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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | Fang, Chong Wang, Yanli Campbell, Robert E. Oscar, Breland G. Tang, Longteng Zhao, Yongxin Liu, Weimin |
| Description | Author Affiliation: Oscar BG ( Department of Chemistry, Oregon State University, Corvallis, OR 97331); Liu W ( Department of Chemistry, Oregon State University, Corvallis, OR 97331); Zhao Y ( Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada.); Tang L ( Department of Chemistry, Oregon State University, Corvallis, OR 97331); Wang Y ( Department of Chemistry, Oregon State University, Corvallis, OR 97331); Campbell RE ( Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada.); Fang C ( Department of Chemistry, Oregon State University, Corvallis, OR 97331); |
| 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. |
| ISSN | 00278424 |
| e-ISSN | 10916490 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Issue Number | 28 |
| Volume Number | 111 |
| Language | English |
| Publisher | National Academy of Sciences |
| Publisher Date | 2014-07-01 |
| Publisher Place | United States |
| Access Restriction | Open |
| Subject Keyword | Biosensing Techniques Calcium Calmodulin Chemistry Green Fluorescent Proteins Recombinant Fusion Proteins Animals Genetics Cations, Divalent Spectrometry, Fluorescence Spectrum Analysis, Raman Research Support, Non-U.S. Gov't Multidisciplinary |
| Content Type | Text |
| Resource Type | Article |
| Subject | Multidisciplinary |
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