Effects on the conformation of FVIIa by sTF and Ca²⁺ binding: studies of fluorescence resonance energy transfer and quenching

The apparent length of FVIIa in solution was estimated by a FRET analysis. Two fluorescent probes, fluorescein (Fl-FPR) and a rhodamine derivative (TMR), were covalently attached to FVIIa. The binding site of Fl-FPR was in the protease domain whereas TMR was positioned in the Gla domain, thus allowing a length measure over virtually the whole extension of the protein. From the FRET measurements, the distances between the two probes were determined to be 61.4 for free FVIIa and 65.5? for FVIIa bound to soluble tissue factor (sTF). These seemingly short distances, compared to those anticipated based on the complex crystal structure, require that the probes stretch towards each other. Thus, the apparent distance from the FRET analysis was shown to increase with 4? upon formation of a complex with sTF in solution. However, considering how protein dynamics, based on recent molecular dynamics simulations of FVIIa and sTF:FVIIa (Y.Z. Ohkubo, J.H. Morrissey, E. Tajkhorshid, J. Thromb. Haemost. 8 (2010) 1044-1053), can influence the apparent fluorescence signal our calculations indicated that the global average conformation of active-site inhibited FVIIa is nearly unaltered upon ligation to sTF. It is known from amidolytic activity measurements that Ca(2+) binding leads to activation of FVIIa, but we have for the first time directly demonstrated conformational changes in the environment of the active site upon Ca(2+) binding. Interestingly, this Ca(2+)-induced conformational change can be noted even in the presence of an inhibitor. Forming a complex with sTF further stabilized this conformational change, leading to a more inaccessible active-site located probe.     

Effects on the conformation of FVIIa by sTF and Ca binding: Studies of fluorescence resonance energy transfer and quenching

The apparent length of FVIIa in solution was estimated by a FRET analysis. Two fluorescent probes, fluorescein (Fl-FPR) and a rhodamine derivative (TMR), were covalently attached to FVIIa. The binding site of Fl-FPR was in the protease domain whereas TMR was positioned in the Gla domain, thus allowing a length measure over virtually the whole extension of the protein. From the FRET measurements, the distances between the two probes were determined to be 61.4 for free FVIIa and 65.5 Å for FVIIa bound to soluble tissue factor (sTF). These seemingly short distances, compared to those anticipated based on the complex crystal structure, require that the probes stretch towards each other. Thus, the apparent distance from the FRET analysis was shown to increase with 4 Å upon formation of a complex with sTF in solution. However, considering how protein dynamics, based on recent molecular dynamics simulations of FVIIa and sTF:FVIIa (Y.Z. Ohkubo, J.H. Morrissey, E. Tajkhorshid, J. Thromb. Haemost. 8 (2010) 1044–1053), can influence the apparent fluorescence signal our calculations indicated that the global average conformation of active-site inhibited FVIIa is nearly unaltered upon ligation to sTF.It is known from amidolytic activity measurements that Ca²⁺ binding leads to activation of FVIIa, but we have for the first time directly demonstrated conformational changes in the environment of the active site upon Ca²⁺ binding. Interestingly, this Ca²⁺-induced conformational change can be noted even in the presence of an inhibitor. Forming a complex with sTF further stabilized this conformational change, leading to a more inaccessible active-site located probe.     

Rational design of genetically encoded fluorescence resonance energy transfer-based sensors of cellular Cdc42 signaling

