Liver-type fatty acid binding protein interacts with hepatocyte nuclear factor 4α

Hepatocyte nuclear factor 4α (HNF4α) regulates liver type fatty acid binding protein (L-FABP) gene expression. Conversely as shown herein, L-FABP structurally and functionally also interacts with HNF4α. Fluorescence resonance energy transfer (FRET) between Cy3-HNF4α (donor) and Cy5-L-FABP (acceptor) as well as FRET microscopy detected L-FABP in close proximity (∼80 Å) to HNF4α, binding with high affinity K_d ∼250–300 nM. Circular dichroism (CD) determined that the HNF4α/L-FABP interaction altered protein secondary structure. Finally, L-FABP potentiated transactivation of HNF4α in COS7 cells. Taken together, these data suggest that L-FABP provides a signaling path to HNF4α activation in the nucleus.     

A fluorescence resonance energy transfer sensor based on maltose binding protein

A fluorescence resonance energy-transfer (FRET) sensing system for maltose based on E. coli maltose binding protein (MBP) is demonstrated. The FRET donor portion of the sensing system consists of MBP modified with long wavelength-excitable cyanine dyes (Cy3 or Cy3.5). The novel acceptor portion of the sensor consists of beta-cyclodextrin (beta-CD) modified with either the cyanine dye Cy5 or the dark quencher QSY9. Binding of the modified beta-CD to dye-conjugated MBP results in assembly of the FRET complex. Added maltose displaces the beta-CD-dye adduct and disrupts the FRET complex, resulting in a direct change in fluorescence of the donor moiety. In the use of these FRET pairs, MBP dissociation values for maltose were estimated (0.14-2.90 microM). Maltose limits of detection were in the 50-100 nm range.     

Fluorescence labeling, purification, and immobilization of a double cysteine mutant calmodulin fusion protein for single-molecule experiments

We present a method of labeling and immobilizing a low-molecular-weight protein, calmodulin (CaM), by fusion to a larger protein, maltose binding protein (MBP), for single-molecule fluorescence experiments. Immobilization in an agarose gel matrix eliminates potential interactions of the protein and the fluorophore(s) with a glass surface and allows prolonged monitoring of protein dynamics. The small size of CaM hinders its immobilization in low-weight-percentage agarose gels; however, fusion of CaM to MBP via a flexible linker provides sufficient restriction of translational mobility in 1% agarose gels. Cysteine residues were engineered into MBP.CaM (MBP-T34C,T110C-CaM) and labeled with donor and acceptor fluorescent probes yielding a construct (MBP.CaM-DA) which can be used for single-molecule single-pair fluorescence resonance energy transfer (spFRET) experiments. Mass spectrometry was used to verify the mass of MBP.CaM-DA. Assays measuring the activity of CaM reveal minimal activity differences between wild-type CaM and MBP.CaM-DA. Single-molecule fluorescence images of the donor and acceptor dyes were fit to a two-dimensional Gaussian function to demonstrate colocalization of donor and acceptor dyes. FRET is demonstrated both in bulk fluorescence spectra and in fluorescence trajectories of single MBP.CaM-DA molecules. The extension of this method to other biomolecules is also proposed.     

Cyclic AMP-dependent protein kinase binding to A-kinase anchoring proteins in living cells by fluorescence resonance energy transfer of green fluorescent protein fusion proteins.

A-kinase anchoring proteins tether cAMP-dependent protein kinase (PKA) to specific subcellular locations. The purpose of this study was to use fluorescence resonance energy transfer to monitor binding events in living cells between the type II regulatory subunit of PKA (RII) and the RII-binding domain of the human thyroid RII anchoring protein (Ht31), a peptide containing the PKA-binding domain of an A-kinase anchoring protein. RII was linked to enhanced yellow fluorescent protein (EYFP), Ht31 was linked to enhanced cyan fluorescent protein (ECFP), and these constructs were coexpressed in Chinese hamster ovary cells. Upon excitation of the donor fluorophore, Ht31.ECFP, an increase in emission of the acceptor fluorophore, RII.EYFP, and a decrease in emission from Ht31.ECFP were observed. The emission ratio (acceptor/donor) was increased 2-fold (p < 0.05) in cells expressing Ht31.ECFP and RII.EYFP compared with cells expressing Ht31P.ECFP, the inactive form of Ht31, and RII.EYFP. These results provide the first in vivo demonstration of RII/Ht31 interaction in living cells and confirm previous in vitro findings of RII/Ht31 binding. Using surface plasmon resonance, we also showed that the green fluorescent protein tags did not significantly alter the binding of Ht31 to RII. Thus, fluorescence resonance energy transfer can be used to directly monitor protein-protein interactions of the PKA signaling pathway in living cells.     

