In-depth characterization of ubiquitin turnover in mammalian cells by fluorescence tracking.

Despite almost 40 years having passed from the initial discovery of ubiquitin (Ub), fundamental questions related to its intracellular metabolism are still enigmatic. Here we utilized fluorescent tracking for monitoring ubiquitin turnover in mammalian cells, resulting in obtaining qualitatively new data. In the present study we report (1) short Ub half-life estimated as 4 h; (2) for a median of six Ub molecules per substrate as a dynamic equilibrium between Ub ligases and deubiquitinated enzymes (DUBs); (3) loss on average of one Ub molecule per four acts of engagement of polyubiquitinated substrate by the proteasome; (4) direct correlation between incorporation of Ub into the distinct type of chains and Ub half-life; and (5) critical influence of the single lysine residue K27 on the stability of the whole Ub molecule. Concluding, our data provide a comprehensive understanding of ubiquitin-proteasome system dynamics on the previously unreachable state of the art.

Color Tuning of Fluorogens for FAST Fluorogen-Activating Protein.

Using benzylidene imidazolone core, we created a panel of color-shifted fluorogenic ligands for FAST protein without compromise to the binding efficiency and the utility for live-cell protein labeling. This study highlights the potential of benzylidene imidazolones derivatives for rapid expansion of a pallet of live-cell fluorogenic labeling tools.

Short Duplex Module Coupled to G-Quadruplexes Increases Fluorescence of Synthetic GFP Chromophore Analogues.

Aptasensors became popular instruments in bioanalytical chemistry and molecular biology. To increase specificity, perspective signaling elements in aptasensors can be separated into a G-quadruplex (G4) part and a free fluorescent dye that lights up upon binding to the G4 part. However, current systems are limited by relatively low enhancement of fluorescence upon dye binding. Here, we added duplex modules to G4 structures, which supposedly cause the formation of a dye-binding cavity between two modules. Screening of multiple synthetic GFP chromophore analogues and variation of the duplex module resulted in the selection of dyes that light up after complex formation with two-module structures and their RNA analogues by up to 20 times compared to parent G4s. We demonstrated that the short duplex part in TBA25 is preferable for fluorescence light up in comparison to parent TBA15 molecule as well as TBA31 and TBA63 stabilized by longer duplexes. Duplex part of TBA25 may be partially unfolded and has reduced rigidity, which might facilitate optimal dye positioning in the joint between G4 and the duplex. We demonstrated dye enhancement after binding to modified TBA, LTR-III, and Tel23a G4 structures and propose that such architecture of short duplex-G4 signaling elements will enforce the development of improved aptasensors.

Red-Shifted Substrates for FAST Fluorogen-Activating Protein Based on the GFP-Like Chromophores.

A genetically encoded fluorescent tag for live cell microscopy is presented. This tag is composed of previously published fluorogen-activating protein FAST and a novel fluorogenic derivative of green fluorescent protein (GFP)-like chromophore with red fluorescence. The reversible binding of the novel fluorogen and FAST is accompanied by three orders of magnitude increase in red fluorescence (580-650 nm). The proposed dye instantly stains target cellular proteins fused with FAST, washes out in a minute timescale, and exhibits higher photostability of the fluorescence signal in confocal and widefield microscopy, in contrast with previously published fluorogen:FAST complexes.

Designing redder and brighter fluorophores by synergistic tuning of ground and excited states.

We strategically modified the GFP core via chemical synthesis to make redder and brighter biomimetic fluorophores. Based on quantum calculations, solvatochromism analysis, and femtosecond Raman, we unveiled the additive effect of tuning the electronic ground and excited states, respectively, to achieve a dramatic emission redshift with a "double-donor-one-acceptor" structure.

Excited-state locked amino analogues of the green fluorescent protein chromophore with a giant Stokes shift.

We design a novel class of excited-state locked GFP chromophores by introducing an amine group at the double exo-bond and a difluoroboryl bridge. We show that these chromophores intrinsically exhibit a very large Stokes shift of 1 eV. Further tuning through chemical modifications of their aryl substituents makes them environmentally sensitive.