry course were assigned a homolog of model proteins any particular protein of students choice to study and characterize using Pymol in semester. During first phase, each undergraduate worked independently using established guidelines. Student's exploration progress was periodically reviewed in pilot phase with majority of students who perceived it as challenging task successfully completed the assignment. Using the PyMol application to reinforce visual understanding of protein structure was highly satisfying experience that greatly enriched undergraduates understanding and appreciation. This article reports a session from the virtual international 2021 IUBMB/ASBMB workshop, "Teaching Science on BigData."

Bidirectional Photochemistry of Antarctic Microbial Rhodopsin: Emerging Trend of Ballistic Photoisomerization from the 13-cis Resting State.

The decades-long ultrafast examination of nearly a dozen microbial retinal proteins, ion pumps, and sensory photoreceptors has not identified structure–function indicators which predict photoisomerization dynamics, whether it will be sub-picosecond and ballistic or drawn out with complex curve-crossing kinetics. Herein, we report the emergence of such an indicator. Using pH control over retinal isomer ratios, photoinduced transient absorption is recorded in an inward proton pumping Antarctic microbial rhodopsin (AntR) for 13-cis and all-trans retinal resting states. The all-trans fluorescent state decays with 1 ps exponential kinetics. In contrast, in 13-cis it decays within ∼300 fs accompanied by continuous spectral evolution, indicating ballistic internal conversion. The coherent wave packet nature of 13-cis isomerization in AntR matches published results for bacteriorhodopsin (BR) and Anabaena sensory rhodopsin (ASR), which also accommodate both all-trans and 13-cis retinal resting states, marking the emergence of a first structure–photodynamics indicator which holds for all three tested pigments.

Fragment Localized Molecular Orbitals

We introduce the concept of fragment localized molecular orbitals (FLMOs), which are Hartree–Fock molecular orbitals localized in specific fragments constituting a molecular system. In physical terms, we minimize the local electronic energies of the different fragments, at the cost of maximizing the repulsion between them. To showcase the approach, we rationalize the main interactions occurring in large biological systems in terms of interactions between the fragments of the system. In particular, we study an anticancer drug intercalated within DNA and retinal in anabaena sensory rhodopsin as prototypes of molecular systems embedded in biological matrixes. Finally, the FLMOs are exploited to rationalize the formation of two oligomers, prototypes of amyloid diseases, such as Parkinson and Alzheimer.

HwMR is a novel magnesium-associated protein

Microbial rhodopsins (MRho) are vital proteins in Haloarchaea for solar light sensing in extreme living environments. Among them, Haloquadratum walsbyi (Hw) is a species known to survive high MgCl2 concentrations, with a total of three MRhos identified, including a high-acid-tolerance light-driven proton outward pump, HwBR, a chloride-insensitive chloride pump, HwHR, and a functionally unknown HwMR. Here, we showed that HwMR is the sole magnesium-sensitive MRho among all tested MRho proteins from Haloarchaea. We identified at least D84 as one of the key residues mediating such magnesium ion association in HwMR. Sequence analysis and molecular modeling suggested HwMR to have an extra H8 helix in the cytosolic region like those in signal-transduction-type MRho of deltarhodopsin-3 (dR-3) and Anabaena sensory rhodopsin (ASR). Further, HwMR showed a distinctly prolonged M-state formation under a high concentration of Mg2+. On the other hand, an H8 helix truncated mutant preserved photocycle kinetics like the wild type, but it led to missing M-state structure. Our findings clearly suggested not only that HwMR is a novel Mg2+-associated protein but that the association with both Mg2+ and the H8 domain stabilizes M-state formation in HwMR. We conclude that Mg2+ association and H8 are crucial in stabilizing HwMR M state, which is a well-known photoreceptor signaling state.

Kinetics of DNA looping by Anabaena sensory rhodopsin transducer (ASRT) by using DNA cyclization assay

DNA cyclization assay together with single-molecule FRET was employed to monitor protein-mediated bending of a short dsDNA (~ 100 bp). This method provides a simple and easy way to monitor the structural change of DNA in real-time without necessitating prior knowledge of the molecular structures for the optimal dye-labeling. This assay was applied to study how Anabaena sensory rhodopsin transducer (ASRT) facilitates loop formation of DNA as a possible mechanism for gene regulation. The ASRT-induced DNA looping was maximized at 50 mM of Na+, while Mg2+ also played an essential role in the loop formation.

