A non-natural amino acid for efficient incorporation into proteins as a sensitive fluorescent probe.

A small and highly fluorescent non-natural amino acid that contains an anthraniloyl group (atnDap) was incorporated into various positions of streptavidin. The positions were directed by a CGGG/CCCG four-base codon/anticodon pair. The non-natural mutants were obtained in excellent yields and some of them retained strong biotin-binding activity. The fluorescence wavelength as well as the intensity of the anthraniloyl group at position 120 were sensitive to biotin binding. These unique properties indicate that the atnDap is the most suitable non-natural amino acid for a position-specific fluorescent labeling of proteins that is highly sensitive to microenvironmental changes.     

A small-molecule-linked DNA–graphene oxide-based fluorescence-sensing system for detection of biotin

In this paper, we establish a novel fluorescence-sensing system for the detection of biotin based on the interaction between DNA and graphene oxide and on protection of the terminal of the biotinylated single-stranded DNA fluorescent probe by streptavidin. In this system, streptavidin binds to the biotinylated DNA, which protects the DNA from hydrolysis by exonuclease I. The streptavidin–DNA conjugate is then adsorbed to the graphene oxide resulting in the fluorescence being quenched. Upon the addition of free biotin, it competes with the labeled biotin for the binding sites of streptavidin and then the exonuclease I digests the unbound DNA probe from the 3′ to the 5′ terminal, releasing the fluorophore from the DNA. Because of the weak affinity between the fluorophore and graphene oxide, the fluorescence is recovered. Under optimal conditions, the fluorescence intensity is proportional to the concentration of biotin in the concentration range of 0.5–20 nmol/L. The detection limit for biotin is 0.44 nmol/L. The proposed fluorescence-sensing system was applied to the determination of biotin in some real samples with satisfactory reproducibility and accuracy. This work could provide a common platform for detecting small biomolecules based on protein–small molecule ligand binding.     

Temperature control of biotin binding and release with A streptavidin-poly(N-isopropylacrylamide) site-specific conjugate.

The many laboratory and diagnostic applications utilizing streptavidin as a molecular adaptor rely on its high affinity and essentially irreversible interaction with biotin. However, there are many situations where recovery of the biotinylated molecules is desirable. We have previously shown that poly(N-isopropylacrylamide) (PNIPAAm), a temperature-sensitive polymer, can reversibly block biotin association as the polymer's conformation changes at its lower critical solution temperature (LCST). Here, we have constructed a streptavidin-PNIPAAm conjugate which is able to bind biotin at room temperature or lower and release bound biotin at 37 degrees C. The conjugate can repeatedly bind and release biotin as temperature is cycled through the LCST. A genetically engineered streptavidin mutant, E116C, which has only one cysteine residue, was conjugated site specifically via the sulfhydryl groups with a PNIPAAm that has pendent sulfhydryl-reactive vinyl sulfone groups. The conjugation site is near the tryptophan 120 residue, which forms a van der Waals contact with biotin that is important in generating the large binding free energy. The temperature-induced conformational change of the polymer at position 116 may lead to structural changes in the region of tryptophan 120 that are responsible for the reversible binding between biotin and the conjugated streptavidin.     

