S-Nitroso compounds interfere with zinc probing by Zinquin

The intracellular homeostasis of zinc is postulated to be controlled by signaling through nitric oxide (NO). Administration of the NO donor S-nitrosocysteine (SNOC) caused a rapid drop in the fluorescence of the zinc-specific fluorescence of the zinc probe zinquin in C6 glioma cells. Tentatively, a strong effect of NO on the level of mobile intracellular zinc ions was concluded. However, zinc analysis with atomic absorption spectrometry demonstrated that the total cellular zinc level was not changed under these conditions. Sodium nitrite or an NO donor devoid of sulfhydryl groups (diethylamine NONOate) exerted no degrading effect on the Zn/zinquin fluorescence, but cysteine alone evoked a similar decline as SNOC. Hence, the sulfhydryl groups of cysteine seem to compete for zinc from the Zn/zinquin complex. Analysis of the reaction products by mass spectrometry demonstrated that cysteine caused a depletion of zinc from the Zn/zinquin complex, whereas an NO donor without sulfhydryl groups (diethylamine NONOate) did not. It is concluded that great caution should be employed when using S-nitroso compounds together with zinquin in investigations of intracellular zinc homeostasis.     

Correction for incomplete labeling in the measurement of distance distributions by frequency-domain fluorometry.

Measurements of time-resolved fluorescence are now being used to recover conformational distributions of biological macromolecules. The fluorescence data of the donor are easily corrupted by incomplete labeling of the macromolecules by the acceptor. In the present paper we describe a general procedure to correct for incomplete acceptor labeling in the determination of distance distributions from frequency-domain measurements of the donor fluorescence decay kinetics. The method can also be used to determine the extent of acceptor labeling. Simulated data were used to determine the effect of incomplete labeling on resolution of the distance distribution and the effect on the recovered distributions if one fails to account for incomplete labeling by the acceptor. The expressions and implemented algorithm were verified using known mixtures of donor-control and donor-acceptor pair molecules, which simulated the presence of a donor population lacking the acceptor. Finally, we present data on the distance distributions between two labeled sites in myosin S1 (Cys-697 to Cys-707) where it was not possible to obtain complete labeling of the acceptor site.     

Metal ion-independent association of factor VIII subunits and the roles of calcium and copper ions for cofactor activity and inter-subunit affinity

Factor VIII circulates as a divalent metal ion-dependent heterodimer comprised of a light chain (LC) and a heavy chain (HC). Reassociation of factor VIII subunits was assessed using fluorescence energy transfer where LC and HC were labeled with acrylodan (Ac; fluorescence donor) and fluorescein-5-maleimide (Fl; fluorescence acceptor), respectively. The reduction of donor fluorescence due to the acceptor was used as an indicator of binding. Subunits associated with high affinity (K(d) = 53.8 nM) in the absence of metal ion and presence of EDTA. However, this product showed no cofactor activity, as measured by a factor Xa generation assay. In the presence of 25 mM Ca(2+), no increase in the intersubunit affinity was observed (K(d) = 48.7 nM) but specific activity of the cofactor was approximately 30% that of native factor VIII. At saturating levels of Fl-HC relative to Ac-LC, donor fluorescence decreased to 79.3 and 73.5% of its original value in the absence and presence of Ca(2+), respectively. Thrombin cleaved the heterodimers that were associated in the absence or presence of Ca(2+) with similar efficiency, indicating that the lack of activity was not the result of a defect in activation. Cu(2+) (0.5 microM) increased the intersubunit affinity by approximately 100 fold (K(d) = 0.52 nM) and the specific activity to approximately 60% of native factor VIII. The former effect was independent of Ca(2+), whereas the latter effect required Ca(2+). These results indicate that the intersubunit association in factor VIII is primarily metal-ion independent while divalent metal ions serve specific roles. Ca(2+) appears essential to promote the active conformation of factor VIII while Cu(2+) primarily enhances the intersubunit affinity.     

Fluorescence energy transfer between Ca2+ transport ATPase molecules in artificial membranes.

