Browsing by Author "Brown, Alex"

Filter results by typing the first few letters
Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    A tandem green–red heterodimeric fluorescent protein with high FRET efficiency
    (2016) Alsayed Alaraby Abdelsalam, Mohamad; D Wiens, Matthew; Shen, Yi; Li, Xi; Smisdom, Nick; Zhang, Wei; Brown, Alex; E Campbell, Robert
    The tetrameric red fluorescent protein from Discosoma sp. coral (DsRed) has previously been engineered to produce dimeric and monomeric fluorescent variants with excitation and emission profiles that span the visible spectrum. The brightest of the effectively monomeric DsRed variants is tdTomato—a tandem fusion of a dimeric DsRed variant. Here we describe the engineering of brighter red (RRvT), green (GGvT), and green–red heterodimeric (GRvT) tdTomato variants. GRvT exhibited 99 % intramolecular FRET efficiency, resulting in long Stokes shift red fluorescence. These new variants could prove useful for multicolor live‐cell imaging applications.
  • Thumbnail Image
    Item
    Two-photon absorption in fluorescent protein chromophores: TDDFT and CC2 results
    (American Chemical Society, 2014) Alsayed Alaraby Abdelsalam, Mohamad; Brown, Alex
    Two-photon spectroscopy of fluorescent proteins is a powerful bioimaging tool. Considerable effort has been made to measure absolute two-photon absorption (TPA) for the available fluorescent proteins. Being a technically involved procedure, there is significant variation in the published experimental measurements even for the same protein. In this work, we present a time-dependent density functional theory (TDDFT) study on isolated chromophores comparing the ability of four functionals (PBE0, B3LYP, CAM-B3LYP, and LC-BLYP) combined with the 6-31+G(d,p) basis set to reproduce averaged experimental TPA energies and cross sections. The TDDFT energies and TPA cross sections are also compared to corresponding CC2/6-31+G(d,p) results for excitation to S1 for the five smallest chromophores. In general, the computed TPA energies are less functional dependent than the TPA cross sections. The …
  • Thumbnail Image
    Item
    Two-photon absorption of fluorescent protein chromophores incorporating non-canonical amino acids: TD-DFT screening and classical dynamics
    (Royal Society of Chemistry, 2015) Alsayed Alaraby Abdelsalam, Mohamad; Brown, Alex
    الوصف Two-photon spectroscopy of fluorescent proteins is a powerful bio-imaging tool characterized by deep tissue penetration and little damage. However, two-photon spectroscopy has lower sensitivity than one-photon microscopy alternatives and hence a protein with a large two-photon absorption cross-section is needed. We use time-dependent density functional theory (TD-DFT) at the B3LYP/6-31+G(d,p) level of theory to screen twenty-two possible chromophores that can be formed upon replacing the amino-acid Tyr66 that forms the green fluorescent protein (GFP) chromophore with a non-canonical amino acid. A proposed chromophore with a nitro substituent was found to have a large two-photon absorption cross-section (29 GM) compared to other fluorescent protein chromophores as determined at the same level of theory. Classical molecular dynamics are then performed on a nitro-modified fluorescent protein …