The biotin-thyroxin conjugate as a bifunctional ligand of binding proteins

The conjugate of the residue of vitamin H (biotin, Bt) with the hormone of thyroid gland thyroxin (T₄) was prepared by N-acylation of N-(3-aminopropyl)biotin amide with N-hydroxysuccinimide ester of N-acetyl thyroxin. The interactions of the Bt-T₄ conjugate with one or simultaneously with two binding proteins with affinity to Bt or T₄ in solution and on a solid phase were studied by electron spectroscopy, enzyme immunoassay, and computer modeling. Bt-T₄ was specifically fixed in the Bt-binding site of the streptavidin molecule via a large number of hydrogen bonds and hydrophobic interactions. The maximum of the streptavidin fluorescence shifted to a long-wave area and its intensity decreased as a result of complex formation. The degree of quenching of the protein emission was significantly higher than that of the streptavidin-Bt complex. Additional fluorescence quenching resulted from interactions which were sensitive to pH, ionic strength, and detergents and stabilized the position of the thyroxin part of the conjugate near Trp120 of streptavidin in its complex with Bt-T₄. The Bt-T₄ conjugate also formed a specific equimolar complex with T₄-binding human globulin (TBG) by the same mechanism as that for T₄. The Bt residue did not participate in the interactions which changed characteristics of the TBG fluorophores. The Bt-T₄ conjugate was bound to avidin on a solid phase in the solid phase enzyme immunoassay owing to its biotin function, whereas its thyroxin part was exposed to a solution and interacted with polyclonal antibodies to T₄. The intact T₄ competitively inhibited this interaction after its addition to the system. Bt-T₄ also exhibited its bifunctional activity in other immune analytic system. The conjugate bound streptavidin was labeled with Eu³⁺-chelate and subsequently formed a three component complex with participation of a monoclonal antibody to T₄ immobilized on a solid phase. Free T₄ inhibited the thyroxin function of the conjugate bound to the labeled streptavidin proportionally to its concentration in a sample of human blood serum. Parameters of the immunofluorescent analysis demonstrated that the streptavidin-Bt-T₄ complex was actively bound to the T₄-antibody, but had practically no interaction with serum T₄-binding proteins, including TBG. Probably, nonspecific interactions of the T₄ residue with streptavidin in its complex with Bt-T₄, along with steric factors, complicated penetration of thyroxin in this complex into active sites of TBG and other T₄-binding proteins of blood serum. The Bt-T₄ stable conjugate was synthesized according to a plain scheme and could be used as a bifunctional ligand of binding proteins in biochemical studies and immune analytical systems for medicinal diagnostics.     

Screening scheme based on measurement of fluorescence lifetime in the nanosecond domain

The authors demonstrate that the fluorescence lifetime of certain fluorescent labels is a useful parameter to detect affinity binding between biotin and streptavidin, as well as between biotinylated bovine serum albumin and streptavidin. The assay is performed in a microplate format, and lifetimes are determined using dye laser-induced fluorescence. Four fluorescent labels are presented that undergo a significant change in their lifetime upon affinity binding. The scheme, referred to as the fluorescence lifetime affinity assay, has several attractive features in that it requires single labeling only, represents a homogeneous assay, allows each of the 2 binding partners to be labeled, and is compatible with the standard microwell formats used in high-throughput screening.     

Biotin binding changes the conformation and decreases tryptophan accessibility of streptavidin

Biotin binding reduces the tryptophan fluorescence emissions of streptavidin by 39%, blue shifts the emission peak from 333 to 329 nm, and reduces the bandwidth at half height from 53 to 46 nm. The biotin-induced emission difference spectrum resembles that of a moderately polar tryptophan. Streptavidin fluorescence can be described by two lifetime classes: 2.6 nsec (34%) and 1.3 nsec (66%). With biotin bound, lifetimes are 1.3 nsec (26%) and 0.8 nsec (74%). Biotin binding reduces the average fluorescence lifetime from 1.54 to 0.88 nsec. Biotin does not quench the fluorescence of indoles. The fluorescence changes are consistent with biotin binding causing a conformational change which moves tryptophans into proximity to portions of streptavidin which reduce the quantum yield and lifetimes. Fluorescence quenching by acrylamide revealed two classes of fluorophores. Analysis indicated a shielded component comprising 20–28% of the initial fluorescence with (K_SV+V)0.55 M^–1. The more accessible component has a predominance of static quenching. Measurements of fluorescence lifetimes at different acrylamide concentrations confirmed the strong static quenching. Since static quenching could be due to acrylamide binding to streptavidin, a dye displacement assay for acrylamide binding was constructed. Acrylamide does bind to streptavidin (Ka=5 M^–1), and probably binds within the biotin-binding site. In the absence of biotin, none of streptavidin's fluorescence is particularly accessible to iodide. In the presence of biotin, iodide neither quenches fluorescence nor alters emission spectra, and acrylamide access is dramatically reduced. We propose that the three tryptophans which always line the biotin site are sufficiently close to the surface of the binding site to be quenched by bound acrylamide. These tryptophans are shielded from iodide, most probably due to steric or ionic hindrances against diffusion into the binding site. Most of the shielding conferred by biotin binding can be attributed to the direct shielding of these residues and of a fourth tryptophan which moves into the binding site when biotin binds, as shown by X-ray studies (Weberet al., 1989).     