The purified ATPase of sarcoplasmic reticulum was covalently labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS) or with iodoacetamidofluorescein (IAF). In reconstituted vesicles containing both types of ATPase molecules fluorescence energy transfer was observed from the IAEDANS (donor) to the IAF (acceptor) fluorophore as determined by the ratio of donor and acceptor fluorescence intensities, and by nanosecond decay measurements of donor fluorescence in the presence or absence of the acceptor. The observed energy transfer may arise by random collisions between ATPase molecules due to Brownian motion or by formation of complexes containing several ATPase molecules. Experimental distinction between these two models of energy transfer is possible based on predictions derived from mathematical models. Up to tenfold dilution of the lipid phase of reconstituted vesicles with egg lecithin had no measurable effect upon the energy transfer, suggesting that random collision between ATPase molecules in the lipid phase is not the principal cause of the observed effect. Addition of unlabeled ATPase in five- to tenfold molar excess over the labeled molecules abolished energy transfer. These observations together with electron microscopic and chemical cross-linking studies support the existence of ATPase oligomers in the membrane with sufficiently long lifetimes for energy transfer to occur. A hypothetical equilibrium between monomeric and tetrameric forms of the ATPase governed by the membrane potential is proposed as the structural basis of the regulation of Ca uptake and release by sarcoplasmic reticulum membranes during muscle contraction and relaxation.     

Changes in polyphasic chlorophyll a fluorescence induction curve upon inhibition of donor or acceptor side of photosystem II in isolated thylakoids

The action of various inhibitors affecting the donor and acceptor sides of photosystem II (PSII) on the polyphasic rise of chlorophyll (Chl) fluorescence was studied in thylakoids isolated from pea leaves. Low concentrations of diuron and stigmatellin increased the magnitude of J-level of the Chl fluorescence rise. These concentrations barely affected electron transfer from PSII to PSI as revealed by the unchanged magnitude of the fast component (t(1/2) = 24 ms) of P700+ dark reduction. Higher concentrations of diuron and stigmatellin suppressed electron transport from PSII to PSI, which corresponded to the loss of thermal phase, the Chl fluorescence rise from J-level to the maximal, P-level. The effect of various concentrations of carbonylcyanide m-chlorophenylhydrazone (CCCP), which abolishes S-state cycle and binds at the plastoquinone site on QB, the secondary quinone acceptor PSII, on the Chl fluorescence rise was very similar to that of diuron and stigmatellin. Low concentrations of diuron, stigmatellin, or CCCP given on the background of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), which is shown to initiate the appearance of a distinct I-peak in the kinetics of Chl fluorescence rise measured in isolated thylakoids [BBA 1607 (2003) 91], increased J-step yield to I-step level and retarded Chl fluorescence rise from I-step to P-step. The increased J-step fluorescence rise caused by these three types of inhibitors is attributed to the suppression of the non-photochemical quenching of Chl fluorescence by [S2+ S3] states of the oxygen-evolving complex and oxidized P680, the primary donor of PSII reaction centers. In the contrary, the decreased fluorescence yield at P step (J-P, passing through I) is related to the persistence of a "plastoquinone"-type quenching owing to the limited availability of photochemically generated electron equivalents to reduce PQ pool in PSII centers where the S-state cycle of the donor side is modified by the inhibitor treatments.     

Near-infrared fluorescent oligodeoxyribonucleotide reporters for sensing NF-kappaB DNA interactions in vitro

