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Ms. Ai Shinobu, The Hebrew University of Jerusalem
GFP as a model system for mapping proton wires in proteins
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 âproton-wireâ. In this work, we investigate proton shuttling in the Green Fluorescent Protein (GFP), a bioluminescent protein which revolutionalized cell biology as a fluorescent marker by allowing the visualization of cellular processes in-vivo.
We developed a computer algorithm for mapping proton wires in X-ray structures of proteins1 and applied it to a GFP mutant possessing the highest resolution of all GFP structures reported to date (0.85Ã ).2 In this structure, the previously reported active site wire is found to spread and continue on the external protein surface, sending arms of polar amino acid residues into solution. This structure is suggested to act as a proton collecting apparatus that captures protons from solution and delivers them directly to the chromophore which is located in the middle of the GFP-barrel, protected from the external solution.
The finding of the extensive hydrogen bond network for this high resolution mutant is not accidental, a systematic investigation of over 100 mutants shows a clear correlation between the water content of the X-ray structures and their resolution. With increasing water content, the proton wire detected by our program becomes progressively larger. These finding support the scenario in which a photodissociated proton from wild-type GFP can leak outside, and then another proton is recruited via the active site wire. In addition to GFP, we also examine the GFP-like protein DsRed and compare the active site wires and the dependence of cluster size and resolution according to the model built by GFP.
References.
[1] Shinobu, A.; Agmon, N. J. Phys. Chem. A 2009, 113, 7253.
[2] Shinobu, A.; Palm, G. J.; Schierbeek, A. J.; Agmon, N. J. Am. Chem. Soc. 2010, 132, 11093. Cover page of issue 32.
For more information contact Dr. Kyril Solntsev (404-385-3117).