The temporal and spatial control of Rho GTPase signaling pathways is a central issue in understanding the molecular mechanisms that generate complex cellular movements. The Rho protein Cdc42 induces a significant conformational change in its downstream effector, the Wiskott-Aldrich syndrome protein (WASP). On the basis of this conformational change, we have created a series of single-molecule sensors for both active Cdc42 and Cdc42 guanine nucleotide exchange factors (GEFs) that utilize fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent proteins. In vitro, the Cdc42 sensors produce up to 3.2-fold FRET emission ratio changes upon binding active Cdc42. The GEF sensors yield up to 1.7-fold changes in FRET upon exchange of GDP for GTP. The GEF-catalyzed rate of nucleotide exchange for the GEF sensor is indistinguishable from that of wild-type Cdc42, but GAP-catalyzed nucleotide hydrolysis is slowed approximately 16-fold. In vivo, both sensors faithfully report on Cdc42 and/or Cdc42-GEF activity. These results establish the successful creation of rationally designed and genetically encoded tools that can be used to image the activity of biologically and medically important molecules in living systems.     

2-Color calcium pump reveals closure of the cytoplasmic headpiece with calcium binding

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) undergoes conformational changes while transporting calcium, but the details of the domain motions are still unclear. The objective of the present study was to measure distances between the cytoplasmic domains of SERCA2a in order to reveal the magnitude and direction of conformational changes. Using fluorescence microscopy of live cells, we measured intramolecular fluorescence resonance energy transfer (FRET) from a donor fluorescent protein fused to the SERCA N-terminus to an acceptor fluorescent protein fused to either the N-, P-, or transmembrane domain. The "2-color" SERCA constructs were catalytically active as indicated by ATPase activity in vitro and Ca uptake in live cells. All constructs exhibited dynamic FRET changes in response to the pump ligands calcium and thapsigargin (Tg). These FRET changes were quantified as an index of SERCA conformational changes. Intramolecular FRET decreased with Tg for the two N-domain fusion sites (at residue 509 or 576), while the P- (residue 661) and TM-domain (C-terminus) fusions showed increased FRET with Tg. The magnitude of the Tg-dependent conformational change was not decreased by coexpression of phospholamban (PLB), nor did PLB slow the kinetics of Tg binding. FRET in ionophore-permeabilized cells was lower in EGTA than in saturating calcium for all constructs, indicating a decrease in domain separation distance with the structural transition from E2 (Ca-free) to E1 (Ca-bound). The data suggest closure of the cytoplasmic headpiece with Ca-binding. The present results provide insight into the structural dynamics of the Ca-ATPase. In addition, the 2-color SERCA constructs developed for this study may be useful for evaluating candidate small molecule regulators of Ca uptake activity.     

A computational tool for designing FRET protein biosensors by rigid-body sampling of their conformational space

Biosensors relying on the fluorescence resonance energy transfer (FRET) between fluorescent proteins have been used for live-cell imaging of cellular events including Ca(2+) signaling. The efficiency of energy transfer between the donor and acceptor fluorescent proteins depends on the relative distance and orientation between them, which become altered by conformational changes of a fused sensory protein caused by a cellular event. In this way, changes in FRET efficiency of Ca(2+) biosensors can be correlated with Ca(2+) concentrations. The design of these FRET biosensors can be improved by modeling conformational changes before and after a cellular event. Hence, a computational tool called FPMOD was developed to predict FRET efficiency changes by constructing FRET biosensors and sampling their conformational space through rigid-body rotation. We showed with FPMOD that our computational modeling approach can qualitatively predict the FRET efficiencies of a range of biosensors, which had strong agreement with experimental results.     

The use of FRET imaging microscopy to detect protein-protein interactions and protein conformational changes in vivo.

Intermolecular and intramolecular FRET between two spectrally overlapping green fluorescent protein variants fused to two different host proteins or at two different sites within the same protein offers a unique opportunity to monitor real-time protein-protein interactions or protein conformational changes. By using fluorescence digital imaging microscopy, one can visualize the location of green fluorescent proteins within a living cell and follow the time course of the changes in FRET corresponding to cellular events at a millisecond time resolution. The observation of such dynamic molecular events in vivo provides vital insight into the action of biological molecules.     

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.     

