Molecular Modeling of Fluorescent SERCA Biosensors

Svensson, B. G.; Autry, Joseph M.; Thomas, David D.

doi:10.1007/978-1-4939-3179-8_42

Cited by 3 publications

(4 citation statements)

References 23 publications

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“…These include null linker constructs with a flexible peptide between the FPs and a FRET biosensor that monitors the structural changes of the human cardiac calcium pump (SERCA2a), as it pumps and stores calcium ions in the sarcoplasm reticulum, as required for proper heart relaxation. This two-color SERCA (2CS) [20] biosensor has been extensively evaluated using multiple FRET techniques [1,2,19,24] including single-molecule FRET [15]. It serves as a reliable FRET biosensor to evaluate our new FRET technology and assays.…”

Section: Resultsmentioning

confidence: 99%

Red-Shifted FRET Biosensors for High-Throughput Fluorescence Lifetime Screening

Schaaf

Grant

et al. 2018

Biosensors

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We have developed fluorescence resonance energy transfer (FRET) biosensors with red-shifted fluorescent proteins (FP), yielding improved characteristics for time-resolved (lifetime) fluorescence measurements. In comparison to biosensors with green and red FRET pairs (GFP/RFP), FPs that emit at longer wavelengths (orange and maroon, OFP/MFP) increased the FRET efficiency, dynamic range, and signal-to-background of high-throughput screening (HTS). OFP and MFP were fused to specific sites on the human cardiac calcium pump (SERCA2a) for detection of structural changes due to small-molecule effectors. When coupled with a recently improved HTS fluorescence lifetime microplate reader, this red-shifted FRET biosensor enabled high-precision nanosecond-resolved fluorescence decay measurements from microliter sample volumes at three minute read times per 1536-well-plate. Pilot screens with a library of small-molecules demonstrate that the OFP/MFP FRET sensor substantially improves HTS assay quality. These high-content FRET methods detect minute FRET changes with high precision, as needed to elucidate novel structural mechanisms from small-molecule or peptide regulators discovered through our ongoing HTS efforts. FRET sensors that emit at longer wavelengths are highly attractive to the FRET biosensor community for drug discovery and structural interrogation of new therapeutic targets.

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Section: Resultsmentioning

confidence: 99%

Red-Shifted FRET Biosensors for High-Throughput Fluorescence Lifetime Screening

Schaaf

Grant

et al. 2018

Biosensors

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“…Vogel et al ., compares the dependence of <E> for CFP on the assumption of dynamic and static models, and demonstrates that the apparent FRET values observed (between 0.10–0.15) for the SERCA2a-PLB biosensor are similar assuming either model 58 . Further, the κ 2 parameter was calculated by simulation for a SERCA molecule with CFP attached at the N-terminus using a similar length linker as used in this study 59 . The orientation factor is 0.6, which is slightly lower than what is predicted by a dynamic model 59 .…”

Section: Discussionmentioning

confidence: 99%

“…Further, the κ 2 parameter was calculated by simulation for a SERCA molecule with CFP attached at the N-terminus using a similar length linker as used in this study 59 . The orientation factor is 0.6, which is slightly lower than what is predicted by a dynamic model 59 .…”

Section: Discussionmentioning

confidence: 99%

Targeting protein-protein interactions for therapeutic discovery via FRET-based high-throughput screening in living cells

Stroik

Yuen

Janicek

et al. 2018

Sci Rep

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We have developed a structure-based high-throughput screening (HTS) method, using time-resolved fluorescence resonance energy transfer (TR-FRET) that is sensitive to protein-protein interactions in living cells. The membrane protein complex between the cardiac sarcoplasmic reticulum Ca-ATPase (SERCA2a) and phospholamban (PLB), its Ca-dependent regulator, is a validated therapeutic target for reversing cardiac contractile dysfunction caused by aberrant calcium handling. However, efforts to develop compounds with SERCA2a-PLB specificity have yet to yield an effective drug. We co-expressed GFP-SERCA2a (donor) in the endoplasmic reticulum membrane of HEK293 cells with RFP-PLB (acceptor), and measured FRET using a fluorescence lifetime microplate reader. We screened a small-molecule library and identified 21 compounds (Hits) that changed FRET by >3SD. 10 of these Hits reproducibly alter SERCA2a-PLB structure and function. One compound increases SERCA2a calcium affinity in cardiac membranes but not in skeletal, suggesting that the compound is acting specifically on the SERCA2a-PLB complex, as needed for a drug to mitigate deficient calcium transport in heart failure. The excellent assay quality and correlation between structural and functional assays validate this method for large-scale HTS campaigns. This approach offers a powerful pathway to drug discovery for a wide range of protein-protein interaction targets that were previously considered “undruggable”.

