First biphotochromic fluorescent protein moxSAASoti stabilized for oxidizing environment.

Biphotochromic proteins simultaneously possess reversible photoswitching (on-to-off) and irreversible photoconversion (green-to-red). High photochemical reactivity of cysteine residues is one of the reasons for the development of "mox"-monomeric and oxidation resistant proteins. Based on site-saturated simultaneous two-point C105 and C117 mutagenesis, we chose C21N/C71G/C105G/C117T/C175A as the moxSAASoti variant. Since its on-to-off photoswitching rate is higher, off-to-on recovery is more complete and photoconversion rates are higher than those of mSAASoti. We analyzed the conformational behavior of the F177 side chain by classical MD simulations. The conformational flexibility of the F177 side chain is mainly responsible for the off-to-on conversion rate changes and can be further utilized as a measure of the conversion rate. Point mutations in mSAASoti mainly affect the pKa values of the red form and off-to-on switching. We demonstrate that the microscopic measure of the observed pKa value is the C-O bond length in the phenyl fragment of the neutral chromophore. According to molecular dynamics simulations with QM/MM potentials, larger C-O bond lengths are found for proteins with larger pKa. This feature can be utilized for prediction of the pKa values of red fluorescent proteins.

The role of cysteine residues in the allosteric modulation of the chromophore phototransformations of biphotochromic fluorescent protein SAASoti.

Biphotochromic fluorescent protein SAASoti contains five cysteine residues in its sequence and a V127T point mutation transforms it to the monomeric form, mSAASoti. These cysteine residues are located far from the chromophore and might control its properties only allosterically. The influence of individual, double and triple cysteine substitutions of mSAASoti on fluorescent parameters and phototransformation reactions (irreversible green-to-red photoconversion and reversible photoswitching) is studied. A set of mSAASoti mutant forms (C21N, C117S, C71V, C105V, C175A, C21N/C71V, C21N/C175A, C21N/C71G/C175A) is obtained by site-directed mutagenesis. We demonstrate that the C21N variant exists in a monomeric form up to high concentrations, the C71V substitution accelerates photoconversion to the red form and the C105V variant has the maximum photoswitching rate. All C175A-containing variants demonstrate different photoswitching kinetics and decreased photostability during subsequent switching cycles compared with other considered systems. Classical molecular dynamic simulations reveal that the F177 side chain located in the vicinity of the chromophore is considerably more flexible in the mSAASoti compared with its C175A variant. This might be the explanation of the experimentally observed slowdown the thermal relaxation rate, i.e., trans-cis isomerization of the chromophore in mSAASoti upon C175A substitution.

Sensors for Proteolytic Activity Visualization and Their Application in Animal Models of Human Diseases.

Various sensors designed for optical and photo(opto)acoustic imaging in living systems are becoming essential components of basic and applied biomedical research. Some of them including those developed for determining enzyme activity in vivo are becoming commercially available. These sensors can be used for various fluorescent signal detection methods: from whole body tomography to endoscopy with miniature cameras. Sensor molecules including enzyme-cleavable macromolecules carrying multiple quenched near-infrared fluorophores are able to deliver their payload in vivo and have long circulation time in bloodstream enabling detection of enzyme activity for extended periods of time at low doses of these sensors. In the future, more effective "activated" probes are expected to become available with optimized sensitivity to enzymatic activity, spectral characteristics suitable for intraoperative imaging of surgical field, biocompatibility and lack of immunogenicity and toxicity. New in vivo optical imaging methods such as the fluorescence lifetime and photo(opto)acoustic imaging will contribute to early diagnosis of human diseases. The use of sensors for in vivo optical imaging will include more extensive preclinical applications of experimental therapies. At the same time, the ongoing development and improvement of optical signal detectors as well as the availability of biologically inert and highly specific fluorescent probes will further contribute to the introduction of fluorescence imaging into the clinic.

Biodistribution and stability of CdSe core quantum dots in mouse digestive tract following per os administration: advantages of double polymer/silica coated nanocrystals.