Reversible Two‐Enzyme Coimmobilization on pH‐Responsive Imprinted Monolith for Glucose Detection

A new enzymatic glucose biosensor based on reversible co‐immobilization of horseradish peroxidase (HRP) and glucose oxidase (GOx) on a pH‐responsive imprinted monolith is prepared. The poly(4‐vinylphenylboronic acid)‐grafted imprinted polymer using HRP as a template is formed via surface initiated atom transfer radical polymerization within the pores of brominated poly(glycidyl methacrylate‐co‐ethylene dimethacrylate) macroporous monolith contained in a 100 μm I.D. capillary column. The two enzymes conjugate is formed via the strong affinity interaction between biotin‐labeled GOx and streptavidin‐labeled HRP. The modulation of the external pH value enables reusability of the biosensor simply using stripping of the inactive enzymes at a low pH value and subsequent immobilization of fresh enzymes at a high pH value. Under the optimized conditions, the enzymatic biosensor features excellent performance in detection of glucose with a linear range of its concentration from 0.11 to 38.85 mmol/L and a limit of detection of 0.03 mmol/L. A relative standard deviation of 3.7% is calculated from determination of twenty glucose samples. This novel enzymatic sensing system is successfully applied for determination of glucose in human serum, and confirms an enhancement both in selectivity and specificity compared to the more traditionally clinical methods.     

UV resonance Raman study of streptavidin binding of biotin and 2-iminobiotin: comparison with avidin

UV resonance Raman (UVRR) spectroscopy is used to study the binding of biotin and 2-iminobiotin by streptavidin, and the results are compared to those previously obtained from the avidin-biotin complex and new data from the avidin-2-iminobiotin complex. UVRR difference spectroscopy using 244-nm excitation reveals changes to the tyrosine (Tyr) and tryptophan (Trp) residues of both proteins upon complex formation. Avidin has four Trp and only one Tyr residue, while streptavidin has eight Trp and six Tyr residues. The spectral changes observed in streptavidin upon the addition of biotin are similar to those observed for avidin. However, the intensity enhancements observed for the streptavidin Trp Raman bands are less than those observed with avidin. The changes observed in the streptavidin Tyr bands are similar to those observed for avidin and are assigned exclusively to the binding site Tyr 43 residue. The Trp and Tyr band changes are due to the exclusion of water and addition of biotin, resulting in a more hydrophobic environment for the binding site residues. The addition of 2-iminobiotin results in spectral changes to both the streptavidin and avidin Trp bands that are very similar to those observed upon the addition of biotin in each protein. The changes to the Tyr bands are very different than those observed with the addition of biotin, and similar spectral changes are observed in both streptavidin and avidin. This is attributable to hydrogen bond changes to the binding site Tyr residue in each protein, and the similar Tyr difference features in both proteins supports the exclusive assignment of the streptavidin Tyr difference features to the binding site Tyr 43.     

Characterization of low affinity Fcγ receptor biotinylation under controlled reaction conditions by mass spectrometry and ligand binding analysis

Chemical protein biotinylation and streptavidin or anti‐biotin‐based capture is regularly used for proteins as a more controlled alternative to direct coupling of the protein on a biosensor surface. On biotinylation an interaction site of interest may be blocked by the biotin groups, diminishing apparent activity of the protein. Minimal biotinylation can circumvent the loss of apparent activity, but still a binding site of interest can be blocked when labeling an amino acid involved in the binding. Here, we describe reaction condition optimization studies for minimal labeling. We have chosen low affinity Fcγ receptors as model compounds as these proteins contain many lysines in their active binding site and as such provide an interesting system for a minimal labeling approach. We were able to identify the most critical parameters (protein:biotin ratio and incubation pH) for a minimal labeling approach in which the proteins of choice remain most active toward analyte binding. Localization of biotinylation by mass spectrometric peptide mapping on minimally labeled material was correlated to protein activity in binding assays. We show that only aiming at minimal labeling is not sufficient to maintain an active protein. Careful fine‐tuning of critical parameters is important to reduce biotinylation in a protein binding site.     