Two types of reporters for optical sensing of NF-kappaB p50 protein-oligodeoxyribonucleotide (ODN) duplex interactions were designed and compared in vitro. The reporters were based on the effect of fluorescence resonance energy transfer (FRET) between the pair donor Cy5.5 near-infrared (NIR) fluorochrome and either 800CW emitting fluorescence dye acceptor (800CW-Cy), or a nonemitting QSY 21 dye quencher (QSY-Cy). The donor and the acceptor dyes were covalently linked to the complementary oligonucleotides, respectively: Cy dye was conjugated to 3'-thiol, whereas 800CW or QSY21 were conjugated to a hydrophilic internucleoside phosphate amino linker. The reporters were tested initially using recombinant NF-kappaB p50 protein binding assays. Both reporters were binding p50 protein, which protected oligonucleotide duplex from degradation in the presence of exonuclease.The incubation of 800CW-Cy reporter in the presence of control or IL-1beta treated human endothelial cells showed the uptake of the reporter in the cytoplasm and the nucleus. The measurement of NIR fluorescence ratio (i.e. Cy5.5/800CW) showed a partial loss of FRET and the increased Cy5.5 fluorescence in nontreated, control cells. Thus, the specific p50 binding to ODN duplex reporters affected the donor-acceptor fluorochrome pair. NF-kappaB p50 exhibited the protective effect on FRET between NIR fluorochromes linked to the complementary strands of the reporter duplex.     

Scanning near-field fluorescence resonance energy transfer microscopy

A new microscopic technique is demonstrated that combines attributes from both near-field scanning optical microscopy (NSOM) and fluorescence resonance energy transfer (FRET). The method relies on attaching the acceptor dye of a FRET pair to the end of a near-field fiber optic probe. Light exiting the NSOM probe, which is nonresonant with the acceptor dye, excites the donor dye introduced into a sample. As the tip approaches the sample containing the donor dye, energy transfer from the excited donor to the tip-bound acceptor produces a red-shifted fluorescence. By monitoring this red-shifted acceptor emission, a dramatic reduction in the sample volume probed by the uncoated NSOM tip is observed. This technique is demonstrated by imaging the fluorescence from a multilayer film created using the Langmuir-Blodgett (LB) technique. The film consists of L-alpha-dipalmitoylphosphatidylcholine (DPPC) monolayers containing the donor dye, fluorescein, separated by a spacer group of three arachidic acid layers. A DPPC monolayer containing the acceptor dye, rhodamine, was also transferred onto an NSOM tip using the LB technique. Using this modified probe, fluorescence images of the multilayer film reveal distinct differences between images collected monitoring either the donor or acceptor emission. The latter results from energy transfer from the sample to the NSOM probe. This method is shown to provide enhanced depth sensitivity in fluorescence measurements, which may be particularly informative in studies on thick specimens such as cells. The technique also provides a mechanism for obtaining high spatial resolution without the need for a metal coating around the NSOM probe and should work equally well with nonwaveguide probes such as atomic force microscopy tips. This may lead to dramatically improved spatial resolution in fluorescence imaging.     

Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy

Fluorescence resonance energy transfer (FRET) detects the proximity of fluorescently labeled molecules over distances >100 A. When performed in a fluorescence microscope, FRET can be used to map protein-protein interactions in vivo. We here describe a FRET microscopy method that can be used to determine whether proteins that are colocalized at the level of light microscopy interact with one another. This method can be implemented using digital microscopy systems such as a confocal microscope or a wide-field fluorescence microscope coupled to a charge-coupled device (CCD) camera. It is readily applied to samples prepared with standard immunofluorescence techniques using antibodies labeled with fluorescent dyes that act as a donor and acceptor pair for FRET. Energy transfer efficiencies are quantified based on the release of quenching of donor fluorescence due to FRET, measured by comparing the intensity of donor fluorescence before and after complete photobleaching of the acceptor. As described, this method uses Cy3 and Cy5 as the donor and acceptor fluorophores, but can be adapted for other FRET pairs including cyan fluorescent protein and yellow fluorescent protein.     

