Fluorescence Resonance Energy Transfer Distance Research Articles

A Fluorescence Resonance Energy Transfer Aptasensor for Aflatoxin B1 Based on Ligand-Induced ssDNA Displacement

In this study, a fluorescence resonance energy transfer (FRET)-based aptasensor for the detection of aflatoxin B1 (AFB1) was designed using a carboxyfluorescein (FAM)-labeled aptamer and short complementary DNA (cDNA) labeled with low molecular quencher RTQ1. The sensing principle was based on the detection of restored FAM-aptamer fluorescence due to the ligand-induced displacement of cDNA in the presence of AFB1, leading to the destruction of the aptamer/cDNA duplex and preventing the convergence of FAM and RTQ1 at the effective FRET distance. Under optimal sensing conditions, a linear correlation was obtained between the fluorescence intensity of the FAM-aptamer and the AFB1 concentration in the range of 2.5–208.3 ng/mL with the detection limit of the assay equal to 0.2 ng/mL. The assay time was 30 min. The proposed FRET aptasensor has been successfully validated by analyzing white wine and corn flour samples, with recovery ranging from 76.7% to 91.9% and 84.0% to 86.5%, respectively. This work demonstrates the possibilities of labeled cDNA as an effective and easily accessible tool for sensitive AFB1 detection. The homogeneous FRET aptasensor is an appropriate choice for contaminant screening in complex matrices.

Design of a novel FRET based fluorescent chemosensor and their application for highly sensitive detection of nitroaromatics

Abstract We report the development of a Fluorescence Resonance Energy Transfer (FRET) based sensor system for highly sensitive detection of nitro-aromatic compounds. The sensor system is composed of a copolymer (poly(methyl methacrylate (MMA))-co-7-(4-trifluoromethyl)coumarin-N-methacrylamide (TCMA)) (CP) 1 bearing coumarin derivative in the side chain as donor and curcumin (dye) as an acceptor. Primarily, the fluorescence of the above mentioned copolymer is quenched by non-radiative energy transfer to curcumin which occurs when the two molecules are within FRET distance scale. The efficiency of this FRET process was calculated. A noticeable decrease in the FRET signal is observed as nitroaromatics viz. 2,4-dinitrotoluene (DNT) and 2,4,6-trinitrotoluene (TNT) is gradually introduced into the sensor system. Fluorescence lifetime is measured to validate the efficacy of the energy transfer process in CP-curcumin sensor system. The mechanistic details of molecular interactions between CP and curcumin system are established by infrared spectroscopy and UV–vis absorption spectroscopy.

Multi step FRET among three laser dyes Pyrene, Acriflavine and Rhodamine B

Fluorescence Resonance Energy Transfer (FRET) system using three dyes has been demonstrated. It has been observed that multi step energy transfer occurred from Pyrene to Rhodamine B via Acriflavine. Here Acriflavine acts as an antenna to receive energy from Pyrene and transfer the same to Rhodamine B. This multi step FRET system is advantageous compared to the conventional FRET as this can be used to study molecular level interaction beyond conventional FRET distance (1–10nm) as well as studying multi-branched macromolecules. The introduction of clay enhances the FRET efficiencies among the dye pair, which is an advantage to make the multi step system more useful. Similar approach can be used for increasing FRET efficiencies by using other dyes.

Homo- and hetero-dimerization of human UDP-glucuronosyltransferase 2B7 (UGT2B7) wild type and its allelic variants affect zidovudine glucuronidation activity