Determination of diethylstilbestrol based on biotin–streptavidin‐amplified time‐resolved fluoro‐immunoassay

A rapid and sensitive time‐resolved fluoroimmunoassay (TR–FIA) based on the biotin–streptavidin amplification system was developed for the determination of diethylstilbestrol (DES). Europium‐labelled streptavidin derivatives combined with europium and anhydride of diethylene triamine penta‐acetic acid were used to label streptavidin; biotin was coupled with goat anti‐rabbit IgG to form a biotin–goat anti‐rabbit IgG bridge between streptavidin–europium and the anti‐DES antibody in the immunoassay. The DES assay was carried out by measuring the fluorescence of Eu³⁺–SA at 615 nm. The presented method produced a wide linear range, 0.001–1000.0 ng/mL, and a detection limit up to 0.81 pg/mL for DES. The method was applied to determine DES in serum samples, with recoveries of 97.4–107.8% and RSD 1.32–4.04%. The assay results by the present method showed that biotin–streptavidin amplified TR–FIA for DES detection; it may offer high sensitivity and promising alternative special methods in biological samples. Copyright © 2011 John Wiley & Sons, Ltd.     

Fluorescence quenching studies of Trp repressor using single-tryptophan mutants

Time-resolved and steady-state fluorescence have been used to resolve the heterogeneous emission of single-tryptophan-containing mutants of Trp repressors W19F and W99F into components. Using iodide as the quencher, the fluorescence-quenching-resolved spectra (FQRS) have been obtained The FQRS method shows that the fluorescence emission of Trp99 can be resolved into two component spectra characterized by maxima of fluorescence emission at 338 and 328 nm. The redder component is exposed to the solvent and participates in about 21% of the total fluorescence emission of TrpR W19F. The second component is inacessible to iodide, but is quenched by acrylamide. The tryptophan residue 19 present in TrpR W99F can be resolved into two component spectra using the FQRS method and iodide as a quencher. Both components of Trp19 exhibit similar maxima of emission at 322–324 nm and both are quenchable by iodide. The component more quenchable by iodide participates in about 38% of the total TrpR W99F emission. The fluorescence lifetime measurements as a function of iodide concentration support the existence of two classes of Trp99 and Trp19 in the Trp repressor. Our results suggest that the Trp aporepressor can exist in the ground state in two distinct conformational states which differ in the microenvironment of the Trp residues.Abbreviations TrpR tryptophan aporepressor fromE. coli - TrpR W19F TrpR mutant with phenylalanine substituted for tryptophan at position 19 - TrpR W99F TrpR mutant with phenylalanine substituted for tryptophan at position 99 - FQRS fluorescence-quenching-resolved spectra - FPLC fast protein liquid chromatography     

Position-specific incorporation of a highly photodurable and blue-laser excitable fluorescent amino acid into proteins for fluorescence sensing

A new fluorescent amino acid, L-2-acridonylalanine, was incorporated into proteins at specific positions using 4-base codon/anticodon strategy. The efficiency of the incorporation was high enough to obtain enough quantities of the mutants. The acridonyl group was highly fluorescent when it was excited at the wavelengths of blue-lasers and was highly photodurable compared with conventional fluorophores often used for biological analyses. The fluorescence intensity was sensitive to small changes in the polarity of the environment. When the nonnatural amino acid was incorporated into specific positions of streptavidin, the mutant protein worked as a fluorescent sensor to biotin. Similarly, when the amino acid was incorporated into camel single-chain antibody, the mutant protein sensitively responded to the antigen molecule. The high incorporation efficiency, the high photodurability, the excitability with blue-lasers, and high sensitivity to the environment make the acridonylalanine as the promising fluorescent amino acid for sensing small molecules when incorporated into specific positions of various antibodies, receptors, and enzymes.     

A streptavidin-biotin binding system that minimizes blocking by endogenous biotin