Detection of SNARE complexes with FRET using the tetracysteine system

The three proteins synaptosomal-associated protein 25 (SNAP-25), Syntaxin-1a and vesicle-associated membrane protein (VAMP-2) are collectively called SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). By assembling into an exocytic complex, the three SNAREs help in catalyzing membrane fusion. Due to lack of probes that adequately reconstitute the intracellular behavior of endogenous SNAREs, the dynamics of SNARE complexes in living cells is poorly understood. Here we describe a new FRET-based probe, called Cerulean-SNAP-25-C4 (CSNAC), that can track the conformational changes undergone by SNAP-25 as exocytic complexes assemble. The fluorescent protein Cerulean was attached to the N terminus and served as a FRET donor. The biarsenical dye FlAsH served as a FRET acceptor and was attached to a short tetracysteine motif (C4) motif inserted into the so-called linker domain of SNAP-25. CSNAC reported successive FRET changes when first Syntaxin-1a and then VAMP-2 were added in vitro. Small tetracysteine insertions used as a FRET acceptor are expected to have less steric hindrance than previously used GFP-based fluorophores. We propose that genetically-encoded tetracysteine tags can be used to study regulated SNARE complex assembly in vivo.     

Design of FRET-based GFP probes for detection of protease inhibitors

In this study, tandem Green fluorescent protein (GFP) fusion proteins were designed to detect proteolytic activity of thrombin based on the principle of fluorescence resonance energy transfer (FRET). The thrombin-specific recognition sequence, LVPR, was strategically placed in between a cyan-emitting mutant of the green fluorescent protein and an enhanced yellow-emitting fluorescent protein to allow thrombin-specific cleavage with detectable changes of FRET signal. A 4.6-fold increase of fluorescence emission ratio was observed upon addition of thrombin. This FRET-based probe was further tested for dose-dependent effects of thrombin specific inhibitor, hirudin. Our result showed a nice correlation between fluorescence emission ratios and concentrations of hirudin with subnanomolar sensitivity. We propose that FRET-based GFP probes can be used for high-throughput screening of protease inhibitors.     

基于GFP的FRET应用

绿色荧光蛋白（GFP）是一种活性荧光标记,已被用来研究基因表达、分子定位,蛋白质折叠和转运;荧光共振能量转移（FRET）是一种无损伤的光学检测方法,能检测到小于纳米的距离变化。将GFP的活性定位标记功能与FRET的高分辨率相结合。为活体研究生物分子的功能和命运开创了新的篇章。作者在介绍GFP和FRET原理的基础上,综述了基于GFP的FRET在蛋白酶活性,蛋白质间相互作用 构象改变研究中的应用。     

Fluorescent Biosensors of Intracellular Targets from Genetically Encoded Reporters to Modular Polypeptide Probes

With the escalation of drug discovery programmes, it has become essential to visualize and monitor biological activities in healthy and pathological cells, with high spatial and temporal resolution. To this aim, the development of probes and sensors, which can report on the levels and activities of specific intracellular targets, has become essential. Together with the discovery of the Green Fluorescent Protein (GFP), and the development of GFP-based reporters, recent advances in the synthesis of small molecule fluorescent probes, and the explosion of fluorescence-based imaging technologies, the biosensor field has witnessed a dramatic expansion of fluorescence-based reporters which can be applied to complex biological samples, living cells and tissues to probe protein/protein interactions, conformational changes and posttranslational modifications. Here, we review recent developments in the field of fluorescent biosensor technology. We describe different varieties and categories of fluorescent biosensors together with an overview of the technologies commonly employed to image biosensors in cellulo and in vivo. We discuss issues and strategies related to the choice of synthetic fluorescent probes, labelling, quenching, caging and intracellular delivery of biosensors. Finally, we provide examples of some well-characterized genetically encoded FRET reporter systems, peptide and protein biosensors and describe biosensor applications in a wide variety of fields.     

