Near-Infrared Fluorescent Proteins and Their Applications.

High transparency, low light-scattering, and low autofluorescence of mammalian tissues in the near-infrared (NIR) spectral range (~650-900 nm) open a possibility for in vivo imaging of biological processes at the micro- and macroscales to address basic and applied problems in biology and biomedicine. Recently, probes that absorb and fluoresce in the NIR optical range have been engineered using bacterial phytochromes - natural NIR light-absorbing photoreceptors that regulate metabolism in bacteria. Since the chromophore in all these proteins is biliverdin, a natural product of heme catabolism in mammalian cells, they can be used as genetically encoded fluorescent probes, similarly to GFP-like fluorescent proteins. In this review, we discuss photophysical and biochemical properties of NIR fluorescent proteins, reporters, and biosensors and analyze their characteristics required for expression of these molecules in mammalian cells. Structural features and molecular engineering of NIR fluorescent probes are discussed. Applications of NIR fluorescent proteins and biosensors for studies of molecular processes in cells, as well as for tissue and organ visualization in whole-body imaging in vivo, are described. We specifically focus on the use of NIR fluorescent probes in advanced imaging technologies that combine fluorescence and bioluminescence methods with photoacoustic tomography.

[Knotted proteins].

For a long time the presence of knots in a protein structure was not admitted. However, the existence of proteins with various types of knots has now been proven. The functional significance of knotted topology remains unclear despite numerous assumptions. Studing the structure of knots in proteins and their impact on the acquisition of native structure of proteins is important for the understanding of protein folding as a whole. We review the types of knots in the proteins discovered to date, including trefoil knot, figure-of-eight knot, and more complex knots with 5 and 6 crossings of polypeptide chain. We survey the folding of knotted proteins as well.

Histone H3K4me3 binding is required for the DNA repair and apoptotic activities of ING1 tumor suppressor.

Inhibitor of growth 1 (ING1) is implicated in oncogenesis, DNA damage repair, and apoptosis. Mutations within the ING1 gene and altered expression levels of ING1 are found in multiple human cancers. Here, we show that both DNA repair and apoptotic activities of ING1 require the interaction of the C-terminal plant homeodomain (PHD) finger with histone H3 trimethylated at Lys4 (H3K4me3). The ING1 PHD finger recognizes methylated H3K4 but not other histone modifications as revealed by the peptide microarrays. The molecular mechanism of the histone recognition is elucidated based on a 2.1 A-resolution crystal structure of the PHD-H3K4me3 complex. The K4me3 occupies a deep hydrophobic pocket formed by the conserved Y212 and W235 residues that make cation-pi contacts with the trimethylammonium group. Both aromatic residues are essential in the H3K4me3 recognition, as substitution of these residues with Ala disrupts the interaction. Unlike the wild-type ING1, the W235A mutant, overexpressed in the stable clones of melanoma cells or in HT1080 cells, was unable to stimulate DNA repair after UV irradiation or promote DNA-damage-induced apoptosis, indicating that H3K4me3 binding is necessary for these biological functions of ING1. Furthermore, N216S, V218I, and G221V mutations, found in human malignancies, impair the ability of ING1 to associate with H3K4me3 or to induce nucleotide repair and cell death, linking the tumorigenic activity of ING1 with epigenetic regulation. Together, our findings reveal the critical role of the H3K4me3 interaction in mediating cellular responses to genotoxic stresses and offer new insight into the molecular mechanism underlying the tumor suppressive activity of ING1.

[Fluorescent proteins: physical-chemical properties and application in cell biology].

Green fluorescent protein (GFP) from jellyfish Aequorea victoria is the most extensively studied and widely used in cell biology protein. At present novel naturally occurring GFP-like proteins have been discovered and enhanced mutants of Aequorea GFP have been created. These mutants differ from wild-type GFP by stability, value of quantum yield, absorption and fluorescence spectra position and photochemical properties. GFP-like proteins are the fast growing family. This review is an attempt to characterize the main groups of GFP-like proteins, describe their structure and mechanisms of chromophore formation and summarize the main trends of their utilization as markers and biosensors in cell and molecular biology.

[Expression of recombinant actin 5C from Drosophila in the methylotrophic yeast Pichia pastoris].