Most human UDP-glucuronosyltransferase (UGT; EC 2.4.1.17) genes contain non-synonymous single nucleotide polymorphisms (nsSNPs) which cause amino acid substitutions. Allelic variants caused by nsSNPs may exhibit absent or reduced enzyme activity. UGT2B7 is one of the most important UGTs that glucuronidates abundant endobiotics and xenobiotics, such as estriol, morphine, and anticancer drugs. Three nsSNPs, UGT2B7*71S (211G>T), UGT2B7*2 (802C>T) and UGT2B7*5 (1192G>A) are observed in the UGT2B7 gene, and they code for allozymes UGT2B7*71S (A71S), UGT2B7*2 (H268Y), and UGT2B7*5 (D398N). UGT2B7 has been observed to form oligomers that affect its enzymatic activity and in this study, we investigated protein–protein interactions among UGT2B7 allozymes wild type (WT), A71S, H268Y and D398N, by performing a systematic quantitative fluorescence resonance energy transfer (FRET) analysis in combination with co-immunoprecipitation assay. Quantitative FRET analysis revealed that UGT2B7 allozymes formed homo- and hetero-dimers and showed distinct features in donor–acceptor distances. Both codon 71 and codon 268 in the N-terminal domain were involved in the dimeric interaction. Co-immunoprecipitation experiments also proved that UGT2B7 allozymes formed stable dimers. The glucuronidation activities of homo- and hetero-dimers were further tested with zidovudine as the substrate. An increase in activity was observed when WT hetero-dimerized with A71S compared with homo-dimers, while both H268Y and D398N impaired the activity of WT and A71S by forming hetero-dimers. In addition, zidovudine glucuronidation activity is associated with FRET distance. These findings provide insights into the consequences of amino acid substitution in UGT2B7 on zidovudine glucuronidation and the association between protein–protein interaction and glucuronidation activity.

Structural Characteristic of Folding/Unfolding Intermediate of Pokeweed Anti-viral Protein Revealed by Time-resolved Fluorescence

The structural feature of unfolding intermediate of pokeweed anti-viral protein (PAP) was characterized using time-resolved fluorescence spectroscopic methods to elucidate protein folding/unfolding process. CD and fluorescence spectra consistently demonstrated that the unfolding of PAP completed at 4 M of guanidine hydrochloride (GuHCl). The fluorescence resonance energy transfer (FRET) and time-resolve fluorescence depolarization analysis of Trp208 and Trp237 located in the C-terminal domain of PAP suggested that peculiar unfolding intermediate populated before reaching to the unfolding state. The FRET distance of Trp237 to Tyr182 was extended to more than 28 Å with keeping the compact conformation in the unfolding intermediate state populated in the presence of 2 M GuHCl. On the other hand, Trp208 maintained the energy transfer pair with Tyr72 near the active site, although the rotational freedom was increased a little. There results suggest that the most distinguished structural feature of the unfolding intermediate of PAP is the separation of C-terminal domain from N-terminal domain. FRET and fluorescence depolarization studies also showed that C-terminal domain would be more separated to liberate the segmental motions of Trp208 and Trp237 distinctly at the unfolding state.

Interaction of SERCA with the Transmembrane Domain of Phospholamban Measured by FRET in Live Cells

Phospholamban (PLB) is the key regulator of the SERCA calcium pump. Previous quantitative fluorescence resonance energy transfer (FRET) measurements between Cerulean-SERCA and YFP-PLB have shown that calcium and thapsigargin reduce the affinity of the PLB-SERCA binding interaction. Though a structure change in the regulatory complex would account for this reduced affinity, we detected no change in FRET distance. We hypothesized that in the presence of calcium the transmembrane domain of PLB moves to a secondary site on SERCA without altering the positions of the PLB cytosolic domain or fluorescent protein fusion tag. To test this hypothesis, we engineered a truncated PLB construct (tmPLB) composed of a fluorescent protein fused to the transmembrane domain of PLB (residues 28-52). We observed FRET from Cerulean-SERCA to YFP-tmPLB, but with a significantly longer FRET distance compared to full-length PLB. SERCA-tmPLB FRET was not significantly changed by saturating concentrations of calcium or thapsigargin. Notably, we did not detect inhibition of SERCA by YFP-tmPLB using an in-cell calcium uptake assay, even though other groups have previously shown that the PLB transmembrane domain can inhibit SERCA activity. In addition, oligomerization of the tmPLB was detected by intrapentameric FRET. Compared to full-length PLB, the oligomerization affinity of tmPLB was normal, but we measured a shorter FRET distance as a result of deletion of the cytoplasmic domain. The results demonstrate that tmPLB retains some normal protein-protein interactions despite its apparently anomalous interaction with SERCA. Taken together, the results suggest a non-inhibitory binding interaction between YFP-tmPLB and SERCA, perhaps as a result of steric hindrance by the YFP fusion tag. It is also possible that the truncated PLB binds selectively to a non-inhibitory site on SERCA. Experiments are underway to address these unresolved questions.