Fluorescence of Alexa Fluor Dye Tracks Protein Folding

Fluorescence spectroscopy is an important tool for the characterization of protein folding. Often, a protein is labeled with appropriate fluorescent donor and acceptor probes and folding-induced changes in Förster Resonance Energy Transfer (FRET) are monitored. However, conformational changes of the protein potentially affect fluorescence properties of both probes, thereby profoundly complicating interpretation of FRET data. In this study, we assess the effects protein folding has on fluorescence properties of Alexa Fluor 488 (A488), which is commonly used as FRET donor. Here, A488 is covalently attached to Cys69 of apoflavodoxin from Azotobacter vinelandii. Although coupling of A488 slightly destabilizes apoflavodoxin, the three-state folding of this protein, which involves a molten globule intermediate, is unaffected. Upon folding of apoflavodoxin, fluorescence emission intensity of A488 changes significantly. To illuminate the molecular sources of this alteration, we applied steady state and time-resolved fluorescence techniques. The results obtained show that tryptophans cause folding-induced changes in quenching of Alexa dye. Compared to unfolded protein, static quenching of A488 is increased in the molten globule. Upon populating the native state both static and dynamic quenching of A488 decrease considerably. We show that fluorescence quenching of Alexa Fluor dyes is a sensitive reporter of conformational changes during protein folding.     

Determination of fluorescent probe orientations on biomolecules by conformational searching: algorithm testing and applications to the atomic model of myosin.

The ability of a localized conformational searching method to predict probe orientation was tested on model nucleic acid and protein structures and applied to the prediction of skeletal myosin integrity upon chemical modification of its reactive thiols. Double-stranded oligonucleotides were chemically labeled with donor and acceptor resonance energy transfer probes at each end for distance determinations. These measurements were made independently using a terbium chelate as a donor to each of four chemically and spectroscopically distinct acceptor probes from the xanthene and cyanine dye groups. The choice of acceptor significantly affected the separation distance measured. Conformational searching algorithms on the atomic model corrected for the differences to within 0.2 nm on average. Verifying its usefulness on proteins, the localized conformational searching method determined the orientation of a fluorescent probe on RNase A that corresponds closely to available crystallographic models of the labeled protein (RMS deviation = 0.1 nm). Also, analysis of the symmetry of the fluorophores' structures suggests why FRET orientation factors are often closer to their dynamic average value than might normally be expected. Furthermore, the computational method provides insights about FRET data that are important for assessing the stability of the alpha-helix separating the SH1 and SH2 reactive thiols in skeletal myosin.     

Conformational changes accompany phosphorylation of the epidermal growth factor receptor C-terminal domain

The precise regulation of epidermal growth factor receptor (EGFR) signaling is crucial to its function in cellular growth control. Various studies have suggested that the C-terminal phosphorylation domain, itself a substrate for the EGFR kinase activity, exerts a regulatory influence upon it, although the molecular mechanism for this regulation is unknown. The fluorescence resonance energy transfer (FRET) technique was employed to examine how C-terminal domain conformational changes in the context of receptor activation and autophosphorylation might regulate EGFR enzymatic activity. A novel FRET reporter system was devised in which recombinant purified EGFR intracellular domain (ICD) proteins of varying C-terminal lengths were site-specifically labeled at their extreme C termini with blue fluorescent protein (BFP) and a fluorescent nucleotide analog, 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP), binding at their active sites. This novel BFP/TNP-ATP FRET pair demonstrated efficient energy transfer as evidenced by appreciable BFP-donor quenching by bound TNP-ATP. In particular, a marked reduction in energy transfer was observed for the full-length BFP-labeled EGFR-ICD protein upon phosphorylation, likely reflecting its movement away from the active site. The estimated distances from the BFP module to the TNP-ATP-occupied active site for the full-length and C-terminally truncated proteins also reveal the possible folding geometry of this domain with respect to the kinase core. The present studies demonstrate the first use of BFP/TNP-ATP as a FRET reporter system. Furthermore, the results described here provide biophysical evidence for phosphorylation-dependent conformational changes in the C-terminal phosphorylation domain and its likely interaction with the kinase core.     