多聚甲醛固定对荧光蛋白分子间FRET效率的影响

目的：研究多聚甲醛固定对利用荧光共振能量转移(fluorescence resonance energy transfer, FRET)检测细胞中蛋白质相互作用的影响,解决运动能力较强的细胞中FRET效率检测的问题。方法：选用两个已知能够相互作用的蛋白分子TRA和TRB,将荧光蛋白ECFP和EYFP的编码基因通过融合PCR分别标记在其C端;将两个融合基因共转染靶细胞,一组细胞经低浓度（0.5%）多聚甲醛短时（0.5~1h）固定,另一组不固定,利用激光共聚焦扫描显微镜检测两个融合蛋白之间的FRET效率,比较其在两组细胞之间的差异情况。结果：经过统计学分析,在活细胞和经低浓度多聚甲醛短时间固定的细胞中,ECFP与EYFP之间的FRET效率没有显著差异。结论：低浓度短时间的多聚甲醛固定对于荧光蛋白分子之间的相互作用没有显著的影响,因此对于运动能力过强的细胞可以固定后再进行FRET检测。     

Fluorescence resonance energy transfer between residues on troponin and tropomyosin in the reconstituted thin filament: modeling the troponin-tropomyosin complex

Troponin (Tn), in association with tropomyosin (Tm), plays a central role in the calcium regulation of striated muscle contraction. Fluorescence resonance energy transfer (FRET) between probes attached to the Tn subunits (TnC, TnI, TnT) and to Tm was measured to study the spatial relationship between Tn and Tm on the thin filament. We generated single-cysteine mutants of rabbit skeletal muscle α-Tm, TnI and the β-TnT 25-kDa fragment. The energy donor was attached to a single-cysteine residue at position 60, 73, 127, 159, 200 or 250 on TnT, at 98 on TnC and at 1, 9, 133 or 181 on TnI, while the energy acceptor was located at 13, 146, 160, 174, 190, 209, 230, 271 or 279 on Tm. FRET analysis showed a distinct Ca²⁺-induced conformational change of the Tm-Tn complex and revealed that TnT60 and TnT73 were closer to Tm13 than Tm279, indicating that the elongated N-terminal region of TnT extends beyond the beginning of the next Tm molecule on the actin filament. Using the atomic coordinates of the crystal structures of Tm and the Tn core domain, we searched for the disposition and orientation of these structures by minimizing the deviations of the calculated FRET efficiencies from the observed FRET efficiencies in order to construct atomic models of the Tn-Tm complex with and without bound Ca²⁺. In the best-fit models, the Tn core domain is located on residues 160-200 of Tm, with the arrowhead-shaped I-T arm tilting toward the C-terminus of Tm. The angle between the Tm axis and the long axis of TnC is ∼ 75° and ∼ 85° with and without bound Ca²⁺, respectively. The models indicate that the long axis of TnC is perpendicular to the thin filament without bound Ca²⁺, and that TnC and the I-T arm tilt toward the filament axis and rotate around the Tm axis by ∼ 20° upon Ca²⁺ binding.     

Heterodimerization of y(+)LAT-1 and 4F2hc visualized by acceptor photobleaching FRET microscopy

y(+)LAT-1 and 4F2hc are the subunits of a transporter complex for cationic amino acids, located mainly in the basolateral plasma membrane of epithelial cells in the small intestine and renal tubules. Mutations in y(+)LAT-1 impair the transport function of this complex and cause a selective aminoaciduria, lysinuric protein intolerance (LPI, OMIM #222700), associated with severe, complex clinical symptoms. The subunits of an active transporter co-localize in the plasma membrane, but the exact process of dimerization is unclear since direct evidence for the assembly of this transporter in intact human cells has not been available. In this study, we used fluorescence resonance energy transfer (FRET) microscopy to investigate the interactions of y(+)LAT-1 and 4F2hc in HEK293 cells expressing y(+)LAT-1 and 4F2hc fused with ECFP or EYFP. FRET was quantified by measuring fluorescence intensity changes in the donor fluorophore (ECFP) after the photobleaching of the acceptor (EYFP). Increased donor fluorescence could be detected throughout the cell, from the endoplasmic reticulum and Golgi complex to the plasma membrane. Therefore, our data prove the interaction of y(+)LAT-1 and 4F2hc prior to the plasma membrane and thus provide evidence for 4F2hc functioning as a chaperone in assisting the transport of y(+)LAT-1 to the plasma membrane.     

