Subatomic resolution X-ray structures of green fluorescent protein

Area of Science:

• Biophysics
• Structural Biology
• Biochemistry

Background:

• Green fluorescent protein (GFP) is a vital molecular tool in biology.
• Previous studies lacked subatomic detail due to structural polymorphism around the chromophore.

Purpose of the Study:

• To elucidate the subatomic structure of GFP.
• To resolve the structural polymorphism around the chromophore.
• To determine the authentic resonance state of GFP.

Main Methods:

• Subatomic resolution X-ray crystallography.
• Charge-density analysis using atoms-in-molecules theory.
• Noncovalent interaction analysis.

Main Results:

• Determined positions of H atoms and hydrogen-bonding networks for two protonated forms.
• Identified the anionic chromophores representing the authentic resonance state of GFP.
• Highlighted weak but substantial interactions, including lone pair-π interactions between the chromophore and Thr62, crucial for the chromophore's electronic state.

Conclusions:

• The study provides critical fine structural features for understanding GFP properties.
• Revealed the limitations of current quantum-chemical calculations for GFP structure.
• Established a foundation for future research on GFP and similar fluorescent proteins.