Imaging FRET between spectrally similar GFP molecules in single cells

Fluorescence resonance energy transfer (FRET) detection in fusion constructs consisting of green fluorescent protein (GFP) variants linked by a sequence that changes conformation upon modification by enzymes or binding of ligands has enabled detection of physiological processes such as Ca(2+) ion release, and protease and kinase activity. Current FRET microscopy techniques are limited to the use of spectrally distinct GFPs such as blue or cyan donors in combination with green or yellow acceptors. The blue or cyan GFPs have the disadvantages of less brightness and of autofluorescence. Here a FRET imaging method is presented that circumvents the need for spectral separation of the GFPs by determination of the fluorescence lifetime of the combined donor/acceptor emission by fluorescence lifetime imaging microscopy (FLIM). This technique gives a sensitive, reproducible, and intrinsically calibrated FRET measurement that can be used with the spectrally similar and bright yellow and green fluorescent proteins (EYFP/EGFP), a pair previously unusable for FRET applications. We demonstrate the benefits of this approach in the analysis of single-cell signaling by monitoring caspase activity in individual cells during apoptosis.     

Multiple conformational states of DnaA protein regulate its interaction with DnaA boxes in the initiation of DNA replication

DnaA protein is the initiator of genomic DNA replication in prokaryotes. It binds to specific DNA sequences in the origin of DNA replication and unwinds small AT-rich sequences downstream for the assembly of the replisome. The mechanism of activation of DnaA that enables it to bind and organize the origin DNA and leads to replication initiation remains unclear. In this study, we have developed double-labeled fluorescent DnaA probes to analyze conformational states of DnaA protein upon binding DNA, nucleotide, and Soj sporulation protein using Fluorescence Resonance Energy Transfer (FRET). Our studies demonstrate that DnaA protein undergoes large conformational changes upon binding to substrates and there are multiple distinct conformational states that enable it to initiate DNA replication. DnaA protein adopted a relaxed conformation by expanding ~ 15 Å upon binding ATP and DNA to form the ATP·DnaA·DNA complex. Hydrolysis of bound ATP to ADP led to a contraction of DnaA within the complex. The relaxed conformation of DnaA is likely required for the formation of the multi-protein ATP·DnaA·DNA complex. In the initiation of sporulation, Soj binding to DnaA prevented relaxation of its conformation. Soj·ADP appeared to block the activation of DnaA, suggesting a mechanism for Soj·ADP in switching initiation of DNA replication to sporulation. Our studies demonstrate that multiple conformational states of DnaA protein regulate its binding to DNA in the initiation of DNA replication.     

Ratiometric detection of Zn(II) using chelating fluorescent protein chimeras

Fluorescent indicators for the real-time imaging of small molecules or metal ions in living cells are invaluable tools for understanding their physiological function. Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) between fluorescent protein domains have important advantages over synthetic probes, but often suffer from a small ratiometric change. Here, we present a new design approach to obtain sensors with a large difference in emission ratio between the bound and unbound states. De novo Zn(II)-binding sites were introduced directly at the surface of both fluorescent domains of a chimera of enhanced cyan and yellow fluorescent protein, connected by a flexible peptide linker. The resulting sensor ZinCh displayed an almost fourfold change in fluorescence emission ratio upon binding of Zn(II). Besides a high affinity for Zn(II), the sensor was shown to be selective over other physiologically relevant metal ions. Its unique biphasic Zn(II)-binding behavior could be attributed to the presence of two distinct Zn(II)-binding sites and allowed ratiometric fluorescent detection of Zn(II) over a concentration range from 10 nM to 1 mM. Size-exclusion chromatography and fluorescence anisotropy were used to provide a detailed picture of the conformational changes associated with each Zn(II)-binding step. The high affinity for Zn(II) was mainly due to a high effective concentration of the fluorescent proteins and could be understood quantitatively by modeling the peptide linker between the fluorescent proteins as a random coil. The strategy of using chelating fluorescent protein chimeras to develop FRET sensor proteins with a high ratiometric change is expected to be more generally applicable, in particular for other metal ions and small molecules.     

