Tuning the Flexibility of Glycine-Serine Linkers To Allow Rational Design of Multidomain Proteins.
ABSTRACT: Flexible polypeptide linkers composed of glycine and serine are important components of engineered multidomain proteins. We have previously shown that the conformational properties of Gly-Gly-Ser repeat linkers can be quantitatively understood by comparing experimentally determined Förster resonance energy transfer (FRET) efficiencies of ECFP-linker-EYFP proteins to theoretical FRET efficiencies calculated using wormlike chain and Gaussian chain models. Here we extend this analysis to include linkers with different glycine contents. We determined the FRET efficiencies of ECFP-linker-EYFP proteins with linkers ranging in length from 25 to 73 amino acids and with glycine contents of 33.3% (GSSGSS), 16.7% (GSSSSSS), and 0% (SSSSSSS). The FRET efficiency decreased with an increasing linker length and was overall lower for linkers with less glycine. Modeling the linkers using the WLC model revealed that the experimentally observed FRET efficiencies were consistent with persistence lengths of 4.5, 4.8, and 6.2 Å for the GSSGSS, GSSSSS, and SSSSSS linkers, respectively. The observed increase in linker stiffness with reduced glycine content is much less pronounced than that predicted by a classical model developed by Flory and co-workers. We discuss possible reasons for this discrepancy as well as implications for using the stiffer linkers to control the effective concentrations of connected domains in engineered multidomain proteins.
Project description:This article supplies raw data related to a research article entitled "Joint refinement of FRET measurements using spectroscopic and computational tools" (Kyrychenko et al., 2017) , in which we demonstrate the use of molecular dynamics simulations to estimate FRET orientational factors in a benchmark donor-linker-acceptor system of enhanced cyan (ECFP) and enhanced yellow (EYFP) fluorescent proteins. This can improve the recalculation of donor-acceptor distance information from single-molecule FRET measurements.
Project description:ATP-sensitive K(+) (K(ATP)) channels play important roles in the regulation of membrane excitability in many cell types. ATP inhibits channel activity by binding to a specific site formed by the N and C termini of the pore-forming subunit, Kir6.2, but the structural changes associated with this interaction remain unclear. Here, we use fluorescence resonance energy transfer (FRET) to study the ATP-dependent interaction between the N and C termini of Kir6.2 using a construct bearing fused cyan and yellow fluorescent proteins (ECFP-Kir6.2-EYFP). When expressed in human embryonic kidney cells, ECFP-Kir6.2-EYFP/SUR1 channels displayed FRET that was augmented by agonist stimulation and diminished by metabolic poisoning. Addition of ATP to permeabilized cells or isolated plasma membrane sheets increased FRET. FRET changes were abolished by Kir6.2 mutations that altered ATP-dependent channel closure and channel gating. In the wild-type channel, the ATP concentrations, which increased FRET (EC(50) = 1.36 mM), were significantly higher than those causing channel inhibition (IC(50) = 0.29 mM). Demonstrating the existence of intermolecular interactions, a dimeric construct comprising two molecules of Kir6.2 linked head-to-tail (ECFP-Kir6.2-Kir6.2-EYFP) displayed less FRET than the monomer in the absence of nucleotide but still exhibited ATP-dependent FRET increases (EC(50) = 1.52 mM) and channel inhibition. We conclude that binding of ATP to Kir6.2, (i). alters the interaction between the N- and C-terminal domains, (ii). probably involves both intrasubunit and intersubunit interactions, (iii). reflects ligand binding not channel gating, and (iv). occurs in intact cells when subplasmalemmal [ATP] changes in the millimolar range.
Project description:The Ku70/80 heterodimer binds to DNA ends and attracts other proteins involved in the non-homologous end-joining (NHEJ) pathway of DNA double-strand break repair. We developed a novel assay to measure DNA binding and release kinetics using differences in Förster resonance energy transfer (FRET) of the ECFP-Ku70/EYFP-Ku80 heterodimer in soluble and DNA end bound states. We confirmed that the relative binding efficiencies of various DNA substrates (blunt, 3 nucleotide 5' extension, and DNA hairpin) measured in the FRET assay reflected affinities obtained from direct measurements using surface plasmon resonance. The FRET assay was subsequently used to investigate Ku70/80 behavior in the context of a DNA-dependent kinase (DNA-PK) holocomplex. As expected, this complex was much more stable than Ku70/80 alone, and its stability was influenced by DNA-PK phosphorylation status. Interestingly, the Ku80 C-terminal extension contributed to DNA-PK complex stability but was not absolutely required for its formation. The Ku70 C-terminal SAP domain, on the other hand, was required for the stable association of Ku70/80 to DNA ends, but this effect was abrogated in DNA-PK holocomplexes. We conclude that FRET measurements can be used to determine Ku70/80 binding kinetics. The ability to do this in complex mixtures makes this assay particularly useful to study larger NHEJ protein complexes on DNA ends.