Transmembrane Extension and Oligomerization of the CLIC1 Chloride Intracellular Channel Protein upon Membrane Interaction

Chloride intracellular channel proteins (CLICs) differ from most ion channels as they can exist in both soluble and integral membrane forms. The CLICs are expressed as soluble proteins but can reversibly autoinsert into the membrane to form active ion channels. For CLIC1, the interaction with the lipid bilayer is enhanced under oxidative conditions. At present, little evidence is available characterizing the structure of the putative oligomeric CLIC integral membrane form. Previously, fluorescence resonance energy transfer (FRET) was used to monitor and model the conformational transition within CLIC1 as it interacts with the membrane bilayer. These results revealed a large-scale unfolding between the C- and N-domains of CLIC1 as it interacts with the membrane. In the present study, FRET was used to probe lipid-induced structural changes arising in the vicinity of the putative transmembrane region of CLIC1 (residues 24-46) under oxidative conditions. Intramolecular FRET distances are consistent with the model in which the N-terminal domain inserts into the bilayer as an extended α-helix. Further, intermolecular FRET was performed between fluorescently labeled CLIC1 monomers within membranes. The intermolecular FRET shows that CLIC1 forms oligomers upon oxidation in the presence of the membranes. Fitting the data to symmetric oligomer models of the CLIC1 transmembrane form indicates that the structure is large and most consistent with a model comprising approximately six to eight subunits.

Single‐Molecule FRET Ruler Based on Rigid DNA Origami Blocks

Fluorescence resonance energy transfer (FRET) has become a work-horse for distance measurements on the nanometer scale and between single molecules. Recent model systems for the FRET distance dependence such as polyprolines and dsDNA suffered from limited persistence lengths and sample heterogeneity. We designed a series of rigid DNA origami blocks where each block is labeled with one donor and one acceptor at distances ranging between 2.5 and 14 nm. Since all dyes are attached in one plane to the top surface of the origami block, static effects of linker lengths cancel out in contrast to commonly used dsDNA. We used single-molecule spectroscopy to compare the origami-based ruler to dsDNA and found that the origami blocks directly yield the expected distance dependence of energy transfer since the influence of the linkers on the donor-acceptor distance is significantly reduced. Based on a simple geometric model for the inter-dye distances on the origami block, the Förster radius R(0) could directly be determined from the distance dependence of energy transfer yielding R(0)=5.3±0.3 nm for the Cy3-Cy5 pair.

Characterization of the Colicin E1 Channel using Genetically Encoded Fluorescence

From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.Conformational changes in the colicin E1 channel domain upon membrane association (zero membrane potential) were studied by fluorescence resonance energy transfer (FRET). Six single-Cys and single-stop mutants were prepared to probe changes within the inter-helical distances for the soluble and membrane-binding states. The FRET measurement involved the incorporation of a genetically encoded fluorescent amino acid (coumarin) as the fluorescence donor and a selectively labelled cysteine residue tethered with DABMI (4-dimethylaminophenylazophenyl-4’-maleimide) as the FRET acceptor. The fluorescent coumarin residue was incorporated into the protein through the generation of an orthogonal tRNA/aminoacyl-tRNA synthetase pair that allowed the selective incorporation of the coumarin into any site within the colicin channel domain. This powerful approach eliminates the requirement for selective labelling at donor and acceptor sites with two different chromophores. Each of the six mutant channel proteins harbored a stop (TAG) mutation for coumarin incorporation and a cysteine (TGT) mutation for DABMI attachment. A total of 6 inter-helical distances within helices 1 - 6 were estimated using FRET analysis for both the soluble and membrane-bound states. FRET data showed large changes in the inter-helical distances between helices 3 - 6 upon membrane association. Importantly, the FRET distances correlated well with distances from the soluble X-ray crystal structure. In general, the coumarin-DABMI FRET efficiency decreases upon membrane binding, supporting the umbrella model for the colicin closed channel. Furthermore, the membrane-bound state FRET distances showed excellent correlation with the quasi-circular arrangement model. Herein, we report the success of this new approach to the study of a membrane-associated protein, such as colicin E1 [supported by NSERC, ARM].