A system for actin expression in cells of yeast Pichia pastoris was constructed. Drosophila actin 5C, by 90% homologous to beta-actin of higher eukaryotes, was used as a target protein. To improve the procedures of target protein biosynthesis in yeast cells and of extraction and purification of recombinant actin the fusion protein GFP-actin 5C, having fluorescence protein GFP as a reporter part, was expressed and purified. The dimensions and resistance of yeast cells producing recombinant actin were characterized. It was shown that the size and form of cells depended on the accumulation of recombinant protein. The purified fusion protein was used for obtaining polyclonal antibody for testing recombinant actin.

[The role of quarternary structure in fluorescent protein stability].

The stability of fluorescent proteins (FPs) is of great importance for their use as reporters in studies of gene expression, protein dynamics and localization in cell. A comparative analysis of conformational stability of fluorescent proteins, having different association state was done. The list of studied proteins includes EGFP (monomer of green fluorescent protein, GFP), zFP506 (tetramer GFP), mRFP1 and "dimer2" (monomer and dimmer of red fluorescent protein), DsRed1 (red tetramer). The character of fluorescence intensity changes induced by guanidine hydrochloride (GdnHCl) of these proteins differs significantly. Green tetramer zFP506 has been shown to be more stable than green monomer EGFP, red dimmer "dimer2" has been shown to be less stable than red tetramer DsRed1, while red monomer mRFP1 has been shown to be practically as stable as tetramer DsRedl. It is concluded that the quaternary structure, being an important stabilizing factor, does not represent the only circumstance dictating the dramatic variations between fluorescent proteins in their conformational stability.

An enhanced mutant of red fluorescent protein DsRed for double labeling and developmental timer of neural fiber bundle formation.

We developed a new variant of coral-derived red fluorescent protein, DsRed S197Y, which is brighter and essentially free from secondary fluorescence peak. This makes it an ideal reporter for double labeling with green fluorescent protein (GFP). Though purified protein shows only 20% stronger fluorescence emission, culture cells that express DsRed S197Y exhibit a 3-3.5 times higher level of fluorescence than the cells that express wild-type DsRed. The much slower fluorescence maturation of DsRed than that of GFP is a beneficial feature for a fluorescent developmental timer application. When GFP and DsRed S197Y are expressed simultaneously, emissions start at different latency. This provides information about the time after the onset of expression. It reflects the order of cell differentiation if the expression is activated upon differentiation of certain types of cells. We applied this system to the developing brain of Drosophila and visualized, for the first time, the formation order of neural fibers within a large bundle. Our results showed that newly extending fibers of the mushroom body neurons mainly run into the core rather than to the periphery of the existing bundle. DsRed-based timer thus presents an indispensable tool for developmental biology and genetics of model organisms.

Kinetic analysis of maturation and denaturation of DsRed, a coral-derived red fluorescent protein.

The red fluorescent protein DsRed recently cloned from Discosoma coral, with its significantly red-shifted excitation and emission maxima (558 and 583 nm, respectively), has attracted great interest because of its spectral complementation to other fluorescent proteins, including the green fluorescent protein and its enhanced mutant EGFP. We demonstrated that the much slower DsRed fluorescence development could be described by a three-step kinetic model, in contrast to the fast EGFP maturation, which was fitted by a one-step model. At pH below 5.0 DsRed fluorescence gradually decreased, and the rate and degree of this fluorescence inactivation depended on the pH value. The kinetics of fluorescence inactivation under acidic conditions was fitted by a two-exponential function where the initial inactivation rate was proportional to the fourth power of proton concentration. Subsequent DsRed alkalization resulted in partial fluorescence recovery, and the rate and degree of such recovery depended on the incubation time in the acid. Recovery kinetics had a lag-time and was fitted minimally by three exponential functions. The DsRed absorbance and circular dichroism spectra revealed that the fluorescence loss was accompanied by protein denaturation. We developed a kinetic mechanism for DsRed denaturation that includes consecutive conversion of the initial state of the protein, protonated by four hydrogen ions, to the denatured one through three intermediates. The first intermediate still emits fluorescence, and the last one is subjected to irreversible inactivation. Because of tight DsRed tetramerization we have suggested that obligatory protonation of each monomer results in the fluorescence inactivation of the whole tetramer.

Denaturation and partial renaturation of a tightly tetramerized DsRed protein under mildly acidic conditions.

