Environment-sensitive Fluorescent Probe Research Articles

Rationally designed fluorescently labeled sulfate‐binding protein mutants: Evaluation in the development of a sensing system for sulfate

Periplasmic binding proteins from E. coli undergo large conformational changes upon binding their respective ligands. By attaching a fluorescent probe at rationally selected unique sites on the protein, these conformational changes in the protein can be monitored by measuring the changes in fluorescence intensity of the probe which allow the development of reagentless sensing systems for their corresponding ligands. In this work, we evaluated several sites on bacterial periplasmic sulfate-binding protein (SBP) for attachment of a fluorescent probe and rationally designed a reagentless sensing system for sulfate. Eight different mutants of SBP were prepared by employing the polymerase chain reaction (PCR) to introduce a unique cysteine residue at a specific location on the protein. The sites Gly55, Ser90, Ser129, Ala140, Leu145, Ser171, Val181, and Gly186 were chosen for mutagenesis by studying the three-dimensional X-ray crystal structure of SBP. An environment-sensitive fluorescent probe (MDCC) was then attached site-specifically to the protein through the sulfhydryl group of the unique cysteine residue introduced. Each fluorescent probe-conjugated SBP mutant was characterized in terms of its fluorescence properties and Ser171 was determined to be the best site for the attachment of the fluorescent probe that would allow for the development of a reagentless sensing system for sulfate. Three different environment-sensitive fluorescent probes (1,5-IAEDANS, MDCC, and acylodan) were studied with the SBP171 mutant protein. A calibration curve for sulfate was constructed using the labeled protein and relating the change in the fluorescence intensity with the amount of sulfate present in the sample. The detection limit for sulfate was found to be in the submicromolar range using this system. The selectivity of the sensing system was demonstrated by evaluating its response to other anions. A fast and selective sensing system with detection limits for sulfate in the submicromolar range was developed.

An Accessible Hydrophobic Surface Is a Key Element of the Molecular Chaperone Action of Atp11p

Atp11p is a soluble protein of mitochondria that binds unassembled beta subunits of the F(1)-ATPase and prevents them from aggregating in the matrix. In this report, we show that Atp11p protects the insulin B chain from aggregating in vitro and therefore acts as a molecular chaperone. The chaperone action of Atp11p is mediated by hydrophobic interactions. An accessible hydrophobic surface in Atp11p was identified with the environment-sensitive fluorescent probe 1,1'-bis(4-anilino-5-napththalenesulfonic acid (bis-ANS). The spectral changes of bis-ANS in the presence of Atp11p indicate that the probe binds to a nonpolar region of the protein. Furthermore, the dye quenches the fluorescence of Atp11p tryptophan residues in a concentration-dependent manner. Although up to three molecules of bis-ANS can bind cooperatively to Atp11p, the binding of only one dye molecule is sufficient to virtually eliminate the chaperone activity of the protein.

Imaging of conformational changes of proteins with a new environment-sensitive fluorescent probe designed for site-specific labeling of recombinant proteins in live cells.

We demonstrate herein a new method for imaging conformational changes of proteins in live cells using a new synthetic environment-sensitive fluorescent probe, 9-amino-6,8-bis(1,3,2-dithioarsolan-2-yl)-5H-benzo[a]phenoxazin-5-one. This fluorescent probe can be attached to recombinant proteins containing four cysteine residues at the i, i + 1, i + 4, and i + 5 positions of an alpha-helix. The specific binding of the fluorescent probe to this 4Cys motif enables fluorescent labeling inside cells by its extracellular administration. The high sensitivity of the fluorophore to its environment enables monitoring of the conformational changes of the proteins in live cells as changes in its fluorescence intensity. The present method was applied to calmodulin (CaM), a Ca2+-binding protein that was well-known to expose hydrophobic domains, depending on the Ca2+ concentration. A recombinant CaM fused at its C-terminal with a helical peptide containing a 4Cys motif was labeled with the fluorescent probe inside live cells. The fluorescence intensity changed reversibly depending on the intracellular Ca2+ concentration, which reflected the conformational change of the recombinant CaM in the live cells.