Production of transgenic bovine embryos by transfer of transfected granulosa cells into enucleated oocytes

Adult granulosa donor cells used in the nuclear transfer (NT) procedure can result in cloned cattle. Subsequently, it may be possible to use the same cell type to produce cloned transgenic cattle. Therefore, this study examined the effect of genetic manipulation and serum levels in culture of donor granulosa cells on developmental rates and cell number of bovine NT embryos. A primary cell line was established from granulosa cells collected by aspirating ovarian follicles. Cells transfected with a plasmid containing the enhanced green fluorescence protein (EGFP) gene, and non-transfected cells were used for cloning between passage 10 and 15 as serum-starved and serum-fed donor cells. There were no significant differences (P > 0.1) in cleavage rates or development to the blastocyst stage for NT embryos from transfected (60.4 and 13.5%, respectively) or non-transfected (61.9 and 14.1%, respectively) and serum-starved (60.6 and 13.4%, respectively) or serum-fed (61.3 and 14%, respectively) cells. Development rates to blastocyst stage of embryos produced using cells at passage 15 (27.1%) were significantly higher than those produced with cells at passage 10,11, and 13 (7, 11.5, and 14%, respectively, P < 0.05). Green fluorescence was observed at different intensity levels in all blastocyst stage embryos resulting from transfected donor cells. The results of the present study indicated that genetically modified granulosa cells can be used to produce transgenic NT embryos and primary transgenic adult cells at late passage may be more effective donor cells than earlier passaged cells.     

Electron donor properties of the antitumour drug amsacrine as studied by fluorescence quenching of DNA-bound ethidium

The effect of the antitumour acridine derivative amsacrine [4'-(9-acridinylamino)methanesulphon-m-anisidide] on the fluorescence lifetime of DNA-bound ethidium has been investigated using a synchronously pumped cavity dumped dye laser producing picosecond pulses for sample excitation and a time-correlated single photon counting detection system. As the proportion of DNA-bound amsacrine on the synthetic DNA polymer poly[deoxyadenylic-thymidylic acid] is increased, the fluorescence decay curve of ethidium can be accurately resolved into two exponential components. The short lifetime component, whose proportion increases with increasing proportions of DNA-bound amsacrine, has a lifetime of between 3 and 4 ns, significantly longer than that of ethidium in aqueous solution (1.63 ns). The magnitude of the long lifetime component decreases from 25.4 to 14 ns with increasing proportions of bound amsacrine. It is concluded that a new fluorescence state of ethidium (lifetime 3-4 ns) is present, probably resulting from reversible electron transfer between ethidium and amsacrine. The ability of various 9-anilinoacridine derivatives to quench the fluorescence of DNA-bound ethidium appears to be related to the electron donor properties of the substituents on the anilino ring, as well as to experimental antitumour activity. The electron donor properties of DNA-bound amsacrine may therefore be relevant to its antitumour action.     

Two-photon excitation fluorescence resonance energy transfer with small organic molecule as energy donor for bioassay

A fluorescence resonance energy transfer (FRET) model using two-photon excitable small organic molecule DMAHAS as energy donor has been constructed and tried in an assay for avidin. In the FRET model, biotin was conjugated to the FRET donor, and avidin was labeled with a dark quencher DABS-Cl. Binding of DABS-Cl labeled avidin to biotinylated DMAHAS resulted in the quenching of fluorescence emission of the donor, based on which a competitive assay for free avidin was established. With using such donors that are excited in IR region, it is capable of overcoming some primary shortcomings of conventional one-photon FRET methods, especially in bioassays, such as the interference from background fluorescence or scattering light, the coexcitation of the energy acceptor with the donor. And such small molecules also show advantages over inorganic up-converting particles that also give anti-Stokes photoluminescence and have been applied as FRET donor recently. The results of this work suggest that two-photon excitable small molecules could be a promising energy donor for FRET-based bioassays.     

Simple PbII fluorescent probe based on PbII-catalyzed hydrolysis of phosphodiester

A new fluorescent probe for PbII, p-nitrophenyl 3H-phenoxazin-3-one-7-yl phosphoric acid (NPPA), was designed and synthesized by linking resorufin (serving as a fluorophore and electron acceptor) to p-nitrophenol (serving as a fluorescence quencher and electron donor) through phosphodiester bonds. When NPPA was irradiated with light, intramolecular fluorescence self-quenching took place because of the photoinduced electron transfer from the donor to the acceptor. However, upon the addition of PbII, the phosphate ester bonds in the probe were cleaved and the fluorophore was released, accompanying the retrievement of fluorescence.     