The red fluorescent protein, DsRed, recently cloned from coral Discosoma sp. has one of the longest fluorescence waves and one of the most complex absorbance spectra among the family of fluorescent proteins. In this work we found that with time DsRed fluorescence decreases under mildly acidic conditions (pH 4.0-4.8) in a pH-dependent manner, and this fluorescence inactivation could be partially recovered by subsequent re-alkalization. The DsRed absorbance and circular dichroism spectra under these conditions revealed that the fluorescence changes were caused by denaturation followed by partial renaturation of the protein. Further, analytical ultracentrifugation determined that native DsRed formed a tight tetramer under various native conditions. Quantitative analysis of the data showed that several distinct states of protein exist during the fluorescence inactivation and recovery, and the inactivation of fluorescence can be caused by protonation of a single ionogenic group in each monomer of DsRed tetramer.

Actin dynamics in lamellipodia of migrating border cells in the Drosophila ovary revealed by a GFP-actin fusion protein.

Directional migration of border cells in the Drosophila egg chambers is a developmentally regulated event that requires dynamic cellular functions. In this study, the electron microscopic observation of migrating border cells revealed loose actin bundles in forepart lamellipodia and numerous microvilli extending from nurse cells and providing multiple adhesive contacts with border cells. To analyze the dynamics of actin in migrating border cells in vivo, we constructed a green fluorescent protein-actin fusion protein and induced its expression in Drosophila using the GAL4/UAS system. The green fluorescent protein-actin was incorporated into the actin bundles and it enabled visualization of the rapid cytoskeletal changes in border cell lamellipodia. During the growth of the lamellipodia, the actin bundles that increased in number and size radiated from the bundle-organizing center. Quantification of the fluorescence intensity showed that an accumulation of bundle-associated and spotted green fluorescent protein-actin signals took place during their centripetal movement. Our results favored a treadmilling model for actin behavior in border cell lamellipodia.

Kinetics of ligand binding to a cluster of membrane-associated receptors.

The process of ligand binding to a cluster of membrane-associated receptors is examined theoretically. The theoretical model proposed involves the diffusion of ligands from the solution to the disc-like cluster of receptors on the surface of the spherical cell. When the ligand hits the internal part of the disc-like cluster, it begins to move laterally until it leaves the disc through its outer surface or is bound by one of the receptors inside the disc. If the ligand leaves the cluster, it returns to the solution and hits the disc again after a certain period, etc. According to our model the transition from a diffusion-limited to a reaction-limited process of binding is determined by the dimensionless parameter lambda identical to Dtc/a2, where D is the lateral diffusion coefficient, tc is the characteristic time of reaction, and a is the radius of the disc-like cluster. The forward rate constant kf turns out to be a function of lambda. Comparing the results of our calculations of kf with some experimental data we found that agreement is achieved at high lambda, i.e. the process of ligand binding by clustered receptors is predominantly reaction-limited.

[Mathematical depiction of the kinetics of thrombocyte aggregation]. / Matematicheskoe opisanie kinetiki agregatsii trombotsitov.

A study was made of a model of platelet aggregation in shear flow taking into account the kinetics of intercellular fibrinogen bond formation limited by the turning time of the doublet of collided platelets. The energy curve for two platelets was calculated. One fibrinogen bond was sufficient to form a doublet stable in the flow. The equation for the rate of single platelet disappearance with regard to the kinetics of intercellular fibrinogen bond formation, the stochastic character of bond distribution on the contacts of collided platelets, hydrodynamically controlled time of their interaction was derived. Approximation of the obtained dependencies for the platelet aggregation rate by Hill's equation was suggested and its parameters were determined. A qualitative criterion for kinetic behavior of the system of aggregating platelets was introduced.

[The equation for platelet aggregation rate]. / Uravnenie skorosti agregatsii trombotsitov.

A platelet aggregation model in shear flow taking into account the kinetics of intercellular fibrinogen bond formation limited by aggregated platelets rotation time was considered. For this consideration the average duration of platelets interaction in flow with shear rate value G is shown to be pi/4G. One fibrinogen bond is sufficient to form a solid aggregate between two platelets. The equation for single platelets disappearance rate concerned with intercellular fibrinogen bond formation, stochastic character of bond distribution in collided platelets and hydrodynamically controlled interaction time was obtained. The Hill's approximation for the obtained aggregation rate dependences was suggested and appropriate constants were determined. The qualitative criterion of platelets aggregating systems behavior was introduced.