A team of scientists at Arizona State University (ASU; Tempe, AZ) has shown, for the first time, that a hinge migration mechanism, driven solely by long-range dynamic motions, can be the key for evolution of a green-to-red photoconvertible phenotype in a green fluorescent protein (GFP). This protein, which glows bright green under ultraviolet (UV) light, has found many creative applications in the biosciences.
Rebekka Wachter, a professor in ASU's Department of Chemistry and Biochemistry and the College of Liberal Arts and Sciences and an expert in the field of structural characterization of GFP-like proteins, led the work, which involves collaborations with S. Banu Ozkan from the Center for Biological Physics in the Department of Physics at ASU, and evolutionary biologist Mikhail Matz of the University of Texas at Austin.
GFP has been utilized as an extremely valuable luminous genetic tag for various biological phenomena. Using it, one can observe when proteins are made and where they go. This is done by joining the GFP gene to the gene of the protein of interest so that when the protein is made, it will have GFP hanging off it. Since GFP fluoresces, one can shine light at the cell and wait for the distinctive green fluorescence associated with GFP to appear. The ability of some GFPs to turn red upon prolonged illumination makes them invaluable fluorescent probes in super-resolution microscopy applications.
To fluoresce, GFP-like proteins must adopt a compact, barrel-like shape. The light-triggered red phenotype may have arisen from a common green ancestor by a reversal of firm and soft regions located in opposite corners of the beta-barrel fold.
Although six crystal structures of reconstructed ancestral Kaede-type proteins indicate that the structure is highly conserved, analysis of chain flexibility by Molecular Dynamics and perturbation response scanning, performed in the group of Ozkan, has shown that the individual flexibility of each position (i.e., structural dynamics) alters throughout the evolution of green-to-red photo conversion. So, this study suggests that green-to-red photoconversion may have arisen from a common green ancestor by the shift of the rigid corner near the chromophore to the opposite corner of beta-barrel.
"For the first time, this work establishes a direct experimental link between protein phenotypic change and collective dynamics without any external trigger, such as substrate, product, or effector binding," explains Wachter. "Based on structural, computational, and kinetic data, we propose a novel photoconversion mechanism that provides a plausible path for the irreversible acquisition of red fluorescence."
In spite of intense efforts in a number of laboratories worldwide, the mechanism of photoconversion of Kaede-type proteins has remained largely enigmatic. The present work sheds light on structural, dynamic, and mechanistic features that must be considered when engineering improved fluorescent probes for super-resolution microscopy applications.
Full details of the work appear in the journal Structure; for more information, please visit http://dx.doi.org/10.1016/j.str.2014.11.011.
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