Pretargeted radioimmunotherapy specifically targets radiation to tumors using antibody-streptavidin conjugates followed by radiolabeled biotin. A potential barrier to this cancer therapy is the presence of endogenous biotin in serum, which can block the biotin-binding sites of the antibody-streptavidin conjugate before the administration of radiolabeled biotin. Serum-derived biotin can also be problematic in clinical diagnostic applications. Due to the extremely slow dissociation of the biotin-streptavidin complex, this endogenous biotin can irreversibly block the biotin-binding sites of streptavidin and reduce therapeutic efficacy, as well as reduce sensitivity in diagnostic assays. We tested a streptavidin mutant (SAv-Y43A), which has a 67-fold lower affinity for biotin than wild type streptavidin, and three bivalent bis-biotin constructs as replacements for wild-type streptavidin and biotin used in pretargeting and clinical diagnostics. Biotin dimers were engineered with certain parameters including water solubility, biotinidase resistance, and linker lengths long enough to span the distance between two biotin-binding sites of streptavidin. The bivalent biotins were compared to biotin in exchange, retention, and off-rate assays. The faster off-rate of SAv-Y43A allowed efficient exchange of prebound biotin by the biotin dimers. In fluorescent competition experiments, the biotin dimer ligands displayed high avidity binding and essentially irreversible retention with SAv-Y43A. The off-rate of a biotinidase-stabilized biotin dimer from SAv-Y43A was 4.36 x 10(-)(6) s(-)(1), over 640 times slower compared to biotin. These findings strongly suggest that employing a mutant streptavidin in concert with a bivalent biotin can mitigate the deleterious impact of endogenous biotin, by allowing exchange of bound biotin and retention of the biotin dimer carriers.     

Mutation of a critical tryptophan to lysine in avidin or streptavidin may explain why sea urchin fibropellin adopts an avidin-like domain

Sea urchin fibropellins are epidermal growth factor homologues that harbor a C-terminal domain, similar in sequence to hen egg-white avidin and bacterial streptavidin. The fibropellin sequence was used as a conceptual template for mutation of designated conserved tryptophan residues in the biotin-binding sites of the tetrameric proteins, avidin and streptavidin. Three different mutations of avidin, Trp-110-Lys, Trp-70-Arg and the double mutant, were expressed in a baculovirus-infected insect cell system. A mutant of streptavidin, Trp-120-Lys, was similarly expressed. The homologous tryptophan to lysine (W-->K) mutations of avidin and streptavidin were both capable of binding biotin and biotinylated material. Their affinity for the vitamin was, however, significantly reduced: from K(d) approximately 10(-15) M of the wild-type tetramer down to K(d) approximately 10(-8) M for both W-->K mutants. In fact, their binding to immobilized biotin matrices could be reversed by the presence of free biotin. The Trp-70-Arg mutant of avidin bound biotin very poorly and the double mutant (which emulates the fibropellin domain) failed to bind biotin at all. Using a gel filtration fast-protein liquid chromatography assay, both W-->K mutants were found to form stable dimers in solution. These findings may indicate that mimicry in the nature of the avidin sequence and fold by the fibropellins is not designed to generate biotin-binding, but may serve to secure an appropriate structure for facilitating dimerization.     

Ca2+-dependent conformational changes in the neuronal Ca2+-sensor recoverin probed by the fluorescent dye Alexa647

Recoverin belongs to the superfamily of EF-hand Ca2+-binding proteins and operates as a Ca2+-sensor in vertebrate photoreceptor cells, where it regulates the activity of rhodopsin kinase GRK1 in a Ca2+-dependent manner. Ca2+-dependent conformational changes in recoverin are allosterically controlled by the covalently attached myristoyl group. The amino acid sequence of recoverin harbors a unique cysteine at position 38. The cysteine can be modified by the fluorescent dye Alexa647 using a maleimide-thiol coupling step. Introduction of Alexa647 into recoverin did not disturb the biological function of recoverin, as it can regulate rhodopsin kinase activity like unlabeled recoverin. Performance of the Ca2+-myristoyl switch of labeled recoverin was monitored by Ca2+-dependent association with immobilized lipids using surface plasmon resonance spectroscopy. When the Ca2+-concentration was varied, labeled myristoylated recoverin showed a 37%-change in fluorescence emission and a 34%-change in excitation intensity, emission and excitation maxima shifted by 6 and 18 nm, respectively. In contrast, labeled nonmyristoylated recoverin exhibited only minimal changes. Time-resolved fluorescence measurements showed biexponentiell fluorescence decay, in which the slower time constant of 2 ns was specifically influenced by Ca2+-induced conformational changes. A similar influence on the slower time constant was observed with the recoverin mutant RecE85Q that has a disabled EF-hand 2, but no such influence was detected with the mutant RecE121Q (EF-hand 3 is nonfunctional) that contains the myristoyl group in a clamped position. We conclude from our results that Alexa647 bound to cysteine 38 can monitor the conformational transition in recoverin that is under control of the myristoyl group.     