CdSe-core, ZnS-capped semiconductor quantum dots (QDs) are of great potential for biomedical applications. However, applications in the gastrointestinal tract for in vivo imaging and therapeutic purposes are hampered by their sensitivity to acidic environments and potential toxicity. Here we report the use of coatings with a combination of polythiol ligands and silica shell (QDs PolyT-APS) to stabilize QDs fluorescence under acidic conditions. We demonstrated the stability of water-soluble QDs PolyT-APS both in vitro, in strong acidic solutions, and in vivo. The biodistribution, stability and photoluminescence properties of QDs in the gastrointestinal tract of mice after per os administration were assessed. We demonstrated that QDs coated with current traditional materials - mercapto compounds (QDs MPA) and pendant thiol group (QDs PolyT) - are not capable of protecting QDs from chemically induced degradation and surface modification. Polythiol ligands and silica shell quantum dots (QDs PolyT-APS) are suitable for biological and biomedical applications in the gastrointestinal tract.

Using lanthanide-based resonance energy transfer for in vitro and in vivo studies of biological processes.

This review describes key directions in the development of different probes based on complex compounds of lanthanides for in vitro and in vivo researches. The role of microsecond fluorescence of lanthanides for overcoming problems of background fluorescence is considered. The basic classes of synthetic and genetically encoded complex compounds of lanthanides are summarized. Main principles of designing lanthanide-based molecular sensors, including FRET sensors based on lanthanides and colored fluorescent proteins are described. Their applications in bioanalytical research and cellular bioimaging are described. The main principles of cellular bioimaging using lanthanides are formulated, questions of their delivery into cells are considered, and the problem of their potential toxicity for living organisms is discussed. A technique using multiphoton excitation of lanthanides is described.

Computer modeling of the structure and spectra of fluorescent proteins.

Fluorescent proteins from the family of green fluorescent proteins are intensively used as biomarkers in living systems. The chromophore group based on the hydroxybenzylidene-imidazoline molecule, which is formed in nature from three amino-acid residues inside the protein globule and well shielded from external media, is responsible for light absorption and fluorescence. Along with the intense experimental studies of the properties of fluorescent proteins and their chromophores by biochemical, X-ray, and spectroscopic tools, in recent years, computer modeling has been used to characterize their properties and spectra. We present in this review the most interesting results of the molecular modeling of the structural parameters and optical and vibrational spectra of the chromophorecontaining domains of fluorescent proteins by methods of quantum chemistry, molecular dynamics, and combined quantum-mechanical-molecular-mechanical approaches. The main emphasis is on the correlation of theoretical and experimental data and on the predictive power of modeling, which may be useful for creating new, efficient biomarkers.

Conformation dependence of pKa's of the chromophores from the purple asFP595 and yellow zFP538 fluorescent proteins.

Two members of the green fluorescent protein family, the purple asFP595 and yellow zFP538 proteins, are perspective fluorescent markers for use in multicolor imaging and resonance energy-transfer applications. We report the results of quantum based calculations of the solution pKa values for selected protonation sites of the denatured asFP595 and zFP538 chromophores in the trans- and cis-conformations in order to add in the interpretation of photophysical properties of these proteins. The pKa values were determined from the theromodynamic cycle based on B3LYP/6-311++G(2df,2p) calculations of the gas phase free energies of the molecules and the B3LYP/6-311++G(d,p) calculations of solvation energies. The results show that the pKa's of the protonation sites of the chromophore from asFP595 noticeably depend on the isomer conformation (cis- or trans-), while those of zFP538 are much less sensitive to isomerization.

Assessment of photodynamic destruction of Escherichia coli O157:H7 and Listeria monocytogenes by using ATP bioluminescence.