Quantitative measurement of cAMP concentration using an exchange protein directly activated by a cAMP-based FRET-sensor

Förster resonance energy transfer (FRET)-based biosensors for the quantitative analysis of intracellular signaling, including sensors for monitoring cyclic adenosine monophosphate (cAMP), are of increasing interest. The measurement of the donor/acceptor emission ratio in tandem biosensors excited at the donor excitation wavelength is a commonly used technique. A general problem, however, is that this ratio varies not only with the changes in cAMP concentration but also with the changes of the ionic environment or other factors affecting the folding probability of the fluorophores. Here, we use a spectral FRET analysis on the basis of two excitation wavelengths to obtain a reliable measure of the absolute cAMP concentrations with high temporal and spatial resolution by using an “exchange protein directly activated by cAMP”. In this approach, FRET analysis is simplified and does not require additional calibration routines. The change in FRET efficiency (E) of the biosensor caused by [cAMP] changes was determined as ΔE = 15%, whereas E varies between 35% at low and 20% at high [cAMP], allowing quantitative measurement of cAMP concentration in the range from 150 nM to 15 μM. The method described is also suitable for other FRET-based biosensors with a 1:1 donor/acceptor stoichiometry. As a proof of principle, we measured the specially resolved cAMP concentration within living cells and determined the dynamic changes of cAMP levels after stimulation of the Gs-coupled serotonin receptor subtype 7 (5-HT7).     

Heterodimerization of yLAT-1 and 4F2hc visualized by acceptor photobleaching FRET microscopy

y⁺LAT-1 and 4F2hc are the subunits of a transporter complex for cationic amino acids, located mainly in the basolateral plasma membrane of epithelial cells in the small intestine and renal tubules. Mutations in y⁺LAT-1 impair the transport function of this complex and cause a selective aminoaciduria, lysinuric protein intolerance (LPI, OMIM #222700), associated with severe, complex clinical symptoms. The subunits of an active transporter co-localize in the plasma membrane, but the exact process of dimerization is unclear since direct evidence for the assembly of this transporter in intact human cells has not been available. In this study, we used fluorescence resonance energy transfer (FRET) microscopy to investigate the interactions of y⁺LAT-1 and 4F2hc in HEK293 cells expressing y⁺LAT-1 and 4F2hc fused with ECFP or EYFP. FRET was quantified by measuring fluorescence intensity changes in the donor fluorophore (ECFP) after the photobleaching of the acceptor (EYFP). Increased donor fluorescence could be detected throughout the cell, from the endoplasmic reticulum and Golgi complex to the plasma membrane. Therefore, our data prove the interaction of y⁺LAT-1 and 4F2hc prior to the plasma membrane and thus provide evidence for 4F2hc functioning as a chaperone in assisting the transport of y⁺LAT-1 to the plasma membrane.     

Monitoring of conformational change in maltose binding protein using split green fluorescent protein

In this study, we describe a novel method for the detection of conformational changes in proteins, which is predicated on the reconstitution of split green fluorescent protein (GFP). We employed fluorescence complementation assays for the monitoring of the conformationally altered proteins. In particular, we used maltose binding protein (MBP) as a model protein, as MBP undergoes a characteristic hinge-twist movement upon substrate binding. The common feature of this approach is that GFP, as a reporter protein, splits into two non-fluorescent fragments, which are genetically fused to the N- and C-termini of MBP. Upon binding to maltose, the chromophores move closer together, resulting in the generation of fluorescence. This split GFP method also involves the reconstitution of GFP, which is determined via observations of the degree to which fluorescence intensity is restored. As a result, reconstituted GFP has been observed to generate fluorescence upon maltose binding in vitro, thereby allowing for the direct detection of changes in fluorescence intensity in response to maltose, in a concentration- and time-dependent fashion. Our findings showed that the fluorescence complementation assay can be used to monitor the conformational alterations of a target protein, and this ability may prove useful in a number of scientific and medical applications.     

