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.

Capturing Structural Snapshots during Photochemical Reactions with Ultrafast Raman Spectroscopy: From Materials Transformation to Biosensor Responses.

Chemistry studies the composition, structure, properties, and transformation of matter. A mechanistic understanding of the pertinent processes is required to translate fundamental knowledge into practical applications. The current development of ultrafast Raman as a powerful time-resolved vibrational technique, particularly femtosecond stimulated Raman spectroscopy (FSRS), has shed light on the structure-energy-function relationships of various photosensitive systems.

Dynamic Raman Line Shapes on an Evolving Excited-State Landscape: Insights from Tunable Femtosecond Stimulated Raman Spectroscopy.

Tracking molecular motions in real time remains a formidable challenge in science and engineering fields because the experimental methodology requires simultaneously high spatial and temporal resolutions. Building on early successes and future potential of femtosecond stimulated Raman spectroscopy (FSRS) as a structural dynamics technique, we present a comprehensive study of stimulated Raman line shapes of a photosensitive molecule in solution with tunable Raman pump and probe pulses. Following femtosecond 400 nm electronic excitation, the model photoacid pyranine exhibits dynamic and mode-dependent Raman line shapes when the Raman pump is tuned from the red side toward and across the excited-state absorption (ESA) band (e.

Tracking Ultrafast Vibrational Cooling during Excited-State Proton Transfer Reaction with Anti-Stokes and Stokes Femtosecond Stimulated Raman Spectroscopy.

Energy dissipation following photoexcitation is foundational to photophysics and chemistry. Consequently, understanding such processes on molecular time scales holds paramount importance. Femtosecond stimulated Raman spectroscopy (FSRS) has been used to study the molecular structure-function relationships but usually on the Stokes side.

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.

Panoramic portrait of primary molecular events preceding excited state proton transfer in water.

Photochemistry powers numerous processes from luminescence and human vision, to light harvesting. However, the elucidation of multidimensional photochemical reaction coordinates on molecular timescales remains challenging. We developed wavelength-tunable femtosecond stimulated Raman spectroscopy to simultaneously achieve pre-resonance enhancement for transient reactant and product species of the widely used photoacid pyranine undergoing excited-state proton transfer (ESPT) reaction in solution.

Unraveling ultrafast photoinduced proton transfer dynamics in a fluorescent protein biosensor for Ca(2+) imaging.

Imaging Ca(2+) dynamics in living systems holds great potential to advance neuroscience and cellular biology. G-GECO1.1 is an intensiometric fluorescent protein Ca(2+) biosensor with a Thr-Tyr-Gly chromophore.

Excited state structural events of a dual-emission fluorescent protein biosensor for Ca²⁺ imaging studied by femtosecond stimulated Raman spectroscopy.

Fluorescent proteins (FPs) are luminescent biomolecules that emit characteristic hues upon irradiation. A group of calmodulin (CaM)-green FP (GFP) chimeras have been previously engineered to enable the optical detection of calcium ions (Ca(2+)). We investigate one of these genetically encoded Ca(2+) biosensors for optical imaging (GECOs), GEM-GECO1, which fluoresces green without Ca(2+) but blue with Ca(2+), using femtosecond stimulated Raman spectroscopy (FSRS).

Excited-state structural dynamics of a dual-emission calmodulin-green fluorescent protein sensor for calcium ion imaging.

Fluorescent proteins (FPs) have played a pivotal role in bioimaging and advancing biomedicine. The versatile fluorescence from engineered, genetically encodable FP variants greatly enhances cellular imaging capabilities, which are dictated by excited-state structural dynamics of the embedded chromophore inside the protein pocket. Visualization of the molecular choreography of the photoexcited chromophore requires a spectroscopic technique capable of resolving atomic motions on the intrinsic timescale of femtosecond to picosecond.