Visualizing proton antenna in a high-resolution green fluorescent protein structure.

Area of Science:

• Structural Biology
• Biophysics
• Protein Science

Background:

• Proton-collecting antennas in proteins, proposed to facilitate proton transfer via hydrogen-bond (HB) networks, have lacked direct visualization.
• Understanding proton dynamics is crucial for enzyme mechanisms and cellular processes.

Purpose of the Study:

• To visualize and characterize the proton-collecting apparatus and proton wire network in green fluorescent protein (GFP) at unprecedented resolution.
• To investigate the correlation between protein structure, water content, and proton wire formation.

Main Methods:

• High-resolution X-ray crystallography (0.9 Å) of green fluorescent protein (GFP).
• Computational analysis using an algorithm for mapping proton wires within protein structures.
• Systematic investigation of over 100 GFP mutants to assess water content and resolution.

Main Results:

• The study provides the highest resolution X-ray structure of GFP to date, enabling the identification of key hydrogen atoms and protonation states.
• A novel proton-collecting apparatus, comprising carboxylates, threonines, and water molecules linked by an HB network to Glu5, was identified on the GFP surface.
• An "active site wire" connecting Glu222 to Glu5 was confirmed, alongside newly identified proton exit points via Asn146 and His148.
• Increased water content in GFP structures correlated with higher resolution and larger proton wire networks.

Conclusions:

• The direct visualization of the proton-collecting apparatus in GFP supports the proposed mechanism for proton transfer and collection.
• Findings suggest that photodissociated protons from wild-type GFP can exit the protein and be replenished via the identified collecting apparatus.
• The study highlights the dynamic nature of protein hydration and its critical role in facilitating proton transfer pathways.