Thermostable single domain antibody–maltose binding protein fusion for Bacillus anthracis spore protein BclA detection

We constructed a genetic fusion of a single domain antibody (sdAb) with the thermal stable maltose binding protein from the thermophile Pyrococcus furiosus (PfuMBP). Produced in the Escherichia coli cytoplasm with high yield, it proved to be a rugged and effective immunoreagent. The sdAb–A5 binds BclA, a Bacillus anthracis spore protein, with high affinity (K_D ∼ 50 pM). MBPs, including the thermostable PfuMBP, have been demonstrated to be excellent folding chaperones, improving production of many recombinant proteins. A three-step purification of E. coli shake flask cultures of PfuMBP–sdAb gave a yield of approximately 100 mg/L highly purified product. The PfuMBP remained stable up to 120 °C, whereas the sdAb–A5 portion unfolded at approximately 68 to 70 °C but could refold to regain activity. This fusion construct was stable to heating at 1 mg/ml for 1 h at 70 °C, retaining nearly 100% of its binding activity; nearly one-quarter (24%) activity remained after 1 h at 90 °C. The PfuMBP–sdAb construct also provides a stable and effective method to coat gold nanoparticles. Most important, the construct was found to provide enhanced detection of B. anthracis Sterne strain (34F2) spores relative to the sdAb–A5 both as a capture reagent and as a detection reagent.     

Live cell imaging of protein interactions in poliovirus RNA replication complex using fluorescence resonance energy transfer (FRET)

Poliovirus RNA replication is directed by a replication complex on the rosette-like arrangement of membranous vesicles. Proteins derived from the p3 region of the polioviral genome, such as 3D, 3AB, and 3B (VPg), play key roles in the formation and function of the replication complex. In the present study, by using an acceptor photobleaching protocol for fluorescence resonance energy transfer (FRET) imaging, we visualized the interactions of 3D, 3AB, and VPg in living cells. The interaction of 3AB-VPg was determined by live cell FRET analysis. Quantitative analyses showed that the FRET efficiencies of 3AB-3D, VPg-3D, and 3AB-VPg were 3.9 ± 0.4% (n = 36), 4.5 ± 0.4% (n = 39), and 8.3 ± 0.6% (n = 44), respectively, in the cell cytoplasm where viral replication complexes are formed and function. Poliovirus infection enhanced the protein interactions of VPg-3D and 3AB-3D, with FRET efficiencies in the virus-infected cells of 10.7 ± 1.1% (n = 39) and 9.0 ± 0.9% (n = 37), respectively. This method of live cell analysis of protein interactions in the poliovirus RNA replication complex lays the foundation for further understanding of the real-time process of poliovirus RNA replication.     

Improving the flexibility of genetically encoded voltage indicators via intermolecular FRET

A new family of genetically encoded voltage indicators (GEVIs) has been developed based on intermolecular Förster resonance energy transfer (FRET). To test the hypothesis that the GEVI ArcLight functions via interactions between the fluorescent protein (FP) domains of neighboring probes, the FP of ArcLight was replaced with either a FRET donor or acceptor FP. We discovered relatively large FRET signals only when cells were cotransfected with both the FRET donor and acceptor GEVIs. Using a cyan fluorescent protein donor and an RFP acceptor, we were able to observe a voltage-dependent signal with an emission peak separated by over 200 nm from the excitation wavelength. The intermolecular FRET strategy also works for rhodopsin-based probes, potentially improving their flexibility as well. Separating the FRET pair into two distinct proteins has important advantages over intramolecular FRET constructs. The signals are larger because the voltage-induced conformational change moves two FPs independently. The expression of the FRET donor and acceptor can also be restricted independently, enabling greater cell type specificity as well as refined subcellular voltage reporting.     

Terbium, a fluorescent probe for investigation of siderophore pyochelin interactions with its outer membrane transporter FptA

Pyochelin (Pch) is a siderophore and FptA is its outer membrane transporter produced by Pseudomonas aeruginosa to import iron. The fluorescence of the element terbium is affected by coordinated ligands and it can therefore be used as a probe to investigate the pyochelin-iron uptake pathway in P. aeruginosa. At pH 8.0, terbium fluorescence is greatly enhanced in the presence of pyochelin indicating chelation of the metal by the siderophore. Titration curves showed a 2:1 (Pch:Tb³⁺) stoichiometry and an affinity of K =( 2 ± - 1 )× 10¹¹ M^− 2 was determined. Pch-Tb interaction with the transporter FptA could be followed in vitro and in vivo in P. aeruginosa cells, by Fluorescence Resonance Energy Transfer (FRET) between three partners: the tryptophans of FptA (donor), Pch (acceptor for the Trps and donor for Tb³⁺) and Tb³⁺ (acceptor). Pch-Tb binds to the Pch-Fe outer membrane transporter FptA with a dissociation constant (K_d) of 4.6 μM. This three-partner FRET is a potentially valuable tool for investigation of the interactions between FptA and its siderophore Pch.     

