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 (~ 485 nm), green (~ 505 nm), yellow (~ 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

Interconversion of Anthozoa GFP-like fluorescent and non-fluorescent proteins by mutagenesis

BACKGROUND: Within the family of green fluorescent protein (GFP) homologs, one can mark two main groups, specifically, fluorescent proteins (FPs) and non-fluorescent or chromoproteins (CPs). Structural background of differences between FPs and CPs are poorly understood to date. RESULTS: Here, we applied site-directed and random mutagenesis in order to to transform CP into FP and vice versa. A purple chromoprotein asCP (asFP595) from Anemonia sulcata and a red fluorescent protein DsRed from Discosoma sp. were selected as representatives of CPs and FPs, respectively. For asCP, some substitutions at positions 148 and 165 (numbering in accordance to GFP) were found to dramatically increase quantum yield of red fluorescence. For DsRed, substitutions at positions 148, 165, 167, and 203 significantly decreased fluorescence intensity, so that the spectral characteristics of these mutants became more close to those of CPs. Finally, a practically non-fluorescent mutant DsRed-NF was generated. This mutant carried four amino acid substitutions, specifically, S148C, I165N, K167M, and S203A. DsRed-NF possessed a high extinction coefficient and an extremely low quantum yield (< 0.001). These spectral characteristics allow one to regard DsRed-NF as a true chromoprotein. CONCLUSIONS: We located a novel point in asCP sequence (position 165) mutations at which can result in red fluorescence appearance. Probably, this finding could be applied onto other CPs to generate red and far-red fluorescent mutants. A possibility to transform an FP into CP was demonstrated. Key role of residues adjacent to chromophore's phenolic ring in fluorescent/non-fluorescent states determination was revealed

Natural-Target-Mimicking Translocation-Based Fluorescent Sensor for Detection of SARS-CoV-2 PLpro Protease Activity and Virus Infection in Living Cells

Papain-like protease PLpro, a domain within a large polyfunctional protein, nsp3, plays key roles in the life cycle of SARS-CoV-2, being responsible for the first events of cleavage of a polyprotein into individual proteins (nsp1–4) as well as for the suppression of cellular immunity. Here, we developed a new genetically encoded fluorescent sensor, named PLpro-ERNuc, for detection of PLpro activity in living cells using a translocation-based readout. The sensor was designed as follows. A fragment of nsp3 protein was used to direct the sensor on the cytoplasmic surface of the endoplasmic reticulum (ER) membrane, thus closely mimicking the natural target of PLpro. The fluorescent part included two bright fluorescent proteins—red mScarlet I and green mNeonGreen—separated by a linker with the PLpro cleavage site. A nuclear localization signal (NLS) was attached to ensure accumulation of mNeonGreen into the nucleus upon cleavage. We tested PLpro-ERNuc in a model of recombinant PLpro expressed in HeLa cells. The sensor demonstrated the expected cytoplasmic reticular network in the red and green channels in the absence of protease, and efficient translocation of the green signal into nuclei in the PLpro-expressing cells (14-fold increase in the nucleus/cytoplasm ratio). Then, we used PLpro-ERNuc in a model of Huh7.5 cells infected with the SARS-CoV-2 virus, where it showed robust ER-to-nucleus translocation of the green signal in the infected cells 24 h post infection. We believe that PLpro-ERNuc represents a useful tool for screening PLpro inhibitors as well as for monitoring virus spread in a culture

Analysis of alternative splicing of cassette exons at single-cell level using two fluorescent proteins

Alternative splicing plays a major role in increasing proteome complexity and regulating gene expression. Here, we developed a new fluorescent protein-based approach to quantitatively analyze the alternative splicing of a target cassette exon (skipping or inclusion), which results in an open-reading frame shift. A fragment of a gene of interest is cloned between red and green fluorescent protein (RFP and GFP)-encoding sequences in such a way that translation of the normally spliced full-length transcript results in expression of both RFP and GFP. In contrast, alternative exon skipping results in the synthesis of RFP only. Green and red fluorescence intensities can be used to estimate the proportions of normal and alternative transcripts in each cell. The new method was successfully tested for human PIG3 (p53-inducible gene 3) cassette exon 4. Expected pattern of alternative splicing of PIG3 minigene was observed, including previously characterized effects of UV light irradiation and specific mutations. Interestingly, we observed a broad distribution of normal to alternative transcript ratio in individual cells with at least two distinct populations with ∼45% and >95% alternative transcript. We believe that this method is useful for fluorescence-based quantitative analysis of alternative splicing of target genes in a variety of biological models

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure &lt; 100 mmHg (n = 1127), estimated glomerular filtration rate &lt; 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Anti-fading media for live cell GFP imaging.

Photostability is one of the most important characteristic of a dye for fluorescence microscopy. Recently we demonstrated that vitamins present in imaging media dramatically accelerate photobleaching of Enhanced Green Fluorescent Protein (EGFP) and many other green fluorescent and photoactivatable proteins. Here we tested all vitamins of commonly used media (such as Dulbecco's Modified Eagle Medium, DMEM) one-by-one and found that only two vitamins, riboflavin and pyridoxal, decrease photostability of EGFP. Thus, DMEM without riboflavin and pyridoxal can be used as an imaging medium, which ensures high photostability of GFPs at the expense of minimal biochemical disturbance. Then, we tested some antioxidants and found that a plant flavonoid rutin greatly enhances photostability of EGFP during live cell microscopy. In complete DMEM, rutin increased EGFP photostability up to the level of vitamin-depleted DMEM. Moreover, being added to vitamin-depleted DMEM, rutin was able to further suppress EGFP photobleaching. Potentially, new medium formulations can be widely used for fluorescence microscopy of GFP-expressing cells and model multicellular organisms in a variety of imaging applications, where photostability represents a challenge

Influence of cell growth conditions and medium composition on EGFP photostability in live cells

Photostability is a key characteristic of fluorescent proteins. It was recently demonstrated that green fluorescent protein (GFP) photobleaching in live cells can be suppressed by changes in medium composition. Here we show that Ham's F12 medium provides very high enhanced GFP (EGFP) photostability during fluorescence microscopy of live cells. This property of Ham's F12 medium is associated with decreased concentrations of riboflavin and pyridoxine, and increased concentrations of FeSO4, cyanocobalamine, lipoic acid, hypoxanthine, and thymidine compared with DMEM. We also found that the rate of EGFP photobleaching strongly depends on cell growth conditions such as cell density and the concentration of serum. We conclude that both imaging medium composition and the physiological state of the cells can strongly affect the photostability of fluorescent proteins. Thus, accurate comparison of the photostabilities of fluorescent proteins should be performed only in side-by-side analysis in identical cell growth conditions and media