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

Abstract

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. We use femtosecond stimulated Raman spectroscopy to study the excited-state conformational dynamics of a recently developed FP-calmodulin biosensor, GEM-GECO1, for calcium ion (Ca 2+ ) sensing. This study reveals that, in the absence of Ca 2+ , the dominant skeletal motion is a ∼170 cm −1 phenol-ring in-plane rocking that facilitates excited-state proton transfer (ESPT) with a time constant of ∼30 ps (6 times slower than wild-type GFP) to reach the green fluorescent state. The functional relevance of the motion is corroborated by molecular dynamics simulations. Upon Ca 2+ binding, this in-plane rocking motion diminishes, and blue emission from a trapped photoexcited neutral chromophore dominates because ESPT is inhibited. Fluorescence properties of site-specific protein mutants lend further support to functional roles of key residues including proline 377 in modulating the H-bonding network and fluorescence outcome. These crucial structural dynamics insights will aid rational design in bioengineering to generate versatile, robust, and more sensitive optical sensors to detect Ca 2+ in physiologically relevant environments.calcium-sensing fluorescent protein | femtosecond Raman spectroscopy | fluorescence modulation mechanism | molecular movie G reen fluorescent protein (GFP) first emerged as a revolutionary tool for bioimaging and molecular and cellular biology about 20 years ago (1-3), and the quest to discover and engineer biosensors with improved and expanded functionality has yielded exciting advances. Recently, the color palette of genetically encoded Ca 2+ sensors for optical imaging (the GECO series) has been expanded to include blue, improved green, red intensiometric, and emission ratiometric sensors (4-7). The GECO proteins belong to the GCaMP family of Ca 2+ sensors that are chimeras of a circularly permutated (cp)GFP, calmodulin (CaM), and a peptide derived from myosin light chain kinase (M13) (8). The CaM unit undergoes large-scale structural changes upon Ca 2+ binding as it wraps around M13. These changes, especially at the interfacial region where CaM interacts with cpGFP, allosterically alter the local environment of the tyrosine-derived chromophore and lead to dramatic fluorescence change in the presence of Ca 2+ (9, 10). Because GCaMP and GECO proteins are genetically encodable, show sensitivity to physiologically relevant Ca 2+ concentrations, and respond to Ca 2+ concentration changes rapidly, they have gained increasing popularity for in vivo imaging of Ca 2+ in neur...

Femtosecond stimulated Raman spectroscopy by six-wave mixing

“…One of these polarization components is given in Eq. (10). The remaining 15 polarization components are…”

“…[1][2][3][4][5][6][7][8][9][10][11][12] Recent applications have revealed new insights into systems ranging from proteins 9,10,12 to organic photovoltaic materials. 4,11 The FSRS technique is essentially a sequence of two events: (i) an electronically resonant (actinic) pump pulse initiates a photochemical process; (ii) a stimulated Raman spectrum is obtained at various delay times using a combination of narrowband and broadband laser pulses.…”

Unraveling Ultrafast Photoinduced Proton Transfer Dynamics in a Fluorescent Protein Biosensor for Ca2+ Imaging

“…To enabler ational designa nd optimization of the fluorescent protein (FP)-basedb iosensors from the bottom up, ad etailedu nderstanding of fluorescence mechanism is warranted. [5] In our recent report, the green-blue dual-emission CaM-GFP sensor GEM-GECO1 was shown to exhibit different initial structural dynamics from the wild-type (wt)GFPd espite having the same serine-tyrosine-glycine (SYG) chromophore. This level of information remains lacking for the threonine-tyrosine-glycine (TYG) chromophore that is ac ommon motif in some of the most widely used FP variants (e.g.,E GFP).…”

Sum-Frequency-Generation-Based Laser Sidebands for Tunable Femtosecond Raman Spectroscopy in the Ultraviolet

“…The FSRS technology has been successfully applied to a number of important photosensitive molecular systems including rhodopsin [27], bacteriorhodopsin [28], phytochrome [29], organic dyes in solar cells [30], Fe(II) spin crossover in solution [31], fluorescent proteins [7,32,33], and calcium-ion-sensing protein biosensors [34][35][36]. The main goal of this work is to construct a versatile, tunable FSRS setup that extends the wavelength detection window to the UV regime with desired resonance Raman enhancement.…”