Structural Changes Induced by a Cardiomyopathy Myosin Essential Light Chain Mutation in Muscle Fibers using Fluorescence Resonance Energy Transfer (FRET)

The myosin ventricular essential light chain (vELC) mutation, Arg154His, causes familial hypertrophic cardiomyopathy (FHC). The classical phenotype of left ventricular hypertrophy is accompanied by mid left ventricular chamber thickening in patients with this ELC mutation. We engineered a cysteine in vELC (A187C) and in vRLC (A93C) to measure the distance changes that may be induced by the R154H-FHC-ELC mutation in isometric muscle fibers using FRET. The donor probe, IAEDANS, was placed on C187-vELC (D) and the acceptor probe, IAF, was placed on C93-vRLC (DA). Both labeled LCs were reconstituted into rabbit skeletal muscle fibers. In rigor wildtype fibers, the FRET distance between the probes is consistent with that determined from the crystal structure of the chicken myosin ELC and RLC at the same sites. Relaxation decreased the rigor distance, while contraction induced a distance intermediate between rigor and relaxation. These results suggest that the LC domain undergoes bending or flexible motions during relaxation and contraction. We then introduced the R154H-FHC mutation in the C187-vELC for distance measurements with C93-vRLC in muscle fibers. The presence of the R154H mutation decreased the distance in the wildtype fibers in rigor, relaxation, and contraction. The maximum change occurred in rigor, while the smallest change occurred during contraction. The decrease in the distances indicates that the FHC mutation induced a conformational change in ELC, thus allowing it to move closer to the RLC, suggesting a bending movement in the LC domain. These results support a hypothesis that the FHC-ELC mutation causes a structural change in ELC that may lead to dysfunction in the muscle, to cause the phenotype of the disease. This work is supported by funds from a NIH grant AR052360 to OR.

Expanding the Scope of Single Molecule FRET with DNA Origami

Typical dimensions of cellular components are on the order of a few nanometers and their diverse cellular functions often involve conformational changes that require movements ranging from fractions of a nanometer up to tens of nanometers. The current state of the art tool for studying conformational dynamics is single molecule Fluorescence Resonance Energy Transfer (FRET), whereby energy transfer between two fluorescent dyes is correlated to their spatial separation; however, distance predictions made by FRET theory are only accurate in the range of ∼ 3-7 nm, and the working range is limited to below 10 nm. Furthermore, quantitative distance predictions require case-specific calibrations.Here we present a nanoscale device constructed by DNA origami that improves the quantitative accuracy of FRET distance predictions and expands its potential working range. The device integrates 3d structures built from self-assembled DNA, attachment sites for molecules of interest, sites for surface immobilization, and fluorescent markers for the direct visualization of biomolecular dimensions and dynamics by FRET. The device takes advantage of a distance calibration that can be generally applied to any molecule of interest. We have demonstrated that the device can easily achieve 1 nm distance resolution. For the purpose of proof-of-concept studies we specifically integrated a piece of double stranded DNA (dsDNA) containing a recognition sequence for Catabolite Activator Protein (CAP), which is know to bend dsDNA upon binding, between the arms of this device. The bending angle will be evaluated with single particle electron microscopy (EM), and FRET microscopy will be employed in solution to resolve real-time kinetics and deformations of CAP-DNA binding.

Coclustering of ErbB1 and ErbB2 Revealed by FRET-Sensitized Acceptor Bleaching

Classical theory states that ligand binding induces the dimerization of ErbB proteins, leading to their activation. Although we and other investigators have shown the existence of preformed homoclusters of ErbB receptors and analyzed their composition, the stoichiometry of their heteroclusters has not been quantitatively described. Here, we report the development of the fluorescence resonance energy transfer (FRET)-sensitized acceptor bleaching (FSAB) technique to quantitate the ratio of ErbB1 and ErbB2 in their heteroclusters. In FSAB, photolabile acceptors within FRET distance from photostable donors are excited and photobleached by FRET, and the fraction of acceptors that are participating in FRET is determined. In quiescent SKBR-3 breast cancer cells, ∼35% of ErbB1 and ∼10% of ErbB2 have been found in heteroclusters. Epidermal growth factor (ligand of ErbB1) increased the fraction of ErbB2 heteroclustering with ErbB1, whereas the ratio of heteroclustered ErbB1 did not change significantly. The fractions of heteroclustered ErbB1 and ErbB2 were independent of their expression levels, indicating that the formation of these clusters is not driven by the law of mass action. In contrast, the FRET efficiency depended on the donor/acceptor ratio as expected. We present a model in which preformed receptor clusters are rearranged upon ligand stimulation, and report that the composition of these clusters can be quantitatively described by the FSAB technique.