Control of a Redox Reaction on Lipid Bilayer Surfaces by Membrane Dipole Potential

Nitro-2,1,3-benzoxadiazol-4-yl (NBD) group is a widely used, environment-sensitive fluorescent probe. The negatively charged dithionite rapidly reduces the accessible NBD-labeled lipids in liposomes to their corresponding nonfluorescent derivatives. In this study both the phospholipid headgroup and acyl chain NBD-labeled l- α-1,2-dipalmitoyl- sn-glycero-3-phospho-[ N-(4-nitrobenz-2-oxa-1,3-diazole)-ethanolamine] (DPPN) and 1-acyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]dodecanoyl]- sn-glycero-3-phosphocholine (NBD-PC), respectively, were employed. The correlation of both the rate coefficient k 1 of the redox reaction and the fluorescence properties of the two probes with the membrane dipole potential Ψ in fluid dipalmitoylglycerophosphocholine (DPPC) liposomes is demonstrated. When Ψ of the bilayer was varied (decreased by phloretin or increased by 6-ketocholestanol), the value for k 1 decreased for both DPPN and NBD-PC with increasing Ψ. For both fluorophores a positive correlation to Ψ was evident for the relative fluorescence emission intensity (RFI, normalized to the emission of the fluorophore in a DPPC matrix). The relative changes in emission intensity as a function of Ψ were approximately equal for both NBD derivatives. Changes similar to those caused by phloretin were seen when dihexadecylglycerophosphocholine (DHPC) was added to DPPC liposomes, in keeping with the lower dipole potential for the former lipid compound compared with DPPC. These effects of Ψ on NBD fluorescence should be taken into account when interpreting data acquired using NBD-labeled lipids as fluorescent probes.

Mechanical and chemical properties of cysteine-modified kinesin molecules.

To probe the structural changes within kinesin molecules, we made the mutants of motor domains of two-headed kinesin (4-411 aa) in which either all the five cysteines or all except Cys45 were mutated. A residual cysteine (Cys45) of the kinesin mutant was labeled with an environment-sensitive fluorescent probe, acrylodan. ATPase activity, mechanical properties, and fluorescence intensity of the mutants were measured. Upon acrylodan-labeled kinesin binding to microtubules in the presence of 1 mM AMPPNP, the peak intensity was enhanced by 3.4-fold, indicating the structural change of the kinesin head by the binding. Substitution of cysteines decreased both the maximum microtubule-activated ATPase and the sliding velocity to the same extent. However, the maximum force and the step size were not affected; the force produced by a single molecule was 6-6.5 pN, and a step size due to the hydrolysis of one ATP molecule by kinesin molecules was about 10 nm for all kinesins. This step size was close to a unitary step size of 8 nm. Thus, the mechanical events of kinesin are tightly coupled with the chemical events.

人工膜と生体膜の接点を探る 最近の膜研究の方法 環境感受性色素によるリポソームと細胞の膜物性のイメージング

The environment-sensitive fluorescent probes, such as prodan, acrylodan and laurdan, change their emission wavelength depending on the polarity of their environment. For example, laurdan changes its emission peak from 440 nm in the gel phase of lipid bilayer to 490 nm in the liquid-crystalline phase. Time-resolved fluorescence spectra of laurdan in liposomes suggested that the 490 nm component of fluorescence was a emission from dipolar-relaxed states of excited fluorophores and that the dipolar relaxation was due to water molecules penetrated into the hydrophobic region of membranes. The generalized polarization (GP), defined by two fluorescence intensities at these characteristic emission wavelengths, is a measure of membrane fluidity and polarity. We have constructed an imaging system which enables us to obtain spatial distribution of the GP. The system is composed of a fluorescence microscope, monochromatic filters, a cooled-CCD camera, and an image-analysis system. It was found that the average GP value in a liposome and its temperature dependence were almost the same as those obtained by a conventional fluorescence spectrometer, by appending only one instrumental constant which described the difference of relative sensitivity between the two wavelength channels. By using this apparatus to the study of membrane heterogeneity of CHO (chinese hamster ovary) cells, it was found that plasma membranes were less polar than the membranes of organelles.

Movement of a loop in domain 3 of aerolysin is required for channel formation.