Structural changes of yellow Cameleon domains observed by quantitative FRET analysis and polarized fluorescence correlation spectroscopy

Förster resonance energy transfer (FRET) is a widely used method for monitoring interactions between or within biological macromolecules conjugated with suitable donor-acceptor pairs. Donor fluorescence lifetimes in absence and presence of acceptor molecules are often measured for the observation of FRET. However, these lifetimes may originate from interacting and noninteracting molecules, which hampers quantitative interpretation of FRET data. We describe a methodology for the detection of FRET that monitors the rise time of acceptor fluorescence on donor excitation thereby detecting only those molecules undergoing FRET. The large advantage of this method, as compared to donor fluorescence quenching method used more commonly, is that the transfer rate of FRET can be determined accurately even in cases where the FRET efficiencies approach 100% yielding highly quenched donor fluorescence. Subsequently, the relative orientation between donor and acceptor chromophores is obtained from time-dependent fluorescence anisotropy measurements carried out under identical conditions of donor excitation and acceptor detection. The FRET based calcium sensor Yellow Cameleon 3.60 (YC3.60) was used because it changes its conformation on calcium binding, thereby increasing the FRET efficiency. After mapping distances and orientation angles between the FRET moieties in YC3.60, cartoon models of this FRET sensor with and without calcium could be created. Independent support for these representations came from experiments where the hydrodynamic properties of YC3.60 under ensemble and single-molecule conditions on selective excitation of the acceptor were determined. From rotational diffusion times as found by fluorescence correlation spectroscopy and consistently by fluorescence anisotropy decay analysis it could be concluded that the open structure (without calcium) is flexible as opposed to the rather rigid closed conformation. The combination of two independent methods gives consistent results and presents a rapid and specific methodology to analyze structural and dynamical changes in a protein on ligand binding.     

Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization

A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to improve the FRET signals to handle in vivo applications. Consequently, we found that a double-labeled donor probe labeled with Bodipy dye yielded a remarkable increase in fluorescence intensity compared to a single-labeled donor probe used in an ordinary FRET. This double-labeled donor system can be easily applied to improve various FRET probes since the dependence upon sequence and label position in enhancement is not as strict. Furthermore this method could be applied to other nucleic acid substances, such as oligo RNA and phosphorothioate oligonucleotides (S-oligos) to enhance FRET signal. Although the double-labeled donor probes labeled with a variety of fluorophores had unexpected properties (strange UV-visible absorption spectra, decrease of intensity and decay of donor fluorescence) compared with single-labeled ones, they had no relation to FRET enhancement. This signal amplification mechanism cannot be explained simply based on our current results and knowledge of FRET. Yet it is possible to utilize this double-labeled donor system in various applications of FRET as a simple signal-enhancement method.     

Action of salicylaldoxime on electron transport reactions,fluorescence yield,and light-induced field changes in spinach chloroplasts: A new mode of inhibition in photosystem II

Salicylaldoxime (1–10 mm) inhibits chloroplast electron transport reactions by a reversible and an irreversible modification of photosystem II. The irreversible inhibition correlates with removal of the loosely bound pool of manganese associated with the water-splitting mechanism. The reversible inhibition is characterized by (i) a suppression of artificial donor reactions, (ii) a high initial fluorescence yield, and (iii) a decline in the amplitude of the flash-induced electric field across the membrane. After removal of the inhibitor, the initial fluorescence yield declines to near-control levels, but the variable portion of the fluorescence rise remains missing. Addition of an artificial donor restores the variable fluorescence yield and normal electron transport rates to 2,6-dichlorophenolindophenol. Characteristics of the reversible inhibition suggest that salicylaldoxime causes suppression of photochemical charge separation in photosystem II.     