Antimicrobial photodynamic therapy was shown to be effective against a wide range of bacterial cells, as well as for fungi, yeasts, and viruses. It was shown previously that photodestruction of yeast cells treated with photosensitizers resulted in cell destruction and leakage of ATP. Three photosensitizers were used in this study: tetra(N-methyl-4-pyridyl)porphine tetratosylate salt (TMPyP), toluidine blue O (TBO), and methylene blue trihydrate (MB). A microdilution method was used to determine MICs of the photosensitizers against both Escherichia coli O157:H7 and Listeria monocytogenes. To evaluate the effects of photodestruction on E. coli and L. monocytogenes cells, a bioluminescence method for detection of ATP leakage and a colony-forming assay were used. All tested photosensitizers were effective for photodynamic destruction of both bacteria. The effectiveness of photosensitizers (in microgram-per-milliliter equivalents) decreased in the order TBO > MB > TMPyP for both organisms. The MICs were two- to fourfold higher for E. coli O157:H7 than for L. monocytogenes. The primary effects of all of the photosensitizers tested on live bacterial cells were a decrease in intracellular ATP and an increase in extracellular ATP, accompanied by elimination of viable cells from the sample. The time courses of photodestruction and intracellular ATP leakage were different for E. coli and L. monocytogenes. These results show that bioluminescent ATP-metry can be used for investigation of the first stages of bacterial photodestruction.

Color transitions in coral's fluorescent proteins by site-directed mutagenesis.

BACKGROUND: Green Fluorescent Protein (GFP) cloned from jellyfish Aequorea victoria and its homologs from corals Anthozoa have a great practical significance as in vivo markers of gene expression. Also, they are an interesting puzzle of protein science due to an unusual mechanism of chromophore formation and diversity of fluorescent colors. Fluorescent proteins can be subdivided into cyan (approximately 485 nm), green (approximately 505 nm), yellow (approximately 540 nm), and red (>580 nm) emitters. RESULTS: Here we applied site-directed mutagenesis in order to investigate the structural background of color variety and possibility of shifting between different types of fluorescence. First, a blue-shifted mutant of cyan amFP486 was generated. Second, it was established that cyan and green emitters can be modified so as to produce an intermediate spectrum of fluorescence. Third, the relationship between green and yellow fluorescence was inspected on closely homologous green zFP506 and yellow zFP538 proteins. The following transitions of colors were performed: yellow to green; yellow to dual color (green and yellow); and green to yellow. Fourth, we generated a mutant of cyan emitter dsFP483 that demonstrated dual color (cyan and red) fluorescence. CONCLUSIONS: Several amino acid substitutions were found to strongly affect fluorescence maxima. Some positions primarily found by sequence comparison were proved to be crucial for fluorescence of particular color. These results are the first step towards predicting the color of natural GFP-like proteins corresponding to newly identified cDNAs from corals.

Alternative cyclization in GFP-like proteins family. The formation and structure of the chromophore of a purple chromoprotein from Anemonia sulcata.

Anemonia sulcata purple protein (asFP595) belongs to a family of green fluorescent protein (GFP)-like proteins from the Anthozoa species. Similar to GFP, asFP595 apparently forms its chromophore by modifying amino acids within its polypeptide chain. Until now, the GFP-like proteins from Anthozoa were thought to contain chromophores with the same imidazolidinone core as GFP. Mass spectral analysis of a chromophore-containing tryptic pentapeptide from asFP595 demonstrates that chromophore formation in asFP595 is stoichiometrically the same as that in GFP: one H(2)O and two H(+) are released while a Schiff base and dehydrotyrosine are formed. However, structural studies of this asFP595 chromopeptide show that in contrast to GFP, the other peptide bond nitrogen and carbonyl carbon are required for chromophore cyclization, a reaction that yields the six-membered heterocycle 2-(4-hydroxybenzylidene)-6-hydroxy-2,5-dihydropyrazine. Spectrophotometric titration reveals three pH-dependent forms of the asFP595 chromopeptide: yellow (absorption maximum = 430 nm) at pH 3.0; red (absorption maximum = 535 nm) at pH 8.0; and colorless (absorption maximum = 380 nm) at pH 14.0. The pK(a) values for these spectral transitions (6.8 and 10.9) are consistent with the ionization of the phenolic group of dehydrotyrosine and deprotonation of the amidinium cation in the chromophore heterocycle, respectively. The amidinium group in asFP595 accounts for the unique absorption spectrum of the protein, which is substantially red-shifted relative to that of GFP. When the asFP595 chromophore cyclizes, the Cys-Met bond adjacent to the chromophore hydrolyzes, splitting the chromoprotein into 8- and 20-kDa fragments. High performance liquid chromatography analysis of a tryptic digest of denatured asFP595 shows that a pentapeptide with the cleaved Cys-Met bond is the only fragment associated with the red-shifted absorbance. These results imply that fragmentation of asFP595 is a critical step in protein maturation.