Metamorphic Response of the CLIC1 Chloride Intracellular Ion Channel Protein upon Membrane Interaction

A striking feature of the CLIC (chloride intracellular channel) protein family is the ability of its members to convert between a soluble state and an integral membrane channel form. Direct evidence of the structural transition required for the CLIC protein to autonomously insert into the membrane is lacking, largely because of the challenge of probing the conformation of the membrane-bound protein. However, insights into the CLIC transmembrane form can be gained by biophysical methods such as fluorescence resonance energy transfer (FRET) spectroscopy. This approach was used to measure distances from tryptophan 35, located within the CLIC1 putative N-domain transmembrane region, to three native cysteine residues within the C-terminal domain. These distances were computed both in aqueous solution and upon the addition of membrane vesicles. The FRET distances were used as constraints for modeling of a structure for the CLIC1 integral membrane form. The data are suggestive of a large conformational unfolding occurring between the N- and C-domains of CLIC1 upon interaction with the membrane. Consistent with previous findings, the N-terminal domain of CLIC1 is likely to insert into the lipid bilayer, while the C-domain remains in solution on the extravesicular side of the membrane.

Single-Molecule and FRET Fluorescence Correlation Spectroscopy Analyses of Phage DNA Packaging: Colocalization of Packaged Phage T4 DNA Ends within the Capsid

Linear DNAs of any sequence can be packaged into empty viral procapsids by the phage T4 terminase with high efficiency in vitro. Packaging substrates of 5 kbp and 50 kbp, terminated by energy transfer dye pairs, were constructed from plasmid and λ phage DNAs. Nuclease and fluorescence correlation spectroscopy (FCS) assays showed that ∼ 20% of the substrate DNA was packaged and that the DNA dye ends of the packaged DNA were protected from nuclease digestion. Upon packaging, both 5-kbp and 50-kbp DNAs produced comparable fluorescence resonance energy transfer (FRET) between Cy5 and Cy5.5 double-dye terminated DNAs. Single-molecule FRET (sm-FRET) and photobleaching analysis shows that FRET is intramolecular rather than intermolecular upon packaging of most procapsids and demonstrates that single-molecule detection allows mechanistic analysis of packaging in vitro. FRET-FCS and sm-FRET measurements are comparable and show that both the 5-kbp and the 50-kbp packaged DNA ends are held within 8–9 nm of each other, within the dimensions of the long axis of the procapsid portal. The calculated distribution of FRET distances is relatively narrow for both FRET-FCS and sm-FRET, suggesting that the two packaged DNA ends are held at the same fixed distance relative to each other in most capsids. Because one DNA end is known to be positioned for ejection through the portal, it can be inferred that both DNAs ends are held in proximity to the portal entrance and ejection channel. The analysis suggests that a DNA loop, rather than a DNA end, is translocated by the packaging motor to fill the procapsid.

Binding Studies Between Cofilin And Actin Using Fluorescence Resonance Energy Transfer And Molecular Modeling

The actin cytoskeleton is a 3-dimensional network within all eukaryotic cells. The assembly of actin monomers (G-actin) to form filaments (F-actin) is regulated by a number of actin binding proteins (ABPs). Cofilin is a principle regulator of actin dynamics and is essential for cell division and changes in cell shape.A full understanding of the functional effects of cofilin on actin is not possible due to the lack of an atomic-resolution model of the actin-cofilin complex. To date, several predicted models have been proposed based on the putative cofilin-actin interface. The actin-binding surface of cofilin was identified from structural homology with other ABPs, mutagenesis and NMR. Recently a new model was proposed (Kamal JKA et al, 2007, Proc Nat Acad Sci USA 104:7910) which identified the cofilin-binding interface on actin using radiolytic footprinting. Additionally, we have generated 16 possible cofilin-actin complexes ab initio using RosettaDock software (http://www.rosettacommons.org) by inputting the atomic models of actin and cofilin.In vitro mutagenesis within cofilin has been undertaken to provide sites suitable for modification with extrinsic fluorescent probes, while preserving the capacity of cofilin to bind to G-actin. Selective mutation of several amino acids in cofilin has allowed us to identify mutants that remain functional. To test the proposed actin-cofilin models, intermolecular fluorescence resonance energy transfer (FRET) spectroscopy has been performed to calculate distances between several sites on cofilin and actin. These FRET distances will be used as constraints to generate a molecular model of the cofilin-actin complex.