Optical sensors for measuring dynamic changes of cytosolic metabolite levels in yeast

Optical sensors allow dynamic quantification of metabolite levels with subcellular resolution. Here we describe protocols for analyzing cytosolic glucose levels in yeast using genetically encoded F?rster resonance energy transfer (FRET) sensors. FRET glucose sensors with different glucose affinities (K(d)) covering the low nano- to mid- millimolar range can be targeted genetically to the cytosol or to subcellular compartments. The sensors detect the glucose-induced conformational change in the bacterial periplasmic glucose/galactose binding protein MglB using FRET between two fluorescent protein variants. Measurements can be performed with a single sensor or multiple sensors in parallel. In one approach, cytosolic glucose accumulation is measured in yeast cultures in a 96-well plate using a fluorimeter. Upon excitation of the cyan fluorescent protein (CFP), emission intensities of CFP and YFP (yellow fluorescent protein) are captured before and after glucose addition. FRET sensors provide temporally resolved quantitative data of glucose for the compartment of interest. In a second approach, reversible changes of cytosolic free glucose are measured in individual yeast cells trapped in a microfluidic platform, allowing perfusion of different solutions while FRET changes are monitored in a microscope setup. By using the microplate fluorimeter protocol, 96 cultures can be measured in less than 1 h; analysis of single cells of a single genotype can be completed in <2 h. FRET-based analysis has been performed with glucose, maltose, ATP and zinc sensors, and it can easily be adapted for high-throughput screening using a wide spectrum of sensors.     

Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA

We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution through quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). The localization of fl-tRNA to active translation sites was confirmed through its co-localization with cellular factors and its dynamic alterations upon inhibition of protein synthesis. Moreover, fluorescence resonance energy transfer (FRET) signals, generated when fl-tRNAs, separately labeled as a FRET pair occupy adjacent sites on the ribosome, quantitatively reflect levels of protein synthesis in defined cellular regions. In addition, FRET signals enable detection of intra-populational variability in protein synthesis activity. We demonstrate that FtTM allows quantitative comparison of protein synthesis between different cell types, monitoring effects of antibiotics and stress agents, and characterization of changes in spatial compartmentalization of protein synthesis upon viral infection.     

Homomultimerization of the coxsackievirus 2B protein in living cells visualized by fluorescence resonance energy transfer microscopy

The 2B protein of enteroviruses is the viral membrane-active protein that is responsible for the modifications in host cell membrane permeability that take place in enterovirus-infected cells. The 2B protein shows structural similarities to the group of lytic polypeptides, polypeptides that permeate membranes either by forming multimeric membrane-integral pores or, alternatively, by lying parallel to the lipid bilayer and disturbing the curvature and symmetry of the membrane. Our aim is to gain more insight into the molecular architecture of the 2B protein in vivo. In this study, the possible existence of multimers of the coxsackie B3 virus 2B protein in single living cells was explored by fluorescence resonance energy transfer (FRET) microscopy. FRET between fusion proteins 2B-ECFP and 2B-EYFP (enhanced cyan and yellow fluorescent variants of green fluorescent protein) was monitored by using spectral imaging microscopy (SPIM) and fluorescence lifetime imaging microscopy (FLIM). Both techniques revealed the occurrence of intermolecular FRET between 2B-ECFP and 2B-EYFP, providing evidence for the formation of protein 2B homomultimers. Putative models for the mode of action of the membrane-active 2B protein and the formation of membrane-integral pores by 2B multimers are discussed.