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“…The horse SERCA1a protein sequence (this work) and the rabbit SERCA1a protein sequence (GenBank accession number ABW96358.1 6 ) were aligned manually, identifying 48 residue variations and a 1-residue deletion at position 504 in horse (Figure S1). The Modeller 9.17 software package (161,162) was used to construct the homology model of horse SERCA1a (Figure 3), as previously utilized to construct molecular models of rabbit SERCA1a labelled with small-molecule fluorophore or GFP derivative (98,163). During the modeling procedure, only the horse residues that were substituted from the rabbit amino acid sequence were allowed to be optimized, plus the two residues on each side of the deletion 504 in horse SERCA1a.…”

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confidence: 99%

Horse gluteus is a null-sarcolipin muscle with enhanced sarcoplasmic reticulum calcium transport

Karim

Svensson

et al. 2019

Preprint

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We have analyzed gene transcription, protein expression, and enzymatic activity of the Ca 2+ -transporting ATPase (SERCA) in horse gluteal muscle. Horses are bred for peak athletic performance but exhibit a high incidence of exertional rhabdomyolysis, with myosolic Ca 2+ suggested as a correlative linkage. To assess Ca 2+ regulation in horse gluteus, we developed an improved protocol for isolating horse sarcoplasmic reticulum (SR) vesicles. RNA-seq and immunoblotting determined that the ATP2A1 gene (protein product SERCA1) is the predominant Ca 2+ -ATPase expressed in horse gluteus, as in rabbit muscle. Gene expression was assessed for four regulatory peptides of SERCA, finding that sarcolipin (SLN) is the predominant regulatory peptide transcript expressed in horse gluteus, as in rabbit muscle. Surprisingly, the RNA transcription ratio of SLN-to-ATP2A1 in horse gluteus is an order of magnitude higher than in rabbit muscle, but conversely, the protein expression ratio of SLN-to-SERCA1 in horse gluteus is an order of magnitude lower than in rabbit. Thus, the SLN gene is not translated to a stable protein in horse gluteus, yet the suprahigh level of SLN RNA suggests a non-coding role. Gel-stain analysis revealed that horse SR expresses calsequestrin (CASQ) protein abundantly, with a CASQ-to-SERCA ratio ~3-fold greater than rabbit SR. The Ca 2+ transport rate of horse SR vesicles is ~2-fold greater than rabbit SR, suggesting horse myocytes have enhanced luminal Ca 2+ stores that increase intracellular Ca 2+ release and muscular performance. The absence of SLN inhibition of SERCA and the abundant expression of CASQ may potentiate horse muscle contractility and susceptibility to exertional rhabdomyolysis. The horse has been selectively bred for thousands of years to achieve remarkable athletic ability, in part conferred by a naturally-high proportion (75-95%) of fast-twitch myofibers in locomotor muscles that provide powerful contractions and high speed (1). Calcium (Ca 2+ ) 1 is the signaling molecule that controls muscle contraction by activating the actomyosin sliding-filament mechanism (2). In turn, myosolic Ca 2+ is controlled predominantly by the sarcoplasmic reticulum (SR), a membrane organelle that connects with transverse tubules as membrane junctions and also surrounds actomyosin myofibrils as longitudinal sheaths (3). Thus, SR is a dual structure/function membrane system comprising junctional SR with the ryanodine receptor Ca 2+ release channel (RYR) acting to initiate contraction, and longitudinal SR with the Ca 2+ transporting ATPase (SERCA) acting to induce relaxation (4,5). For muscle contraction, the bolus of released Ca 2+ originates almost solely from SR through the RYR channel, and this released Ca 2+ is subsequently re-sequestered in the SR lumen by SERCA (6). The strength of muscle contraction is controlled by the amount of Ca 2+ released from SR, which is in turn modulated by the Ca 2+ load in the SR lumen (7). Calsequestrin (CASQ), which binds up to 80 Ca 2+ ions (mol/mol), is the high-capacity ...

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Molecular Modeling of Fluorescent SERCA Biosensors

Cited by 3 publications

References 23 publications

Red-Shifted FRET Biosensors for High-Throughput Fluorescence Lifetime Screening

Red-Shifted FRET Biosensors for High-Throughput Fluorescence Lifetime Screening

Targeting protein-protein interactions for therapeutic discovery via FRET-based high-throughput screening in living cells

Horse gluteus is a null-sarcolipin muscle with enhanced sarcoplasmic reticulum calcium transport

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