The Increase in the Peroxidase Activity of the Cytochrome with Substitutions in the Universal Binding Site Is Associated with Changes in the Ability to Interact with External Ligands.

Cytochrome (CytC), a one-electron carrier, transfers electrons from complex to cytochrome oxidase (CcO) in the electron-transport chain. Electrostatic interaction with the partners, complex and CcO, is ensured by a lysine cluster near the heme forming the Universal Binding Site (UBS). We constructed three mutant variants of mitochondrial CytC with one (2Mut), four (5Mut), and five (8Mut) Lys->Glu substitutions in the UBS and some compensating Glu->Lys substitutions at the periphery of the UBS for charge compensation.

Hybrid protein-peptide system for the selective pH-dependent binding and photodynamic ablation of cancer cells.

Creating new tools for the early diagnosis and treatment of cancer is one of the most important and intensively developing areas of modern medicine. Currently, photodynamic cancer therapy (PDT) is attracting increasing attention as a unique modality of minimally invasive treatment and due to the absence of acquired resistance. However, PDT is associated with undesirable activities, such as non-specific photodynamic effects of sunlight on healthy tissues.

Mutant Cytochrome C as a Potential Detector of Superoxide Generation: Effect of Mutations on the Function and Properties.

Cytochrome c (CytC) is a single-electron carrier between complex bc1 and cytochrome c-oxidase (CcO) in the electron transport chain (ETC). It is also known as a good radical scavenger but its participation in electron flow through the ETC makes it impossible to use CytC as a radical sensor. To solve this problem, a series of mutants were constructed with substitutions of Lys residues in the universal binding site (UBS) which interact electrostatically with negatively charged Asp and Glu residues at the binding sites of CytC partners, bc1 complex and CcO.

Ratiometric Singlet Oxygen Sensor Based on BODIPY-DPA Dyad.

Compounds sensitive to reactive oxygen species are widely used in the study of processes in living cells and in the development of therapeutic agents for photodynamic therapy. In the present work, we have synthesized a dyad in which the BODIPY dye is chemically bound to 9,10-diphenylanthracene (DPA). Here, DPA acts as a specific sensor of singlet oxygen and BODIPY as a reference dye.

Co-expression of different proteins in Escherichia coli using plasmids with identical origins of replication.

It is generally accepted that the use of two different plasmids with the identical origins of replication in bacteria is not desirable due to their "incompatibility". The utilization of the same bacterial enzymatic apparatus for replication of different plasmids is thought to cause a significant redistribution in favor of one of them. In the present work, examining co-expression of two different fluorescent proteins in Escherichia coli, we have shown that the use of highly homologous plasmids with identical origins of replication and providing resistance to different antibiotics results in high representation of both plasmids in bacteria.

Modulation of Membrane Microviscosity by Protein-Mediated Carotenoid Delivery as Revealed by Time-Resolved Fluorescence Anisotropy.

Carotenoids are potent antioxidants with a wide range of biomedical applications. However, their delivery into human cells is challenging and relatively inefficient. While the use of natural water-soluble carotenoproteins capable to reversibly bind carotenoids and transfer them into membranes is promising, the quantitative estimation of the delivery remains unclear.

Cancer cells targeting with genetically engineered constructs based on a pH-dependent membrane insertion peptide and fluorescent protein.

The targeted delivery of nanodrugs to malignant neoplasm is one of the most pressing challenges in the development of modern medicine. It was reported earlier that a bacteriorhodopsin-derived pH low insertion peptide (pHLIP) targets acidic tumors and has the ability to translocate low molecular weight cargoes across the cancer cell membrane. Here, to better understand the potential of pHLIP-related technologies, we used genetically engineered fluorescent protein (EGFP) as a model protein cargo and examined targeting efficiencies of EGFP-pHLIP hybrid constructs in vitro with the HeLa cell line at different pHs.

FLIM-Based Intracellular and Extracellular pH Measurements Using Genetically Encoded pH Sensor.

The determination of pH in live cells and tissues is of high importance in physiology and cell biology. In this report, we outline the process of the creation of SypHerExtra, a genetically encoded fluorescent sensor that is capable of measuring extracellular media pH in a mildly alkaline range. SypHerExtra is a protein created by fusing the previously described pH sensor SypHer3s with the neurexin transmembrane domain that targets its expression to the cytoplasmic membrane.

Multiscale classification of heart failure phenotypes by unsupervised clustering of unstructured electronic medical record data.

As a leading cause of death and morbidity, heart failure (HF) is responsible for a large portion of healthcare and disability costs worldwide. Current approaches to define specific HF subpopulations may fail to account for the diversity of etiologies, comorbidities, and factors driving disease progression, and therefore have limited value for clinical decision making and development of novel therapies. Here we present a novel and data-driven approach to understand and characterize the real-world manifestation of HF by clustering disease and symptom-related clinical concepts (complaints) captured from unstructured electronic health record clinical notes.

Impact of external amino acids on fluorescent protein chromophore biosynthesis revealed by molecular dynamics and mutagenesis studies.

