Rational Design for High Bioorthogonal Fluorogenicity of Tetrazine-Encoded Green Fluorescent Proteins.

The development of bioorthogonal fluorogenic probes constitutes a vital force to advance life sciences. Tetrazine-encoded green fluorescent proteins (GFPs) show high bioorthogonal reaction rate and genetic encodability, but suffer from low fluorogenicity. Here, we unveil the real-time fluorescence mechanisms by investigating two site-specific tetrazine-modified superfolder GFPs via ultrafast spectroscopy and theoretical calculations.

Switching between Ultrafast Pathways Enables a Green-Red Emission Ratiometric Fluorescent-Protein-Based Ca Biosensor.

Ratiometric indicators with long emission wavelengths are highly preferred in modern bioimaging and life sciences. Herein, we elucidated the working mechanism of a standalone red fluorescent protein (FP)-based Ca biosensor, REX-GECO1, using a series of spectroscopic and computational methods. Upon 480 nm photoexcitation, the Ca-free biosensor chromophore becomes trapped in an excited dark state.

Dissecting Optical Response and Molecular Structure of Fluorescent Proteins With Non-canonical Chromophores.

Tracking the structural dynamics of fluorescent protein chromophores holds the key to unlocking the fluorescence mechanisms in real time and enabling rational design principles of these powerful and versatile bioimaging probes. By combining recent chemical biology and ultrafast spectroscopy advances, we prepared the superfolder green fluorescent protein (sfGFP) and its non-canonical amino acid (ncAA) derivatives with a single chlorine, bromine, and nitro substituent at the site to the phenolate oxygen of the embedded chromophore, and characterized them using an integrated toolset of femtosecond transient absorption and tunable femtosecond stimulated Raman spectroscopy (FSRS), aided by quantum calculations of the vibrational normal modes. A dominant vibrational cooling time constant of ~4 and 11 ps is revealed in Cl-GFP and Br-GFP, respectively, facilitating a ~30 and 12% increase of the fluorescent quantum yield vs.

Preferential Binding of Cytochrome c to Anionic Ligand-Coated Gold Nanoparticles: A Complementary Computational and Experimental Approach.

Membrane-bound proteins can play a role in the binding of anionic gold nanoparticles (AuNPs) to model bilayers; however, the mechanism for this binding remains unresolved. In this work, we determine the relative orientation of the peripheral membrane protein cytochrome c in binding to a mercaptopropionic acid-functionalized AuNP (MPA-AuNP). As this is nonrigid binding, traditional methods involving crystallographic or rigid molecular docking techniques are ineffective at resolving the question.

MHC class I loaded ligands from breast cancer cell lines: A potential HLA-I-typed antigen collection.

Unlabelled: To build a catalog of peptides presented by breast cancer cells, we undertook systematic MHC class I immunoprecipitation followed by elution of MHC class I-loaded peptides in breast cancer cells. We determined the sequence of 3196 MHC class I ligands representing 1921 proteins from a panel of 20 breast cancer cell lines. After removing duplicate peptides, i.

Ultrafast intermolecular proton transfer to a proton scavenger in an organic solvent.

Proton transfer reactions are functionally important in numerous chemical and biological processes. To unravel proton scavengers in action with atomistic details, we studied excited-state proton transfer (ESPT) from photoacid pyranine to the weak base acetate in methanol using transient absorption and wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS). Proton transfer is inhibited in neat methanol, but coherent proton motions and the formation of a charge-separated state occur on the sub-picosecond (sub-ps) timescale, accompanied by chromophore solvation wherein the longitudinal relaxation time of methanol (∼9 ps) dominates.