Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 111, Issue 28, Pages 10191-10196Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1403712111
Keywords
calcium-sensing fluorescent protein; femtosecond Raman spectroscopy; fluorescence modulation mechanism; molecular movie
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Funding
- Oregon State University Faculty Startup Research Grant
- Oregon State University General Research Fund Award
- Natural Sciences and Engineering Research Council of Canada
- Canadian Institutes of Health Research
- University of Alberta fellowship
- Alberta Innovates scholarship
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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 (Ca2+) sensing. This study reveals that, in the absence of Ca2+, the dominant skeletal motion is a similar to 170 cm(-1) phenol-ring in-plane rocking that facilitates excited-state proton transfer (ESPT) with a time constant of similar to 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 Ca2+ 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 Ca2+ in physiologically relevant environments.
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