A photoreversible conformational change in 124 kDa Avena phytochrome

Area of Science:

• Biochemistry
• Photochemistry
• Molecular Biology

Background:

• Phytochrome is a photoreceptor crucial for plant development.
• Understanding the molecular changes between Pr and Pfr forms is key to its function.

Purpose of the Study:

• To investigate molecular differences between phytochrome's Pr and Pfr forms using tryptophan fluorescence quenching.
• To elucidate conformational changes during the Pr to Pfr phototransformation.

Main Methods:

• Tryptophan fluorescence quenching assays using anionic (I-), cationic (Cs+), and neutral (acrylamide) quenchers.
• Analysis of quenching data using Stern-Volmer and modified Stern-Volmer kinetics.
• Measurement of tryptophan fluorescence lifetimes in Pr and Pfr forms.

Main Results:

• Cationic quencher (Cs+) revealed a 2-fold difference in quenching constants (Ksv) between Pr and Pfr, indicating altered tryptophan environments.
• Most tryptophan residues were accessible to acrylamide, but quenching was similar for both forms initially.
• Combined quenching with Cs+ and acrylamide showed a >40% increase in Ksv for Pfr, suggesting distinct environments.
• Two fluorescence lifetime components (major ~1 ns, minor ~4.6 ns) were observed, differing slightly between Pr and Pfr.
• Fluorescence quenching exhibited both static and dynamic components, with steady-state Ksv higher than dynamic Ksv.

Conclusions:

• The Pr to Pfr phototransformation involves significant conformational changes in the phytochrome molecule.
• These changes are predominantly located within the 74 kDa chromophore-bearing domain.
• Tryptophan fluorescence quenching provides insights into the distinct molecular environments of Pr and Pfr states.