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Cytochrome c folding pathway: kinetic native-state hydrogen exchange
Linh Hoang1, Sabrina Bedard, Mallela M G Krishna
1The Johnson Research Foundation, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6059, USA. lhoang@mail.med.upenn.edu
Summary
Native-state hydrogen exchange experiments reveal a previously poorly characterized cytochrome c intermediate early in its unfolding pathway. This study identifies distinct unfolding steps and their kinetic order, advancing our understanding of protein dynamics.
Area of Science:
- Protein folding and dynamics
- Biophysical chemistry
- Structural biology
Background:
- Understanding protein unfolding pathways is crucial for comprehending protein function and misfolding diseases.
- Native-state hydrogen exchange (NSHX) is a powerful technique for probing protein structure and dynamics.
Purpose of the Study:
- To characterize transient, partially unfolded intermediates in cytochrome c unfolding.
- To determine the kinetic order of unfolding steps using NSHX under EX1 conditions.
Main Methods:
- Utilizing native-state hydrogen exchange (NSHX) mass spectrometry.
- Applying EX1 kinetic conditions to monitor amide hydrogen exchange rates.
- Analyzing hydrogen exchange patterns to identify exposed and protected amide hydrogens.
Main Results:
- A distinct cytochrome c intermediate, previously poorly characterized, was identified early in the major unfolding pathway.
- Formation rates of partially unfolded intermediates were determined.
- Four distinct unfolding steps were found to be kinetically ordered, consistent with previous inferences.
Conclusions:
- NSHX under EX1 conditions effectively distinguishes partially unfolded protein intermediates.
- The identified intermediate provides new insights into the initial stages of cytochrome c unfolding.
- The study confirms and refines the kinetic model of cytochrome c unfolding.