Dataset Information

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.

SUBMITTER: Oscar BG

PROVIDER: S-EPMC4104898 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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

Oscar Breland G BG Liu Weimin W Zhao Yongxin Y Tang Longteng L Wang Yanli Y Campbell Robert E RE Fang Chong C

Proceedings of the National Academy of Sciences of the United States of America 20140701 28

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 time ...[more]

PMID: 24987121

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Dataset Information

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

Publications

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

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