Quantitative Comparison of Different Fluorescent Protein Couples for Fast FRET-FLIM Acquisition

The fluorescent-protein based fluorescence resonance energy transfer (FRET) approach is a powerful method for quantifying protein-protein interactions in living cells, especially when combined with fluorescence lifetime imaging microscopy (FLIM). To compare the performance of different FRET couples for FRET-FLIM experiments, we first tested enhanced green fluorescent protein (EGFP) linked to different red acceptors (mRFP1-EGFP, mStrawberry-EGFP, HaloTag (TMR)-EGFP, and mCherry-EGFP). We obtained a fraction of donor engaged in FRET (f_D) that was far from the ideal case of one, using different mathematical models assuming a double species model (i.e., discrete double exponential fixing the donor lifetime and double exponential stretched for the FRET lifetime). We show that the relatively low f_D percentages obtained with these models may be due to spectroscopic heterogeneity of the acceptor population, which is partially caused by different maturation rates for the donor and the acceptor. In an attempt to improve the amount of donor protein engaged in FRET, we tested mTFP1 as a donor coupled to mOrange and EYFP, respectively. mTFP1 turned out to be at least as good as EGFP for donor FRET-FLIM experiments because 1), its lifetime remained constant during light-induced fluorescent changes; 2), its fluorescence decay profile was best fitted with a single exponential model; and 3), no photoconversion was detected. The f_D value when combined with EYFP as an acceptor was the highest of all tandems tested (0.7). Moreover, in the context of fast acquisitions, we obtained a minimal f_D (mf_D) for mTFP1-EYFP that was almost two times greater than that for mCherry-EGFP (0.65 vs. 0.35). Finally, we compared EGFP and mTFP1 in a biological situation in which the fusion proteins were highly immobile, and EGFP and mTFP1 were linked to the histone H4 (EGFP-H4 and mTFP1-H4) in fast FLIM acquisitions. In this particular case, the fluorescence intensity was more stable for EGFP-H4 than for mTFP1-H4. Nevertheless, we show that mTFP1/EYFP stands alone as the best FRET-FLIM couple in terms of f_D analysis.     

The interaction between human PEX3 and PEX19 characterized by fluorescence resonance energy transfer (FRET) analysis

The process of peroxisome biogenesis involves several PEX genes that encode the machinery required to assemble the organelle. Among the corresponding peroxins the interaction between PEX3 and PEX19 is essential for early peroxisome biogenesis. However, the intracellular site of this protein interaction is still unclear. To address this question by fluorescence resonance energy transfer (FRET) analysis, we engineered the enhanced yellow fluorescent protein (EYFP) to the C-terminus of PEX3 and the enhanced cyan fluorescent protein (ECFP) to the N-terminus of PEX19. Functionality of the fusion proteins was shown by transfection of human PEX3- and PEX19-deficient fibroblasts from Zellweger patients with tagged versions of PEX3 and PEX19. This led to reformation of import-competent peroxisomes in both cell lines previously lacking detectable peroxisomal membrane structures. The interaction of PEX3-EYFP with ECFP-PEX19 in a PEX3-deficient cell line during peroxisome biogenesis was visualized by FRET imaging. Although PEX19 was predominantly localized to the cytoplasma, the peroxisome was identified to be the main intracellular site of the PEX3-PEX19 interaction. Results were confirmed and quantified by donor fluorescence photobleaching experiments. PEX3 deletion proteins lacking the N-terminal peroxisomal targeting sequence (PEX3 34-373-EYFP) or the PEX19-binding domain located in the C-terminal half of the protein (PEX3 1-140-EYFP) did not show the characteristic peroxisomal localization of PEX3, but were mislocalized to the cytoplasm (PEX3 34-373-EYFP) or to the mitochondria (PEX3 1-140-EYFP) and did not interact with ECFP-PEX19. We suggest that FRET is a suitable tool to gain quantitative spatial information about the interaction of peroxins during the process of peroxisome biogenesis in single cells. These findings complement and extend data from conventional in vitro protein interaction assays and support the hypothesis of PEX3 being an anchor for PEX19 at the peroxisomal membrane.     

