Trigger Factor-Induced Nascent Chain Dynamics Changes Suggest Two Different Chaperone-Nascent Chain Interactions...

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Protein biogenesis is complex and difficult to study due to ribosome interference with nascent chain measurements.
• Nascent chain dynamics offer insights into co-translational processes like protein folding and chaperone interactions.

Purpose of the Study:

• To develop a platform for studying nascent chain dynamics using site-specifically labeled ribosome-bound nascent chains (RNCs) and time-resolved fluorescence anisotropy.
• To quantitatively model fluorescence depolarization and its relation to nascent chain backbone motion.
• To investigate the co-translational mechanism of bacterial trigger factor (TF) binding to nascent chains.

Main Methods:

• Site-specific labeling of RNCs with fluorescent dyes.
• Time-resolved fluorescence anisotropy measurements.
• Utilizing intrinsically disordered proteins and zinc-finger-domain-containing RNCs for model development.
• Studying the synthesis of Entner-Doudoroff aldolase with and without TF.

Main Results:

• Established a quantitative model linking sub-nanosecond fluorescence depolarization to nascent chain backbone motion.
• Observed decreased sub-nanosecond motion in zinc-finger domains upon zinc addition.
• Demonstrated that TF addition elicits distinct responses in nascent chain dynamics depending on the translation stage.
• Proposed steric hindrance as a mechanism for TF dissociation from the ribosome.

Conclusions:

• Site-specific labeling and time-resolved anisotropy are effective for studying nascent chain dynamics.
• Gained insights into chaperone binding events during translation.
• Provided evidence for a co-translational mechanism involving TF and nascent chains.