Preparation of fluorescent DNA probe by solid-phase organic synthesis

Fluorescent DNA probe based on fluorescence resonance energy transfer (FRET) was prepared by solid-phase organic synthesis when CdTe quantum dots (QDs) were as energy donors and Au nanoparticles (AuNPs) were as energy accepters. The poly(divinylbenzene) core/poly(4-vinylpyridine) shell microspheres, as solid-phase carriers, were prepared by seeds distillation-precipitation polymerization with 2,2′-azobisisobutyronitrile (AIBN) as initiator in neat acetonitrile. The CdTe QDs and AuNPs were self-assembled on the surface of core/shell microspheres, and then the linkage of CdTe QDs with oligonucleotides (CdTe-DNA) and AuNPs with complementary single-stranded DNA (Au-DNA) was on the solid-phase carriers instead of in aqueous solution. The hybridization of complementary double stranded DNA (dsDNA) bonded to the QDs and AuNPs (CdTe-dsDNA-Au) determined the FRET distance of CdTe QDs and AuNPs. Compared with the fluorescence of CdTe-DNA, the fluorescence of CdTe-dsDNA-Au conjugates (DNA probes) decreased extremely, which indicated that the FRET occurred between CdTe QDs and AuNPs. The probe system would have a certain degree recovery of fluorescence when the complementary single stranded DNA was introduced into this system, which showed that the distance between CdTe QDs and AuNPs was increased.

Use of Multidimensional Fluorescence Resonance Energy Transfer To Establish the Orientation of Cholecystokinin Docked at the Type A Cholecystokinin Receptor

Fluorescence resonance energy transfer (FRET) represents a powerful tool to establish relative distances between donor and acceptor fluorophores. By utilizing several donors situated in distinct positions within a docked full agonist ligand and several acceptors distributed at distinct sites within its receptor, multiple interdependent dimensions can be determined. These can provide a unique method to establish or confirm three-dimensional structure of the molecular complex. In this work, we have utilized full agonist analogues of cholecystokinin (CCK) with Aladan distributed throughout the pharmacophore in positions 24, 29, and 33, along with receptor constructs derivatized with Alexa (546) at positions 94, 102, 204, and 341 in the helical bundle and first, second, and third extracellular loops, respectively. These provided 12 FRET distances to overlay on working models of the CCK-occupied receptor. These established that the carboxyl terminus of CCK resides at the external surface of the lipid bilayer, adjacent to the receptor amino-terminal tail, rather than being inserted into the helical bundle. They also provide important experimentally derived constraints for understanding spatial relationships between the docked ligand and the flexible extracellular loop regions. Multidimensional FRET provides a new independent method to establish and refine structural insights into ligand-receptor complexes.

Adaption of Au Nanoparticles and CdTe Quantum Dots in DNA Detection

A DNA fluorescence probe system based on fluorescence resonance energy transfer (FRET) from CdTe quantum dot (QD) donors to Au nanoparticle (AuNP) acceptors is presented. CdTe QDs, 2.5nm in diameter, as energy donors, were prepared in water. Au nanoparticles, 16nm in diameter, as energy acceptors, were prepared from gold chloride by reduction. CdTe QDs were linked to 5'-NH 2-DNA through 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC) as a linker, and the 3'-SH-DNA was self-assembled onto the surface of AuNPs. The hybridization of complementary double stranded DNA (dsDNA) bound to the QDs and AuNPs (CdTe-dsDNA-Au) determined the FRET distance of CdTe QDs and Au nanoparticles. Compared to the fluorescence of CdTe-DNA, the fluorescence of CdTe-DNA-Au conjugates decreased extremely, which indicated that the FRET occurred between CdTe QDs and Au nanoparticles. The fluorescence change of this conjugate depended on the ratio of Au-DNA to CdTe-DNA. When the AuNPs-DNA to QD-DNA ratio was 10:1, the FRET efficiency reached a maximum. The probe system would have a certain degree of fluorescence recovery when a complementary single stranded DNA was introduced into this system, which showed that the distance between CdTe QDs and Au nanoparticles was increased.