An endo-(1→3)-β-d-glucanase from the scallop Chlamys albidus: catalytic properties, cDNA cloning and secondary-structure characterization

An endo-(1→3)-β-d-glucanase (L₀) with molecular mass of 37 kDa was purified to homogeneity from the crystalline style of the scallop Chlamys albidus. The endo-(1→3)-β-d-glucanase was extremely thermolabile with a half-life of 10 min at 37 °C. L₀ hydrolyzed laminaran with K_m ∼ 0.75 mg/mL, and catalyzed effectively transglycosylation reactions with laminaran as donor and p-nitrophenyl β d-glucoside as acceptor (K_m ∼ 2 mg/mL for laminaran) and laminaran as donor and as acceptor (K_m ∼ 5 mg/mL) yielding p-nitrophenyl β d-glucooligosaccharides (n = 2-6) and high-molecular branching (1→3),(1→6)-β-d-glucans, respectively. Efficiency of hydrolysis and transglycosylation processes depended on the substrate structure and decreased appreciably with the increase of the percentage of β-(1→6)-glycosidic bonds, and laminaran with 10% of β-(1→6)-glycosidic bonds was the optimal substrate for both reactions. The CD spectrum of L₀ was characteristic for a protein with prevailing β secondary-structural elements. Binding L₀ with d-glucose as the best acceptor for transglycosylation was investigated by the methods of intrinsic tryptophan fluorescence and CD. Glucose in concentration sufficient to saturate the enzyme binding sites resulted in a red shift in the maximum of fluorescence emission of 1-1.5 nm and quenching the Trp fluorescence up to 50%. An apparent association constant of L₀ with glucose (K_a = 7.4 × 10⁵ ± 1.1 × 10⁵ M⁻¹) and stoichiometry (n = 13.3 ± 0.7) was calculated. The cDNA encoding L₀ was sequenced, and the enzyme was classified in glycoside hydrolases family 16 on the basis of the amino acid sequence similarity.     

The new fluorogenic substrates of neutrophil proteinase 3 optimized in prime site region

Previously selected by the combinatorial chemistry approach, potent fluorogenic substrate of proteinase 3 was used as the starting structure to design new substrates. The general formula of the synthesized peptides is as follows: ABZ-Tyr-Tyr-Abu-ANB-X-NH₂, where ANB (5-amino-2-nitrobenzoic acid) served as a chromophore and an acceptor of fluorescence, ABZ (aminobenzoic acid) is a donor of fluorescence in these fluorescence resonance energy transfer (FRET) peptides, and X is a proteinogenic amino acid (except Cys). The introduced modifications influenced substrate activity of the synthesized peptides. The highest value of specificity constant for proteinase 3 was obtained for the single peptide with Gln in the discussed position (k_cat/K_M = 275,000 M⁻¹ s⁻¹), which was nearly twice as active as the reference compound (lacking a substituent in the X position). In addition, more efficient energy transfer was observed, due mainly to the bathochromic effect for the introduced modification. This approach opens a new possibility to design potent and highly specific substrates of proteinase 3 and other proteinases optimized in the prime site region.     

Förster distances for fluorescence resonant energy transfer between mCherry and other visible fluorescent proteins

We present, for the red fluorescent protein mCherry acting as both fluorescence resonant energy transfer (FRET) donor and acceptor, Förster critical distance (r₀) values with five important visible fluorescent protein (VFP) variants as well as with itself. The pair EYFP-mCherry exhibits an r₀ of 5.66 nm, equaling or exceeding any combination of VFPs reported previously. Moreover, mCherry should be an excellent chromophore for homo-FRET with an r₀ of 5.10 nm for energy transfer between two mCherry moieties. Finally, mCherry exhibits higher r₀ values than does DsRed. These characteristics, combined with mCherry’s rapid folding and excellent spectral properties, suggest that mCherry constitutes a valuable long-wavelength hetero-FRET acceptor and probe for homo-FRET experiments.     

An ISFET biosensor for the monitoring of maltose-induced conformational changes in MBP

Here we describe an ion sensitive field effect transistor (ISFET) biosensor, which was designed to monitor directly the surface charge of structurally altered maltose binding protein (MBP) upon stimulation with maltose. This study is the first report of the application of a FET biosensor to the monitoring of conformationally changed proteins. Consequently, a significant drop in current on the basis of the charge-dependent capacitance measurement has been clearly observed in response to maltose, but not for the glucose control, thereby indicating that the substrate-specific conformational properties of the target protein could be successfully monitored using the ISFET. Collectively, our results clearly suggest that ISFET provide a high fidelity system for the detection of maltose-induced structural alterations in MBP.