Aerolysin is a channel-forming toxin that must oligomerize in order to become insertion-competent. Modeling based on the crystal structure of the proaerolysin dimer and electron microscopic images of the oligomer indicated that a loop in domain 3 must move away from the beta-sheet that forms the main body of the protein before oligomerization can proceed. In order to determine if movement actually occurs, strategically located amino acids in the loop and in the sheet were replaced with cysteines by site-directed mutagenesis. A double mutant was produced in which the new cysteines, at position 253 on the loop and position 300 in the sheet, were close enough together to allow formation of a disulfide bridge. The double mutant was unable to oligomerize, and it was completely inactive, showing not only that the bridge had formed but also that movement of the loop was essential for formation of the oligomer. The existence of the bridge was confirmed by X-ray crystallography. The reduced form of the protein and the single mutants T253C and A300C were as active as wild type, indicating that the amino acid replacements themselves had no functional consequences. Labeling studies using an environment-sensitive fluorescent sulfhydryl-reactive probe confirmed that the structure of the protein changes in the loop region as a consequence of proteolytic activation of proaerolysin, a step which also must precede oligomerization.

An operator-induced conformational change in the C-terminal domain of the lambda repressor.

4,4'-bis(1-anilino-8-naphthalenesulfonic acid (Bis-ANS), an environment-sensitive fluorescent probe for hydrophobic region of proteins, binds specifically to the C-terminal domain of lambda repressor. The binding is characterized by positive cooperativity, the magnitude of which is dependent on protein concentration in the concentration range where dimeric repressor aggregates to a tetramer. In this range, positive cooperativity becomes more pronounced at higher protein concentrations. This suggests a preferential binding of Bis-ANS to the dimeric form of the repressor. Binding of single operator OR1 to the N-terminal domain of the repressor causes enhancement of fluorescence of the C-terminal domain bound Bis-ANS. The binding of single operator OR1 also leads to quenching of fluorescence of tryptophan residues, all of which are located in the hinge or the C-terminal domain. Thus two different fluorescent probes indicate an operator-induced conformational change which affects the C-terminal domain. The significance of this conformational change with respect to the function of lambda repressor has been discussed.

Solubility properties of the alkylmethylglucamide surfactants

The critical micelle concentration (CMC) and the ability to solubilize and form vesicles from phospholipids are important criteria for the selection of a surfactant for reconstitution protocols. The CMC and its temperature dependence were determined for an homologous series of alkylmethylglucamides (MEGA-8, MEGA-9, MEGA-10). Each detergent was added continuously from a concentrated solution to a saline buffer with the environment-sensitive fluorescent probe ANS, held in a thermojacketed cuvette; ANS fluorescence increases at the CMC. The CMCs at 25°C were 51.3, 16.0 and 4.8 mM for MEGA-8, MEGA-9 and MEGA-10. The free energy change for transfer to a micellar environment per -CH 2- was −740 cal/mol, similar to other alkyl series. The CMCs decreased slightly with increasing temperature ( T = 5–40°C) for MEGA-9 and MEGA-10 while that of MEGA-8 was virtually insensitive to temperature in this range. MEGA-9 solubilization of egg PC in aqueous solutions was determined as a function of [PC] and temperature. The lamellar-micellar phase boundaries were determined by simultaneous 90° light scattering and the resonance energy transfer using the headgroup labeled lipid probes NBD-PE and Rho-PE. The [MEGA-9] at solubilization was linear with [PC]; the MEGA-9 to egg PC ratio in the structures at optical clarity was 2.3 while the monomeric [MEGA-9] was 14.3 mM or slightly lower than the CMC at 25°C. Solubilization of egg PC by MEGA-9 was somewhat more temperature-dependent than the CMC of this detergent. Vesicles formed from MEGA-9 tended to be multilamellar. MEGA-9 is clearly different from octyl glucoside, despite its chemical similarity, in terms of its temperature sensitivity and vesicle forming characteristics.

On the possibility of fluorescence from twisted intramolecular charge transfer states of 2-dimethylamino-6-acylnaphthalenes. A quantum-chemical study

MNDO geometry, CNDO/S CI spectra and dipole moments of 2-dimethylamino-6-propionylnaphthalene (PRODAN), a representative of a class of highly environment-sensitive fluorescent probes, were computed. Only the conformer with a twisted dimethylamino group has an excited state dipole moment high enough (27 D) to explain spectral properties of PRODAN. On the basis of the Amos—Burrows theory, fluorescence from the TICT state is predicted in polar solvents.