Controlled three-dimensional immobilization of biomolecules on chemically patterned surfaces

We used electron-beam lithography to fabricate chemical nanostructures, i.e. amino groups in aromatic self-assembled monolayers (SAMs) on gold surfaces. The amino groups are utilized as reactive species for mild covalent attachment of fluorescently labeled proteins. Since non-radiative energy transfer results in strong quenching of fluorescent dyes in the vicinity of the metal surfaces, different labeling strategies were investigated. Spacers of varying length were introduced between the gold surface and the fluorescently labeled proteins. First, streptavidin was directly coupled to the amino groups of the SAMs via a glutaraldehyde linker and fluorescently labeled biotin (X-Biotin) was added, resulting in a distance of approximately 2 nm between the dyes and the surface. Scanning confocal fluorescence images show that efficient energy transfer from the dye to the surface occurs, which is reflected in poor signal-to-background (S/B) ratios of approximately 1. Coupling of a second streptavidin layer increases the S/B-ratio only slightly to approximately 2. The S/B-ratio of the fluorescence signals could be further increased to approximately 4 by coupling of an additional fluorescently labeled antibody layer. Finally, we introduced tetraethylenepentamine as functional spacer molecule to diminish fluorescence quenching by the surface. We demonstrate that the use of this spacer in combination with multiple antibody layers enables the controlled fabrication of highly fluorescent three-dimensional nanostructures with S/B-ratios of >20. The presented technique might be used advantageously for the controlled three-dimensional immobilization of single protein or DNA molecules and the well-defined assembly of protein complexes.     

Fluorescence of native single-Trp mutants in the lactose permease from Escherichia coli: structural properties and evidence for a substrate-induced conformational change.

Six single-Trp mutants were engineered by individually reintroducing each of the native Trp residues into a functional lactose permease mutant devoid of Trp (Trp-less permease; Menezes ME, Roepe PD, Kaback HR, 1990, Proc Natl Acad Sci USA 87:1638-1642), and fluorescent properties were studied with respect to solvent accessibility, as well as alterations produced by ligand binding. The emission of Trp 33, Trp 78, Trp 171, and Trp 233 is strongly quenched by both acrylamide and iodide, whereas Trp 151 and Trp 10 display a decrease in fluorescence in the presence of acrylamide only and no quenching by iodide. Of the six single-Trp mutants, only Trp 33 exhibits a significant change in fluorescence (ca. 30% enhancement) in the presence of the substrate analog beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG). This effect was further characterized by site-directed fluorescent studies with purified single-Cys W33-->C permease labeled with 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS). Titration of the change in the fluorescence spectrum reveals a 30% enhancement accompanied with a 5-nm blue shift in the emission maximum, and single exponential behavior with an apparent KD of 71 microM. The effect of substrate binding on the rate of MIANS labeling of single-Cys 33 permease was measured in addition to iodide and acrylamide quenching of the MIANS-labeled protein. Complete blockade of labeling is observed in the presence of TDG, as well as a 30% decrease in accessibility to iodide with no change in acrylamide quenching. Overall, the findings are consistent with the proposal (Wu J, Frillingos S, Kaback HR, 1995a, Biochemistry 34:8257-8263) that ligand binding induces a conformational change at the C-terminus of helix I such that Pro 28 and Pro 31, which are on one face, become more accessible to solvent, whereas Trp 33, which is on the opposite face, becomes less accessible to the aqueous phase. The findings regarding accessibility to collisional quenchers are also consistent with the predicted topology of the six native Trp residues in the permease.     