Natural animal coloration can Be determined by a nonfluorescent green fluorescent protein homolog.

It is generally accepted that the colors displayed by living organisms are determined by low molecular weight pigments or chromoproteins that require a prosthetic group. The exception to this rule is green fluorescent protein (GFP) from Aequorea victoria that forms a fluorophore by self-catalyzed protein backbone modification. Here we found a naturally nonfluorescent homolog of GFP to determine strong purple coloration of tentacles in the sea anemone Anemonia sulcata. Under certain conditions, this novel chromoprotein produces a trace amount of red fluorescence (emission lambda(max) = 595 nm). The fluorescence demonstrates unique behavior: its intensity increases in the presence of green light but is inhibited by blue light. The quantum yield of fluorescence can be enhanced dramatically by single amino acid replacement, which probably restores the ancestral fluorescent state of the protein. Other fluorescent variants of the novel protein have emission peaks that are red-shifted up to 610 nm. They demonstrate that long wavelength fluorescence is attainable in GFP-like fluorescent proteins.

Fluorescent proteins from nonbioluminescent Anthozoa species.

We have cloned six fluorescent proteins homologous to the green fluorescent protein (GFP) from Aequorea victoria. Two of these have spectral characteristics dramatically different from GFP, emitting at yellow and red wavelengths. All the proteins were isolated from nonbioluminescent reef corals, demonstrating that GFP-like proteins are not always functionally linked to bioluminescence. The new proteins share the same beta-can fold first observed in GFP, and this provided a basis for the comparative analysis of structural features important for fluorescence. The usefulness of the new proteins for in vivo labeling was demonstrated by expressing them in mammalian cell culture and in mRNA microinjection assays in Xenopus embryos.

Modification of monoclonal and polyclonal IgG with palladium (II) coproporphyrin I: stimulatory and inhibitory functional effects induced by two different methods.

Antibodies conjugated with porphyrins and metalloporphyrins have a great potential for applications in fluorescence or phosphorescence immunoassays as well as in photodynamic therapy, radioimaging and internal radiation therapy of cancer. Here we describe how the new preactivated metalloporphyrin, palladium (II) coproporphyrin I-tetra-N-hydroxysuccinimide ester, can be covalently attached to mouse monoclonal and rabbit anti-human ferritin antibodies. The advantages of the proposed reagent over the previously reported carboxylic porphyrins coupled through carbodiimide activation are indicated. Conformational changes in antibodies caused by each of the two methods were assessed from their binding to the antigen (a probe for the antibody Fv domain) and anti-IgG antibodies probing the global conformation of the CH2 domain in the Fc fragment. Porphyrin coupling through carbodiimide activation resulted in a decrease in both functional activities of modified antibodies even at low levels of modification. In contrast, when the N-hydroxysuccinimide (NHS) derivative of porphyrin was used, enhancement of the antigen-binding affinity of porphyrin-antibody conjugates occurred due to an increase in the conformational mobility (flexibility) of the modified antibodies. The stimulatory effect of conjugation was maximal when one porphyrin molecule was coupled to an antibody molecule. Coupling of NHS-activated porphyrin at pH 7.4, 7.8 and pH 8.5 suggested that the high efficiency of the reaction at pH 8.5 could be attributed predominantly to the formation of antibody aggregates, only 50% of which were covalently cross-linked. The lowest percentage of aggregates in porphyrin-antibody conjugates was found at pH 7.4 and a molar reagent-to-protein ratio in the 10:1-40:1 range. Thus, the use of the NHS-activated carboxylic porphyrin provides a mild, simple and convenient procedure for preparing antibody conjugates with enhanced antigen-binding affinity.