Project description:The peroxisome proliferator-activated receptor gamma (PPAR? or PPARG) belongs to the nuclear receptor superfamily, and is a potential drug target for a variety of diseases. In this work, we constructed a series of bacterial biosensors for the identification of functional PPAR? ligands. These sensors entail modified Escherichia coli cells carrying a four-domain fusion protein, comprised of the PPAR? ligand binding domain (LBD), an engineered mini-intein domain, the E. coli maltose binding protein (MBD), and a thymidylate synthase (TS) reporter enzyme. E. coli cells expressing this protein exhibit hormone ligand-dependent growth phenotypes. Unlike our published estrogen (ER) and thyroid receptor (TR) biosensors, the canonical PPAR? biosensor cells displayed pronounced growth in the absence of ligand. They were able to distinguish agonists and antagonists, however, even in the absence of agonist. To improve ligand sensitivity of this sensor, we attempted to engineer and optimize linker peptides flanking the PPAR? LBD insertion point. Truncation of the original linkers led to decreased basal growth and significantly enhanced ligand sensitivity of the PPAR? sensor, while substitution of the native linkers with optimized G(4)S (Gly-Gly-Gly-Gly-Ser) linkers further increased the sensitivity. Our studies demonstrate that the properties of linkers, especially the C-terminal linker, greatly influence the efficiency and fidelity of the allosteric signal induced by ligand binding. Our work also suggests an approach to increase allosteric behavior in this multidomain sensor protein, without modification of the functional LBD.
Project description:Most ion channels are regulated by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) in the cell membrane by diverse mechanisms. Important molecular tools to study ion channel regulation by PtdIns(4,5)P(2) in living cells have been developed in the past. These include fluorescent PH-domains as sensors for Förster resonance energy transfer (FRET), to monitor changes in plasma membrane(.) For controlled and reversible depletion of PtdIns(4,5)P(2), voltage-sensing phosphoinositide phosphatases (VSD) have been demonstrated as a superior tool, since they are independent of cellular signaling pathways. Combining these methods in intact cells requires multiple transfections. We used self-cleaving viral 2A-peptide sequences for adenovirus driven expression of the PH-domain of phospholipase-C?1 (PLC?1) fused to ECFP and EYFP respectively and Ciona intestinalis VSP (Ci-VSP), from a single open reading frame (ORF) in adult rat cardiac myocytes.Expression and correct targeting of ECFP-PH-PLC?1(,) EYFP-PH-PLC?1, and Ci-VSP from a single tricistronic vector containing 2A-peptide sequences first was demonstrated in HEK293 cells by voltage-controlled FRET measurements and Western blotting. Adult rat cardiac myocytes expressed Ci-VSP and the two fluorescent PH-domains within 4 days after gene transfer using the vector integrated into an adenoviral construct. Activation of Ci-VSP by depolarization resulted in rapid changes in FRET ratio indicating depletion of PtdIns(4,5)P(2) in the plasma membrane. This was paralleled by inhibition of endogenous G protein activated K(+) (GIRK) current. By comparing changes in FRET and current, a component of GIRK inhibition by adrenergic receptors unrelated to depletion of PtdIns(4,5)P(2) was identified.Expression of a FRET sensor pair and Ci-VSP from a single ORF provides a useful approach to study regulation of ion channels by phosphoinositides in cell lines and transfection-resistant postmitotic cells. Generally, adenoviral constructs containing self-cleaving 2A-peptide sequences are highly suited for simultaneous transfer of multiple genes in adult cardiac myocytes.
Project description:Neurotensin receptor 1 (NTS1), a Family A G-protein coupled receptor (GPCR), was expressed in Escherichia coli as a fusion with the fluorescent proteins eCFP or eYFP. A fluorophore-tagged receptor was used to study the multimerization of NTS1 in detergent solution and in brain polar lipid bilayers, using fluorescence resonance energy transfer (FRET). A detergent-solubilized receptor was unable to form FRET-competent complexes at concentrations of up to 200 nM, suggesting that the receptor is monomeric in this environment. When reconstituted into a model membrane system at low receptor density, the observed FRET was independent of agonist binding, suggesting constitutive multimer formation. In competition studies, decreased FRET in the presence of untagged NTS1 excludes the possibility of fluorescent protein-induced interactions. A simulation of the experimental data indicates that NTS1 exists predominantly as a homodimer, rather than as higher-order multimers. These observations suggest that, in common with several other Family A GPCRs, NTS1 forms a constitutive dimer in lipid bilayers, stabilized through receptor-receptor interactions in the absence of other cellular signaling components. Therefore, this work demonstrates that well-characterized model membrane systems are useful tools for the study of GPCR multimerization, allowing fine control over system composition and complexity, provided that rigorous control experiments are performed.