Effect of streptavidins with varying biotin binding affinities on the properties of biotinylated gramicidin channels

The pentadecapeptide gramicidin A, which is known to form highly conductive ion channels in a bilayer lipid membrane by assembling as transmembrane head-to-head dimers, can be modified by attaching a biotin group to its C-terminus through an aminocaproyl spacer. Such biotinylated gramicidin A analogues also form ion channels in a hydrophobic lipid bilayer, exposing the biotin group to the aqueous bathing solution. Interaction of the biotinylated gramicidin channels with (strept)avidin has previously been shown to result in the appearance of a long-lasting open state with a doubled transition amplitude in single-channel traces and a deceleration of the macroscopic current kinetics as studied by the sensitized photoinactivation method. Here this interaction was studied further by using streptavidin mutants with weakened biotin binding affinities. The Stv-F120 mutant, having a substantially reduced biotin binding affinity, exhibited an efficacy similar to that of natural streptavidin in inducing both double-conductance channel formation and deceleration of the photoinactivation kinetics of the biotinylated gramicidin having a long linker arm. The Stv-A23D27 mutant with a severely weakened biotin binding affinity was ineffective in eliciting the double-conductance channels, but decelerated noticeably the photoinactivation kinetics of the long linker biotinylated gramicidin. However, the marked difference in the effects of the mutant and natural streptavidins was smaller than expected on the basis of the substantially reduced biotin binding affinity of the Stv-A23D27 mutant. This may suggest direct interaction of this mutant streptavidin with a lipid membrane in the process of its binding to biotinylated gramicidin channels. The role of linker arm length in the interaction of biotinylated gramicidins with streptavidin was revealed in experiments with a short linker gramicidin. This gramicidin analogue appeared to be unable to form double-conductance channels, though several lines of evidence were indicative of its binding by streptavidin. The data obtained show the conditions under which the interaction of streptavidin with biotinylated gramicidin leads to the formation of the double-conductance tandem channels composed of two cross-linked transmembrane dimers.     

Analysis of allosteric signal transduction mechanisms in an engineered fluorescent maltose biosensor

We previously reported the construction of a family of reagentless fluorescent biosensor proteins by the structure-based design of conjugation sites for a single, environmentally sensitive small molecule dye, thus providing a mechanism for the transduction of ligand-induced conformational changes into a macroscopic fluorescence observable. Here we investigate the microscopic mechanisms that may be responsible for the macroscopic fluorescent changes in such Fluorescent Allosteric Signal Transduction (FAST) proteins. As case studies, we selected three individual cysteine mutations (F92C, D95C, and S233C) of Escherichia coli maltose binding protein (MBP) covalently labeled with a single small molecule fluorescent probe, N-((2-iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), each giving rise to a robust FAST protein with a distinct maltose-dependent fluorescence response. The fluorescence emission intensity, anisotropy, lifetime, and iodide-dependent fluorescence quenching were determined for each conjugate in the presence and absence of maltose. Structure-derived solvent accessible surface areas of the three FAST proteins are consistent with experimentally observed quenching data. The D95C protein exhibits the largest fluorescence change upon maltose binding. This mutant was selected for further characterization, and residues surrounding the fluorophore coupling site were mutagenized. Analysis of the resulting mutant FAST proteins suggests that specific hydrogen-bonding interactions between the fluorophore molecule and two tyrosine side-chains, Tyr171 and Tyr176, in the open state but not the closed, are responsible for the dramatic fluorescence response of this construct. Taken together these results provide insights that can be used in future design cycles to construct fluorescent biosensors that optimize signaling by engineering specific hydrogen bonds between a fluorophore and protein.     

Mixed self-assembly of polydiacetylenes for highly specific and sensitive strip biosensors

Polydiacetylenes (PDAs), which possess unique properties that allow them to change color in response to environmental changes such as variations in pH, temperature, and molecular binding, have been widely investigated as signal transducers in biosensor applications. Most PDA-based sensors reported to date have been evaluated largely on the basis of their ability to detect purified samples, however, and their specificity has rarely been tested. In this study, novel PDAs fabricated on polyvinylidene fluoride (PVDF) strips by photoreaction of composite diacetylene self-assemblies were developed as biosensors, and nonspecific binding to off-target biomolecules was assessed. A mixed PDA surface containing biotin and ethanolamide bound the target, i.e., streptavidin, more specifically than did biotin alone. The optimized PDA biosensor exhibited approximately 2850-fold higher selectivity for streptavidin relative to bovine serum albumin controls. A PDA biosensor that was further prepared showed distinctive signals for the urine of diabetic patients compared to urine samples from healthy/non-diabetic person due to the concentration of microalbuminuria. To our knowledge, this is the first strip-type biosensor fabricated with PDAs and the first PDA-based biosensor that can effectively overcome the problem of nonspecific binding.