Identifying lipids tightly bound to an integral membrane protein

Anabaena Sensory Rhodopsin (ASR) is a microbial photosensor from the cyanobacterium Anabaena sp. PCC 7120. It was found in previous studies that ASR co-purifies with several small molecules, although their identities and structural or functional roles remained unclear. Here, we use solid-state nuclear magnetic resonance (SSNMR) spectroscopy and mass spectrometry to characterize these molecules. Numerous correlations atypical for protein amino acids were found and assigned in the SSNMR spectra. The chemical shift patterns correspond to N-acetyl-d-glucosamine, N-acetyl-d-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-d-galactose which are part of the Enterobacterial Common Antigen (ECA). These sugars undergo rapid anisotropic motions and are likely linked flexibly to a rigid anchor that tightly binds ASR. Phosphorus NMR reveals several signals that are characteristic of monophosphates, further suggesting phosphatidylglyceride as the ECA lipid carrier which is anchored to ASR. In addition, NMR signals corresponding to common phospholipid phosphatidylethanolamine (PE) have been detected. The presence of PE tightly interacting with ASR was confirmed using liquid chromatography-mass spectrometry. This article commemorates Professor Michèle Auger and her contributions to membrane biophysics and Nuclear Magnetic Resonance.

CpHMD-Then-QM/MM Identification of the Amino Acids Responsible for the Anabaena Sensory Rhodopsin pH-Dependent Electronic Absorption Spectrum.

Anabaena Sensory Rhodopsin (ASR), a microbial photoactive protein featuring the retinal chromophore in two different conformations, exhibits a pH-dependent electronic absorption spectrum. Using the recently developed CpHMD-then-QM/MM multiscale protocol applied to ASR embedded in a membrane model, the pH-induced changes in its maximum absorption wavelength have been reproduced and analyzed. While the acidic tiny red-shift is essentially correlated with the deprotonation of an aspartic acid located on the ASR extracellular side, the larger blue-shift experimentally reported at pH values larger than 5 involves a cluster of titrating residues sitting on the cytoplasmic side. The ASR pH-dependent spectrum is the consequence of the competitive stabilization of retinal ground and excited states by the protein electrostatic potential.

Receptor Binding and Phosphorylation of Anabaena Sensory Rhodopsin Transducer towards Signaling via Protein‐Protein Cross Talk

Anabaena sensory rhodopsin transducer, ASRT [125 amino acid sequence] is present under the same promoter of its sensory rhodopsin, ASR in Anabaena PCC 7120 a freshwater, filamentous, photosynthetic cynobacteria. The ASRT exists as tetrameric soluble protein indicated to function as downstream signaling partner to sensory rhodopsin. Both solution and crystal structures has reveled that this primarily beta stranded protein exhibit a helical face at carboxyl terminus. Besides receptor binding, ASRT is been shown to serve as a novel eukaryotic-like interaction with DNA. We have demonstrated that carboxyl helical face of tetramer is involved in unusual tetrameric stability. A blast search matches the ASRT to others of unknown function, with 84% similarity and 74% identity to a protein in the myxobacterium Polyangium cellulosum and with 72% similarity and 58% identity to a protein in the legume symbiont Sinorhizobium meliloti. Often the homologues of this family are termed as “DUF”, Domain of Unknown Function member. This DUF1362 family is wide spread over microbial population including pathogens as well. The gene neighbor hood, along with structural fold analysis termed this family as novel small carbohydrate binding protein. We have observed unique xylan binding via electrostatic interaction. Additional bioinformatics analysis has revealed a phosphor transfer along with unique structural fold that may transform it as a unique carbohydrate binding module. However, the signaling state/mechanism of ASRT is obscure. Our initial data with efficient donor, acetyl phosphate, acP using fluorescence quenching of lone Trp-9 suggests that ASRT is involved in phosphorylation. We noticed two notable consensus phosphor transfer residue motif in ASRT, SDKE [53–56] and TRLD [105–108]. Acetyl phosphate has been used as phosphoryl donor in vitro to numerous response regulators [CheY/PhoB/OmpR]. The extent of quenching in presence of Mg2+by increasing concentration of acP yielded lower KM [~21mM] compared to PhoB [~8mM]. It is likely that proximity of phosphor accepting residue (s) to the lone Trp is not comparable to others. Interestingly the ASRT mutant E56Q and D108N indicate the loss of phosphor transfer. We hypothesize that putative position shown as red in figure is involved in ASRT phosphorylation. This motif is in close proximity to receptor binding deduced by CABS docking and GST-assay using various length of ASR [full length, 1–261, and truncated (1–229, 1–239, 1–249)]. Phosphor transfer data along with plausible impact on tetrameric stability will be discussed in this study. We have identified a Serine Threonine Kinase, STK 3169 gene as a likely candidate in phosphor relay. Recent results of STK 3169 cloning, optimized expression, purification and subsequent characterization in phosphorylation assay will be presented. Support or Funding Information Supported by NIH-NIGMS Support for Competitive Research, SCORE SC3 award GM113803 to VT This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Point Mutation of Anabaena Sensory Rhodopsin Enhances Ground-State Hydrogen Out-of-Plane Wag Raman Activity.