Project description:A variety of fusion proteins between the versatile redox partners flavodoxin (FldA) and flavodoxin reductase (Fpr) from Escherichia coli was constructed with the aim to improve the electron transfer properties. The order in which FldA and Fpr were fused and the linker region between them was varied in a systematic manner. A simple molecular tool, designated "DuaLinX", was developed that facilitated the parallel introduction of flexible glycine-rich and rigid proline-rich linkers between the fusion partners in a single cloning event. The fusion constructs were tested for their ability to transfer electrons to cytochrome c and cytochrome P450 109B1 from Bacillus subtilis. With CYP109B1, the performance of the constructs showed, independent of the domain order, a strong dependency on linker length, whereas with cytochrome c this phenomenon was less pronounced. Constructs carrying linkers of ?15 residues effectively supported the CYP109B1-catalysed hydroxylation of myristic acid. Constructs carrying proline-rich linkers generally outperformed their glycine-rich counterparts. The best construct, FldA-Fpr carrying linker ([E/L]PPPP)4, supported CYP109B1 activity equally well as equivalent amounts of the non-fused redox partners, while cytochrome c reductase activity was ~2.7-fold improved. Thus, to functionally connect redox partners, rigid proline-rich linkers may be attractive alternatives to the commonly used flexible glycine-rich linkers.
Project description:There are many multidomain allosteric proteins where an allosteric signal at the allosteric domain modifies the activity of the functional domain. Intrinsically disordered regions (linkers) are widely involved in this kind of regulation process, but the essential role they play therein is not well understood. Here, we investigated the effect of linkers in stabilizing the open or the closed states of multidomain proteins using combined thermodynamic deduction and coarse-grained molecular dynamics simulations. We revealed that the influence of linker can be fully characterized by an effective local concentration [B]0 . When Kd is smaller than [B]0 , the closed state would be favored; while the open state would be preferred when Kd is larger than [B]0 . We used four protein systems with markedly different domain-domain binding affinity and structural order/disorder as model systems to understand the relationship between [B]0 and the linker length as well as its flexibility. The linker length is the main practical determinant of [B]0 . [B]0 of a flexible linker with 40-60 residues was determined to be in a narrow range of 0.2-0.6 mM, while a too short or too long length would dramatically decrease [B]0 . With the revealed [B]0 range, the introduction of a flexible linker makes the regulation of weakly interacting partners possible.
Project description:The objective of the study was to elucidate optical characteristics of the chromophore structures of fluorescent proteins. Raman spectra of commonly used GFP-like fluorescent proteins (FPs) with diverse emission wavelengths (green, yellow, cyan and red), including the enhanced homogenous FPs EGFP, EYFP, and ECFP (from jellyfish) as well as mNeptune (from sea anemone) were measured. High-quality Raman spectra were obtained and many marker bands for the chromophore of the FPs were identified via assignment of Raman spectra bands. We report the presence of a positive linear correlation between the Raman band shift of C5=C6 and the excitation energy of FPs, demonstrated by plotting absorption maxima (cm-1) against the position of the Raman band C5=C6 in EGFP, ECFP, EYFP, the anionic chromophore and the neutral chromophore. This study revealed new Raman features in the chromophores of the observed FPs, and may contribute to a deeper understanding of the optical properties of FPs.
Project description:Ppmar1 and Ppmar2 are two active mariner-like elements (MLEs) cloned from moso bamboo (Phyllostachys edulis (Carrière) J. Houz) genome possessing transposases that harbour nuclear export signal (NES) domain, but not any nuclear localization signal (NLS) domain. To understand the functions of NES in transposon activity, we have conducted two experiments, fluorescence and excision frequency assays in the yeast system. For this, by site-directed mutagenesis, three NES mutants were developed from each of the MLE. In the fluorescence assay, the mutants, NES-1, 2 and 3 along with the wild types (NES-0) were fused with fluorescent proteins, enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) were co-transformed into yeast system. To differentiate protein localisation under the NES influence, ECFP alone was fused to wild and mutant NES domains either on N- or C-terminal and not to EYFP. Fluorescence assay revealed that blue fluorescence of ECFP was more intense than the red fluorescence of the EYFP in the yeast cell matrix. Further, ECFP had a wider localisation in the cellular matrix, but EYFP was largely located in the nucleus. The NES-1 domain was related to the comparatively high spread of ECFP, while NES-2 and NES-3 indicated a low spread, implying that NES activity on nuclear export increased when the NES is made leucine-rich, while the signalling activity was reduced when the leucine content was lowered in the NES domain. In the transposon excision assay, the mutant and wild type NES of both the Ppmar elements were integrated into an Ade2 vector, and within the Ade2 gene. Co-transformation of the vector together with non-autonomous Ppmar transposons and NES-lacking transposases was used to assess the differential excision frequencies of the mutants NES domains. In both the MLEs, NES-1 had the highest excision suppression, which was less than half of the excision frequency of the wild type. NES-2 and NES-3 elements showed, up to three times increase in transposon excision than the wild types. The results suggested that NES is an important regulator of nuclear export of transposase in Ppmar elements and the mutation of the NES domains can either increase or decrease the export signalling. We speculate that in moso bamboo, NESs regulates the transposition activity of MLEs to maintain the genome integrity.