| 概 要 |
Proton shuttling plays a key role in many biological processes.
Inside proteins the transport of a proton takes place in a step-wise manner in which the proton hops through a chain of hydrogen-bonded carrier atoms, a structure referred to as a ‘proton-wire’.
In this work, we investigate proton shuttling in the Green Fluorescent Protein (GFP), a bioluminescent protein which is said to revolutionalize cell biology as a fluorescent marker by allowing the visualization of cellular processes in-vivo.
In a previous work we developed a computer algorithm for mapping proton wires in X-ray structures of proteins. Using this investigation method, we located wires that we suggest to play crucial roles in the proton transfer mechanism of GFP, including an extensive three dimensional network which connects the isolated chromophore with the external solution.
In the current work we propagate the system in time using molecular dynamics (MD) simulations, exploring the behavior of the GFP proton wires under conditions which are more similar to the ones in nature.
We begin our work by focusing on the internal proton wire which was found to be stable under X-ray conditions, and perform analyses to follow the existence and properties of the wire over time.
The wire, which is 13 atoms long (as found in the X-ray), was divided into several sub-paths with the nonwater atoms serving as endpoints. Then, for each sub-path various properties are measured. The fraction of time in which a path is open indicates the ease of proton transfer through it, this, combined with an energy calculation can identify which of the transfer steps is rate determining in the whole pathway. From the length and water fraction of each sub-path, compared to their initial values we can learn about dynamic processes that occur in the protein, such as water molecules entering into the protein barrel and opening alternative pathways for the proton.
The insertion of point mutations is a powerful tool to study the role of specific residues in proteins. Here, we insert point mutations to the Ser72 residue which was suspected as a crucial residue in the proton passage in GFP.
Another analysis that we perform is following the rotation of Serine residues along the path. It had been suggested that Serine residues play an important role in opening and closing paths inside proteins due to the low barrier for their sidechain rotation. Here, we show that the three rotameric states of two Serine residues along the pathway greatly influence the proton transport along the path.
references:
1. Mapping proton-wires in proteins: Carbonic anhydrase and GFP chromophore biosynthesis, Ai Shinobu and Noam Agmon, Journal of Physical Chemistry A,
113 :7253-7266, 2009.
2. Visualizing Proton Antenna in a High Resolution Green Fluorescent Protein Structure, Ai Shinobu, Gottfried J. Palm, Abraham J. Schierbeek, and Noam Agmon, Journal of the American Chemical Society, 132 :11093-11102, 2010.
Cover of issue 32, 18 Aug., 2010, see: http://pubs.acs.org/toc/jacsat/132/32
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