The interaction between the retinal protonated Schiff base (RPSB) and surrounding protein residues inside the retinal pocket is believed to play a major role in the ultrafast isomerization of the former. Coherent time-resolved vibrational spectroscopic techniques are applied to reveal the effect of changes in the protein architecture by point mutations (V112N and L83Q) close to the RPSB in Anabaena sensory rhodopsin (ASR). Our study reveals that such point mutations have a minor effect on the low-frequency (<400 cm-1) torsional modes but dramatically influence the ground-state vibrational Raman activity of the C14-H out-of-plane (HOOP) wag mode (800-820 cm-1). In mutated ASR, the increase of HOOP Raman activity in the ground state is experimentally observed for the all- trans RPSB, which has shorter excited-state lifetime than in wild-type ASR. This indicates that predistortion of the RPSB inside the mutated retinal pocket is a major factor in the acceleration of the isomerization rate.

Anabaena Sensory Rhodopsin: Effect of point mutations on PSBR photo-isomerization speed

We report new experimental results on the ultrafast photo-isomerization of ASR - PSBR where, unlike other retinal proteins, point mutations lead to a 2-fold increase of the photo-isomerization speed for the all-trans isomer. Prominent low-frequency vibrational coherences are reported for both the excited and photo-product ground states.

Mapping the ultrafast vibrational dynamics of all-trans and 13-Cis retinal isomerization in Anabaena Sensory Rhodopsin

The ground and excited state evolution of fingerprint vibrational modes of all-trans-and 13-cis-retinal are mapped by impulsive vibrational spectroscopy. All-trans-retinal shows slower frequency shift dynamics in the excited state in comparison to 13-cis-retinal.

Expression of Anabaena sensory rhodopsin is influenced by different codons of seven residues at the N-terminal region

Microbial rhodopsins are well-known seven-transmembrane proteins that have been extensively studied for their structure and function. These retinal-binding proteins can be divided into two types. Type I is microbial rhodopsin, and type II (visual pigment) is expressed mostly in mammalian eyes. The two primary functions of type I rhodopsin are ion pumping activity and sensory transduction. Anabaena sensory rhodopsin (ASR) is a microbial rhodopsin with a specific function of photosensory transduction. ASR is expressed at moderate levels in Escherichia coli, but its expression level is lower compared to the general green light absorbing proteorhodopsin (GPR). In this study, full-length ASR was used to test the influence of codon usage on expression E. coli. Seven amino acids at the N-terminal region of ASR after the Met start codon were changed randomly using designed primers, which allowed for 8192 different nucleotide combinations. The codon changes were screened for the preferable codons that resulted in higher expression yield. Among the 57 selected mutations, 24 color-enhanced E. coli colonies contained ASR proteins, and they expressed ASR at a higher level than the bacteria with wild-type ASR codon usage. This result strongly suggests that the specific codon usage of only the N-terminal portion of a protein can increase the expression level of the entire protein.

“Ligand Binding and Phosphorylation of Anabaena Sensory Rhodopsin Transducer towards Putative Signaling State in Sensory Rhodopsin Mediated Protein‐Protein Cross Talk”