The precise positioning of catalytic amino acids against the substrate in an enzyme active site is a crucial factor in biocatalysis. Biosynthesis of the chromophores of fluorescent proteins (FPs) is an autocatalytic process that must conform to these requirements. Here, we show that, in addition to the internal amino acid residues in the proximity of the chromophore, chromophore biosynthesis is influenced by the remote amino acids exposed on the outer surface of the β-barrel structure of the FP.

Genetically encoded fluorescent indicators for live cell pH imaging.

Background: Intracellular pH underlies most cellular processes. There is emerging evidence of a pH-signaling role in plant cells and microorganisms. Dysregulation of pH is associated with human diseases, such as cancer and Alzheimer's disease.

Fluorescent protein Dendra2 as a ratiometric genetically encoded pH-sensor.

Fluorescent protein Dendra2 is a monomeric GFP-like protein that belongs to the group of Kaede-like photoconvertible fluorescent proteins with irreversible photoconversion from a green- to red-emitting state when exposed to violet-blue light. In an acidic environment, photoconverted Dendra2 turns green due to protonation of the phenolic group of the chromophore with pKa of about 7.5.

BODIPY-based dye for no-wash live-cell staining and imaging.

In nonpolar solvents, hydrophobic organic fluorophores often show bright fluorescence, whereas in polar media, they usually suffer from aggregation-caused quenching (ACQ). Here, we harnessed this solvatochromic behavior of a 1,3,5,7-tetramethyl-BODIPY derivative for cell staining and applied it to live-cell imaging and flow cytometry. As opposed to commercially available dyes, this BODIPY derivative showed excellent contrast immediately after staining and did not require any wash-off.

Crystal structure of the fluorescent protein from Dendronephthya sp. in both green and photoconverted red forms.

The fluorescent protein from Dendronephthya sp. (DendFP) is a member of the Kaede-like group of photoconvertible fluorescent proteins with a His62-Tyr63-Gly64 chromophore-forming sequence. Upon irradiation with UV and blue light, the fluorescence of DendFP irreversibly changes from green (506 nm) to red (578 nm).

Probing the structural determinants of yellow fluorescence of a protein from Phialidium sp.

Fluorescent proteins homologous to green fluorescent protein (avGFP) display pronounced spectral variability due to different chromophore structures and variable chromophore interactions with the surrounding amino acids. To gain insight into the structural basis for yellow emission, the 3D structure of phiYFP (λ(em)=537 nm), a protein from the sea medusa Phialidium sp., was built by a combined homology modeling - mass spectrometry approach.

GFP family: structural insights into spectral tuning.

Proteins homologous to green fluorescent protein (GFP) span most of the visible spectrum, offering indispensable tools for live cell imaging. Structural transformations, such as posttranslational autocatalytic and photo-induced modifications, chromophore isomerization, and rearrangements in its environment underlie the unique capacity of these proteins to tune their own optical characteristics. A better understanding of optical self-tuning mechanisms would assist in the engineering of more precisely adapted variants and in expanding the palette of GFP-like proteins to the near-infrared region.

Development of transformation vectors based upon a modified plant alpha-tubulin gene as the selectable marker.

A plant transformation and selection system has been developed utilizing a modified tubulin gene as a selectable marker. The vector constructs carrying a mutant alpha-tubulin gene from goosegrass conferring resistance to dinitroaniline herbicides were created for transformation of monocotyledonous and dicotyledonous plants. These constructs contained beta- and/or mutant alpha-tubulin genes driven either by ubiquitin or CaMV 35S promoter.

Chromophore aspartate oxidation-decarboxylation in the green-to-red conversion of a fluorescent protein from Zoanthus sp. 2.

The red fluorescence of a Discosoma coral protein is the result of an additional autocatalytic oxidation of a green fluorescent protein (GFP)-like chromophore. This reaction creates an extra pi-electron conjugation by forming a C=N-C=O substituent. Here we show that the red fluorescence of a protein from Zoanthus sp.

Refined crystal structures of red and green fluorescent proteins from the button polyp Zoanthus.

The three-dimensional structures of the wild-type red (zRFP574) and green (zGFP506) fluorescent proteins (FP) from the button polyp Zoanthus have been determined at 1.51 and 2.2 A resolution, respectively.

Structure and reactivity of the chromophore of a GFP-like chromoprotein from Condylactis gigantea.

Here we present the study of the chromophore structure of the purple chromoprotein from Condylactis gigantea. Tandem mass spectrometry and 1H and 13C NMR of the chromopeptide reveal that the protein contains a chromophore with a chemical structure identical to that of the red fluorescent protein from Discosoma sp. A single A63G substitution demonstrates that the nature of the first amino acid of the XYG chromophore-forming sequence is dispensable for the chromoprotein red shift development.

A purple-blue chromoprotein from Goniopora tenuidens belongs to the DsRed subfamily of GFP-like proteins.

A number of recently cloned chromoproteins homologous to the green fluorescent protein show a substantial bathochromic shift in absorption spectra. Compared with red fluorescent protein from Discosoma sp. (DsRed), mutants of these so-called far-red proteins exhibit a clear red shift in emission spectra as well.