Determination of the size of lipid rafts studied through single-molecule FRET simulations

Fluorescence resonance energy transfer (FRET) is a high-resolution technique that allows the characterization of spatial and temporal properties of biological structures and mechanisms. In this work, we developed an in silico single-molecule FRET methodology to study the dynamics of fluorophores inside lipid rafts. We monitored the fluorescence of a single acceptor molecule in the presence of several donor molecules. By looking at the average fluorescence, we selected events with single acceptor and donor molecules, and we used them to determine the raft size in the range of 5–16 nm. We conclude that our method is robust and insensitive to variations in the diffusion coefficient, donor density, or selected fluorescence threshold.     

Fluorescence resonance energy transfer-based stoichiometry in living cells

Imaging of fluorescence resonance energy transfer (FRET) between fluorescently labeled molecules can measure the timing and location of intermolecular interactions inside living cells. Present microscopic methods measure FRET in arbitrary units, and cannot discriminate FRET efficiency and the fractions of donor and acceptor in complex. Here we describe a stoichiometric method that uses three microscopic fluorescence images to measure FRET efficiency, the relative concentrations of donor and acceptor, and the fractions of donor and acceptor in complex in living cells. FRET stoichiometry derives from the concept that specific donor-acceptor complexes will give rise to a characteristic FRET efficiency, which, if measured, can allow stoichiometric discrimination of interacting components. A first equation determines FRET efficiency and the fraction of acceptor molecules in complex with donor. A second equation determines the fraction of donor molecules in complex by estimating the donor fluorescence lost due to energy transfer. This eliminates the need for acceptor photobleaching to determine total donor concentrations and allows for repeated measurements from the same cell. A third equation obtains the ratio of total acceptor to total donor molecules. The theory and method were confirmed by microscopic measurements of fluorescence from cyan fluorescent protein (CFP), citrine, and linked CFP-Citrine fusion protein, in solutions and inside cells. Together, the methods derived from these equations allow sensitive, rapid, and repeatable detection of donor-, acceptor-, and donor-acceptor complex stoichiometry at each pixel in an image. By accurately imaging molecular interactions, FRET stoichiometry opens new areas for quantitative study of intracellular molecular networks.     

Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data

We studied the fluorescence resonance energy transfer (FRET) efficiency of different donor-acceptor labeled model DNA systems in aqueous solution from ensemble measurements and at the single molecule level. The donor dyes: tetramethylrhodamine (TMR); rhodamine 6G (R6G); and a carbocyanine dye (Cy3) were covalently attached to the 5'-end of a 40-mer model oligonucleotide. The acceptor dyes, a carbocyanine dye (Cy5), and a rhodamine derivative (JA133) were attached at modified thymidine bases in the complementary DNA strand with donor-acceptor distances of 5, 15, 25 and 35 DNA-bases, respectively. Anisotropy measurements demonstrate that none of the dyes can be observed as a free rotor; especially in the 5-bp constructs the dyes exhibit relatively high anisotropy values. Nevertheless, the dyes change their conformation with respect to the oligonucleotide on a slower time scale in the millisecond range. This results in a dynamic inhomogeneous distribution of donor/acceptor (D/A) distances and orientations. FRET efficiencies have been calculated from donor and acceptor fluorescence intensity as well as from time-resolved fluorescence measurements of the donor fluorescence decay. Dependent on the D/A pair and distance, additional strong fluorescence quenching of the donor is observed, which simulates lower FRET efficiencies at short distances and higher efficiencies at longer distances. On the other hand, spFRET measurements revealed subpopulations that exhibit the expected FRET efficiency, even at short D/A distances. In addition, the measured acceptor fluorescence intensities and lifetimes also partly show fluorescence quenching effects independent of the excitation wavelength, i.e. either directly excited or via FRET. These effects strongly depend on the D/A distance and the dyes used, respectively. The obtained data demonstrate that besides dimerization at short D/A distances, an electron transfer process between the acceptor Cy5 and rhodamine donors has to be taken into account. To explain deviations from FRET theory even at larger D/A distances, we suggest that the pi-stack of the DNA double helix mediates electron transfer from the donor to the acceptor, even over distances as long as 35 base pairs. Our data show that FRET experiments at the single molecule level are rather suited to resolve fluorescent subpopulations in heterogeneous mixture, information about strongly quenched subpopulations gets lost.     

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.