Single molecule analyses of the conformational substates of calmodulin bound to the phosphorylase kinase complex

The four integral delta subunits of the phosphorylase kinase (PhK) complex are identical to calmodulin (CaM) and confer Ca(2+) sensitivity to the enzyme, but bind independently of Ca(2+). In addition to binding Ca(2+), an obligatory activator of PhK's phosphoryltransferase activity, the delta subunits transmit allosteric signals to PhK's remaining alpha, beta, and gamma subunits in activating the enzyme. Under mild conditions about 10% of the delta subunits can be exchanged for exogenous CaM. In this study, a CaM double-mutant derivatized with a fluorescent donor-acceptor pair (CaM-DA) was exchanged for delta to assess the conformational substates of PhKdelta by single molecule fluorescence resonance energy transfer (FRET) +/-Ca(2+). The exchanged subunits were determined to occupy distinct conformations, depending on the absence or presence of Ca(2+), as observed by alterations of the compact, mid-length, and extended populations of their FRET distance distributions. Specifically, the combined predominant mid-length and less common compact conformations of PhKdelta became less abundant in the presence of Ca(2+), with the delta subunits assuming more extended conformations. This behavior is in contrast to the compact forms commonly observed for many of CaM's Ca(2+)-dependent interactions with other proteins. In addition, the conformational distributions of the exchanged PhKdelta subunits were distinct from those of CaM-DA free in solution, +/-Ca(2+), as well as from exogenous CaM bound to the PhK complex as delta'. The distinction between delta and delta' is that the latter binds only in the presence of Ca(2+), but stoichiometrically and at a different location in the complex than delta.

Dissecting the Non-specific and Specific Components of the Initial Folding Reaction of Barstar by Multi-site FRET Measurements

Initial polypeptide chain collapse plays a major role in the development of subsequent structure during protein folding, but it has been difficult to elucidate the coupling between its cooperativity and specificity. To better understand this important aspect of protein folding, nine different intramolecular distances in the protein have been measured by fluorescence resonance energy transfer (FRET) in the product(s) of the initial, sub-millisecond collapse reaction during the folding of barstar, under different folding conditions. All nine distances contract in these initial folding products, when the denaturant concentration is reduced. Two of these distances were also measured in peptides corresponding to sequence segments 38–55 and 51–69 of the protein. Surprisingly, both distances do not contract in the peptides which remain fully unfolded when the denaturant concentration is reduced. This suggests that the contraction of at least some segments of the polypeptide chain may be facilitated only by contraction of other segments. In the case of the initial product of folding of the protein, the dependence on denaturant concentration of the relative change in each distance suggests that there are two components to the initial folding reaction. One is a nonspecific component, which appears to be driven by the change in denaturant concentration that is used to initiate refolding. This component corresponds to the collapse of completely unfolded protein ( U) to unfolded protein in refolding conditions ( U C). The extent of nonspecific collapse can be predicted by the response of completely unfolded protein to a change in denaturant concentration. All distances undergo such solvent-induced contraction, but each distance contracts to a different extent. There is also a specific component to initial sub-millisecond folding, in which some distances (but not all) contract more than that predicted by solvent-induced contraction. The observation that only some of the distances undergo contraction over and above solvent-induced contraction, suggest that this specific component is associated with the formation of a specific intermediate ( I E). FRET efficiency and distance change differently for the different donor–acceptor pairs, with a change in denaturant concentration, indicating that the formation or dissolution of structure in U C and I E does not happen in a synchronized manner across different regions of the protein molecule. Also, all nine FRET efficiencies and intramolecular distances in the product(s) of sub-ms folding, change continuously with a change in denaturant concentration. Hence, it appears that the transitions from U to U C and to I E are gradual transformations, and not all-or-none structural transitions. Nevertheless, the product of these gradual transitions, I E, possesses specific structure.