Excitation Ratiometric Chloride Sensing in a Standalone Yellow Fluorescent Protein is Powered by the Interplay between Proton Transfer and Conformational Reorganization

Natural and laboratory-guided evolution has created a rich diversity of fluorescent protein (FP)-based sensors for chloride (Cl−). To date, such sensors have been limited to the Aequorea victoria green fluorescent...

Accurate and efficient structure-based computational mutagenesis for modeling fluorescence levels of Aequorea victoria green fluorescent protein mutants

A computational mutagenesis technique was used to characterize the structural effects associated with over 46 000 single and multiple amino acid variants of Aequorea victoria green fluorescent protein (GFP), whose functional effects (fluorescence levels) were recently measured by experimental researchers. For each GFP mutant, the approach generated a single score reflecting the overall change in sequence-structure compatibility relative to native GFP, as well as a vector of environmental perturbation (EP) scores characterizing the impact at all GFP residue positions. A significant GFP structure–function relationship (P < 0.0001) was elucidated by comparing the sequence-structure compatibility scores with the functional data. Next, the computed vectors for GFP mutants were used to train predictive models of fluorescence by implementing random forest (RF) classification and tree regression machine learning algorithms. Classification performance reached 0.93 for sensitivity, 0.91 for precision and 0.90 for balanced accuracy, and regression models led to Pearson’s correlation as high as r = 0.83 between experimental and predicted GFP mutant fluorescence. An RF model trained on a subset of over 1000 experimental single residue GFP mutants with measured fluorescence was used for predicting the 3300 remaining unstudied single residue mutants, with results complementing known GFP biochemical and biophysical properties. In addition, models trained on the subset of experimental GFP mutants harboring multiple residue replacements successfully predicted fluorescence of the single residue GFP mutants. The models developed for this study were accurate and efficient, and their predictions outperformed those of several related state-of-the-art methods.

A photostable monomeric superfolder GFP

ABSTRACTThe green fluorescent protein GFP from Aequorea victoria has been engineered extensively in the past to generate variants suitable for protein tagging. Early efforts produced the enhanced variant EGFP and its monomeric derivative mEGFP, which have useful photophysical properties, as well as superfolder GFP, which folds efficiently under adverse conditions. We previously generated msGFP, a monomeric superfolder derivative of EGFP. Unfortunately, compared to EGFP, msGFP and other superfolder GFP variants show faster photobleaching. We now describe msGFP2, which retains monomeric superfolder properties while being as photostable as EGFP. msGFP2 contains modified N- and C-terminal peptides that are expected to reduce nonspecific interactions. Compared to EGFP and mEGFP, msGFP2 is less prone to disturbing the functions of certain partner proteins. For general-purpose protein tagging, msGFP2 may be the best available derivative of A. victoria GFP.

Aequorea victoria’s secrets

Using mRNA-Seq and de novo transcriptome assembly, we identified, cloned and characterized nine previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from avGFP. Among these FPs are the brightest GFP homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including two that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing.

Crystal structure of the cyan fluorescent protein Cerulean-S175G

Enhanced cyan fluorescent protein (ECFP) was derived fromAequorea victoriagreen fluorescent protein (avGFP), notably with S65T/Y66W mutations. Its chromophore consists of a tripeptide comprised of Thr65, Trp66 and Gly67 (TWG) residues, while that ofavGFP consists of a Ser65, Tyr66 and Gly67 (SYG) tripeptide. Cerulean and SCFP3A were derived from ECFP-S72A/H148D (a double mutation) with additional Y145A and S175G mutations, respectively, while Cerulean-S175G has both mutations (Y145A and S175G). The crystal structures of these ECFP variants at neutral pH were reported to adopt two distinct major conformations calledECFPandCerulean. In this study, Cerulean-S175G was revealed to adopt only theCeruleanconformation, while Cerulean has been reported to adopt both theECFPand theCeruleanconformations in its crystal structures. Sharing the same S175G mutation with SCFP3A, Cerulean-S175G showed a slightly increased quantum yield, like SCFP3A, but did not adopt theECFPconformation adopted by SCFP3A. Detailed comparison of Cerulean-S175G and other ECFP variants revealed that the notable conformational changes in ECFP variants can be understood mainly in terms of the interaction between the Trp66 residue of the chromophore and residues 145–148 of β-strand 7.

Photoacid behaviour in a fluorinated green fluorescent protein chromophore: ultrafast formation of anion and zwitterion states

The photophysics of the chromophore of the green fluorescent protein in Aequorea victoria (avGFP) are dominated by an excited state proton transfer reaction.

Martin Chalfie y la proteína verde fluorescente

<p>La historia de la proteína verde fluorescente (GFP) se inicia con Osamu Shimomura a principios de la década de 1960, con su descubrimiento de esta proteína a partir de extractos de la medusa <em>Aequorea victoria</em>. Sin embargo, es a principios de la década de 1990 que el neurobiólogo Martin Chalfie, aplicando técnicas de ingeniería genética, logra por primera vez la expresión heteróloga de la GFP en procariotas (<em>Escherichia coli</em>) y eucariotas (<em>Caernohabditis elegans</em>), a partir de un clon del gen de la medusa que codifica a la GFP, preparado por Douglas Prasher.</p> <p>Además de fluorescer en verde, los resultados de este trabajo también demostraron que no se requiere ningún cofactor o enzima de la medusa para su expresión o para la formación misma del fluoróforo. Asimismo, Chalfie propuso que la GFP podría utilizarse para el marcaje de células en anima- les vivos o para el etiquetado de proteínas.</p> <p>Los resultados de Chalfie dispararon las investiga- ciones sobre esta proteína y en la actualidad se usa en diversos campos, tales como la biotecnología, la biología del desarrollo, la química ambiental y la medicina. En la medicina está ayudando a dilucidar los mecanismos celulares por los que se producen muchas enfermedades. A Roger Tsien se le debe, entre otros aportes, la creación de diversas mutan- tes de la GFP.</p>