Anabaena sensory rhodopsin transducer, ASRT [125 amino acid sequence] is present under the same promoter of its sensory rhodopsin, ASR in Anabaena PCC 7120 a freshwater, filamentous, photosynthetic cynobacteria. The ASRT exists as tetrameric soluble protein indicated to function as downstream signaling partner to sensory rhodopsin. Both solution and crystal structures has reveled that this primarily beta stranded protein exhibit a helical face at carboxyl terminus. Besides receptor binding, ASRT is been shown to serve as a novel eukaryotic-like interaction with DNA. We have demonstrated that carboxyl helical face of tetramer is involved in unusual tetrameric stability. A blast search matches the ASRT to others of unknown function, with 84% similarity and 74% identity to a protein in the myxobacterium Polyangium cellulosum and with 72% similarity and 58% identity to a protein in the legume symbiont Sinorhizobium meliloti. Often the homologues of this family are termed as “DUF”, Domain of Unknown Function member. This DUF1362 family is wide spread over microbial population including pathogens as well. The gene neighbor hood, along with structural fold analysis termed this family as novel small carbohydrate binding protein. We have observed unique xylan binding via electrostatic interaction. Additional bioinformatics analysis has revealed a phosphor transfer along with unique structural fold that may transform it as a unique carbohydrate binding module. However, the signaling state/mechanism of ASRT is obscure. Our initial data with efficient donor, acetyl phosphate, acP using fluorescence quenching of lone Trp-9 suggests that ASRT is involved in phosphorylation. We noticed two notable consensus phosphor transfer residue motif in ASRT, SDKE [53–56] and TRLD [105–108]. Acetyl phosphate has been used as phosphoryl donor in vitro to numerous response regulators [CheY/PhoB/OmpR]. The extent of quenching in presence of Mg2+ by increasing concentration of acP yielded lower KM [~21 mM] compared to PhoB [~8 mM]. It is likely that proximity of phosphor accepting residue (s) to the lone Trp is not comparable to others. Interestingly the ASRT mutant E56Q and D108N indicate the loss of phosphor transfer. We hypothesize that putative position shown as red in figure is involved in ASRT phosphorylation. This motif is in close proximity to receptor binding deduced by CABS docking. Phosphor transfer data along with plausible impact on tetrameric stability will be discussed in this study. We have identified a Serine Threonine Kinase, STK 3169 gene as a likely candidate in phosphor relay. We have cloned and optimizing the functional expression, purification for subsequent characterization in phosphorylation assay. Support or Funding Information Supported by NIH-NIGMS SCORE SC3GM113803 & BCU CURE 2017 award to VT Helical Face of ASRT with Phosphor Transfer Site This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Partial solid-state NMR 1H, 13C, 15N resonance assignments of a perdeuterated back-exchanged seven-transmembrane helical protein Anabaena Sensory Rhodopsin.

Anabaena Sensory Rhodopsin (ASR) is a unique photochromic membrane-embedded photosensor which interacts with soluble transducer and is likely involved in a light-dependent gene regulation in the cyanobacterium Anabaena sp. PCC 7120. We report partial spectroscopic 1H, 13C and 15N assignments of perdeuterated and back-exchanged ASR reconstituted in lipids. The reported assignments are in general agreement with previously determined assignments of carbon and nitrogen resonances in fully protonated samples. Because the back-exchange was performed on ASR in a detergent-solubilized state, the location of detected residues reports on the solvent accessibility of ASR in detergent. A comparison with the results of previously published hydrogen/exchange data collected on the ASR reconstituted in lipids, suggests that the protein has larger solvent accessible surface in the detergent-solubilized state.

Effect of point mutations on the ultrafast photo-isomerization of Anabaena sensory rhodopsin.

Anabaena sensory rhodopsin (ASR) is a particular microbial retinal protein for which light-adaptation leads to the ability to bind both the all-trans, 15-anti (AT) and the 13-cis, 15-syn (13C) isomers of the protonated Schiff base of retinal (PSBR). In the context of obtaining insight into the mechanisms by which retinal proteins catalyse the PSBR photo-isomerization reaction, ASR is a model system allowing to study, within the same protein, the protein-PSBR interactions for two different PSBR conformers at the same time. A detailed analysis of the vibrational spectra of AT and 13C, and their photo-products in wild-type ASR obtained through femtosecond (pump-) four-wave-mixing is reported for the first time, and compared to bacterio- and channelrhodopsin. As part of an extensive study of ASR mutants with blue-shifted absorption spectra, we present here a detailed computational analysis of the origin of the mutation-induced blue-shift of the absorption spectra, and identify electrostatic interactions as dominating steric effects that would entail a red-shift. The excited state lifetimes and isomerization reaction times (IRT) for the three mutants V112N, W76F, and L83Q are studied experimentally by femtosecond broadband transient absorption spectroscopy. Interestingly, in all three mutants, isomerization is accelerated for AT with respect to wild-type ASR, and this the more, the shorter the wavelength of maximum absorption. On the contrary, the 13C photo-reaction is slightly slowed down, leading to an inversion of the ESLs of AT and 13C, with respect to wt-ASR, in the blue-most absorbing mutant L83Q. Possible mechanisms for these mutation effects, and their steric and electrostatic origins are discussed.

Spectroscopic characteristics of Rubricoccus marinus xenorhodopsin (RmXeR) and a putative model for its inward H+ transport mechanism.