Monoclonal antibodies against metalloporphyrins. Specificity of interaction with structurally different metalloporphyrins.

Monoclonal antibodies against Pd-coproporphyrin I have been obtained. The antibody specificity for free as well as for conjugated Pd-coproporphyrin I is characterized. Affinity constants are estimated for 3 monoclonal antibodies effectively interacting with free Pd-coproporphyrin I. A comparative study on the binding of monoclonal antibodies with analogues and derivatives of Pd-coproporphyrin I has revealed that the antigen is mainly located inside the antibody paratope. The protein adjoins complementary to the metalloporphyrin in such a manner that antibodies obtained discern only isomer I, and to some degree, isomer III of coproporphyrin.

Kinetic fluorimetric determination of Cu(II) and Zn(II) by their incorporation reactions into a water-soluble porphyrin.

Kinetics of incorporation of Cu, Zn, Fe, Co, Ni and Mn divalent ions into coproporphyrin-I in imidazole buffer solution, pH 7.0, has been studied by monitoring the decrease in fluorescence intensity of the free base porphyrin. All reactions followed simple second-order rate law, the rate constants being decreased in the order Zn > Cu >> Co > Fe >> Mn, Ni. the kinetic fluorimetric method for the determination of Cu(II) and Zn(II) using their incorporation reactions into the porphyrin was developed. Initial rate and fixed-time methods were used to construct calibration graphs over the range 0-1.0 x 10(-5)M of both metals. The analytical characteristics of the method and effect of foreign ions were determined. In the presence of sodium thiosulphate as the masking reagent the determination of micromolar concentrations of Zn in the presence of a 10-fold excess of Cu is possible.

Universal phosphorescence immunoassay.

The aim of this study is to develop a universal phosphorescence immunoassay method using monoclonal antibodies to Pd-coproporphyrin (Pd-CP) and conjugates of various proteins with Pd-CP. Pd-CP and monoclonal antibodies obtained allow a convenient method for the determination of various antigens to be developed. The conditions for immunological reactions with Pd-CP were optimized with respect to the components affecting the nonspecific binding Pd-CP and Pd-CP conjugates.

Kinetics of oxidation of o-dianisidine by hydrogen peroxide in the presence of antibody complexes of iron(III) coproporphyrin.

The complex of iron(III) coproporphyrinI (FeCPI) with antibody D5E3 was studied as an artificial peroxidase, using o-dianisidine as a substrate. At saturation with respect to antibody, the initial rates of o-dianisidine oxidation are practically the same for free and bound FeCPI at a concentration 5 x 10(-9)M, but the catalytic rate constant (kc) for bound FeCPI exceed (kc) for free FeCPI by two- to three-fold. This difference can be explained by a real enhancement of (kc) at the antibody-active site. The dependence of initial rates of the reaction on substrate concentrations obeyed Michaelis-Menten kinetics and revealed substrate activation at high concentrations of o-dianisidine. A comparison of the Stern-Volmer constants for o-dianisidine-induced quenching of the porphyrin fluorescence proves that antibody-bound coproporphyrin is equivalently accessible to the substrate as protoporphyrin bound to apoperoxidase from horseradish peroxidase (HRP). Based on analysis of the (kc) dependence on H2O2 concentrations in the FeCPI-antibody system, we suggest that interaction with hydrogen peroxide is the rate-limiting step for the oxidation reaction.

pH dependence of fluorescence and absorbance spectra of free sulphonated aluminium phthalocyanine and its conjugate with monoclonal antibodies.

Water-soluble phthalocyanines and phthalocyanines linked to targeting monoclonal antibodies are considered to be one of the most promising photosensitizers in photodynamic therapy. Here the spectrum characteristics of sulphonated aluminium phthalocyanine and its protein conjugate in the pH range 1.8-12.0 in solution have been studied. The pK values are determined.