Abstract
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 ortho 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. the parent sfGFP. Similar time constants were also retrieved from the transient absorption spectra, substantiating the correlated electronic and vibrational motions on the intrinsic molecular timescales. Key carbon-halogen stretching motions coupled with phenolate ring motions of the deprotonated chromophores at ca. 908 and 890 cm−1 in Cl-GFP and Br-GFP exhibit enhanced activities in the electronic excited state and blue-shift during a distinct vibrational cooling process on the ps timescale. The retrieved structural dynamics change due to targeted site-specific halogenation of the chromophore thus provides an effective means to design new GFP derivatives and enrich the bioimaging probe toolset for life and medical sciences.
Highlights
The emission profiles of Cl-green fluorescent protein (GFP) and Br-GFP are redshifted vs. superfolder green fluorescent protein (sfGFP), and the shift magnitude increases with the mass of the halogen substituent (Figures 1A–C), the trend matching 3-iodotyrosine-GFP with a red-shifted emission beyond 520 nm (Young et al, 2011)
We prepared and characterized a series of superfolder GFP mutants with non-canonical amino acid (ncAA) chromophores using a combination of fs-Transient absorption (TA) spectroscopy, wavelength-tunable ground and excited-state femtosecond stimulated Raman spectroscopy (FSRS), and density functional theory (DFT) calculations of normal mode frequencies
The single-site halogenated proteins display improved properties that include the redshifted absorption and emission, increased concentration of deprotonated emissive species, and an increased fluorescence quantum yield. Such desirable application properties of the halogenated GFP mutants stem from a solid biophysical chemistry foundation in that they are a direct consequence of the engineerable molecular structure and dynamics of the photosensitive unit inside a protein matrix
Summary
Since its discovery several decades ago, green fluorescent protein (GFP) has been widely used for biolabeling and bioimaging due to its characteristic bright green emission, high fluorescence quantum yield (FQY), and stability (Shimomura et al, 1962; Chalfie et al, 1994; Tsien, 1998; Patterson and Lippincott-Schwartz, 2002; Zimmer, 2002; Betzig et al, 2006; Fang et al, 2009; Jung, 2012b; Dedecker et al, 2013). Point mutation Thr203Tyr of the enhanced yellow fluorescent protein, EYFP, leads to red-shifted absorption and emission due to a π-π interaction between spatially close tyrosine rings (Ormö et al, 1996; Wachter et al, 1998). The red fluorescent proteins are typically formed by an extended conjugation along the chromophore N-acylimine carbonyl (Gross et al, 2000; Shaner et al, 2004; Piatkevich et al, 2010; Subach and Verkhusha, 2012). Because these strategies in tuning GFP spectral properties conventionally involve only 20 standard amino acids, they pose certain limitations in achieving desired properties. The ncAAs can further act as site-specific vibrational probes or spin labels, making them ideal for structural dynamics techniques such as electron paramagnetic resonance (EPR) spectroscopy, NMR, and time-resolved vibrational spectroscopy (Fleissner et al, 2009; Sripakdeevong et al, 2014; Hall et al, 2019)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