A new group of microbial rhodopsins named xenorhodopsins (XeR), which are closely related to the cyanobacterial Anabaena sensory rhodopsin, show a light-driven "inward" proton transport activity, as reported for one representative of this group from Parvularcula oceani (PoXeR). In this study, we functionally and spectroscopically characterized a new member of the XeR clade from a marine bacterium Rubricoccus marinus SG-29T (RmXeR). Escherichia coli cells expressing recombinant RmXeR showed a light-induced alkalization of the cell suspension, which was strongly impaired by a protonophore, suggesting that RmXeR is a light-driven "inward" proton pump as is PoXeR. The spectroscopic properties of purified RmXeR were investigated and compared with those of PoXeR and a light-driven "outward" proton pump, bacteriorhodopsin (BR) from the archaeon Halobacterium salinarum. Action spectroscopy revealed that RmXeR with all-trans retinal is responsible for the light-driven inward proton transport activity, but not with 13-cis retinal. From pH titration experiments and mutational analysis, we estimated the pKa values for the protonated Schiff base of the retinal chromophore and its counterion as 11.1 ± 0.07 and 2.1 ± 0.07, respectively. Of note, the direction of both the retinal composition change upon light-dark adaptation and the acid-induced spectral shift was opposite that of BR, which is presumably related to the opposite directions of ion transport (from outside to inside for RmXeR and from inside to outside for BR). Flash photolysis experiments revealed the appearances of three intermediates (L, M and O) during the photocycle. The proton uptake and release were coincident with the formation and decay of the M intermediate, respectively. Together with associated findings from other microbial rhodopsins, we propose a putative model for the inward proton transport mechanism of RmXeR.

Protonation-state-Coupled Conformational Dynamics in Reaction Mechanisms of Channel and Pump Rhodopsins.

Channel and pump rhodopsins use energy from light absorbed by a covalently bound retinal chromophore to transport ions across membranes of microbial cells. Ion transfer steps, including proton transfer, can couple to changes in protein conformational dynamics and water positions. Although general principles of how microbial rhodopsins function are largely understood, key issues pertaining to reaction mechanisms remain unclear. In this review, we compare the protonation-coupled dynamics of pump and channelrhodopsins, highlighting the roles that water dynamics, protein electrostatics and protein flexibility can have in ion transport mechanisms. We discuss observations supporting important functional roles of inter- and intra-helical carboxylate/hydroxyl hydrogen-bonding motifs. As specific examples, we use the proton pump bacteriorhodopsin, the sodium pump KR2, channelrhodopsins and Anabaena sensory rhodopsin. We outline the usefulness of theoretic biophysics approaches to the study of retinal proteins, challenges in studying the hydrogen-bond dynamics of rhodopsin active sites, and implications for conformational coupling in membrane transporters.

Solid-State NMR Provides Evidence for Small-Amplitude Slow Domain Motions in a Multispanning Transmembrane α-Helical Protein.

Proteins are dynamic entities and populate ensembles of conformations. Transitions between states within a conformational ensemble occur over a broad spectrum of amplitude and time scales, and are often related to biological function. Whereas solid-state NMR (SSNMR) spectroscopy has recently been used to characterize conformational ensembles of proteins in the microcrystalline states, its applications to membrane proteins remain limited. Here we use SSNMR to study conformational dynamics of a seven-helical transmembrane (TM) protein, Anabaena Sensory Rhodopsin (ASR) reconstituted in lipids. We report on site-specific measurements of the 15N longitudinal R1 and rotating frame R1ρ relaxation rates at two fields of 600 and 800 MHz and at two temperatures of 7 and 30 °C. Quantitative analysis of the R1 and R1ρ values and of their field and temperature dependencies provides evidence of motions on at least two time scales. We modeled these motions as fast local motions and slower collective motions of TM helices and of structured loops, and used the simple model-free and extended model-free analyses to fit the data and estimate the amplitudes, time scales and activation energies. Faster picosecond (tens to hundreds of picoseconds) local motions occur throughout the protein and are dominant in the middle portions of the TM helices. In contrast, the amplitudes of the slower collective motions occurring on the nanosecond (tens to hundreds of nanoseconds) time scales, are smaller in the central parts of helices, but increase toward their cytoplasmic sides as well as in the interhelical loops. ASR interacts with a soluble transducer protein on its cytoplasmic surface, and its binding affinity is modulated by light. The larger amplitude of motions on the cytoplasmic side of the TM helices correlates with the ability of ASR to undergo large conformational changes in the process of binding/unbinding the transducer.