Folding of a large protein at high structural resolution

Area of Science:

• Protein folding kinetics
• Biophysics
• Structural biology

Background:

• Maltose binding protein (MBP) is a large, two-domain protein crucial for glucose transport.
• Understanding the folding pathways of large proteins is essential for protein engineering and drug development.

Purpose of the Study:

• To elucidate the kinetic folding pathway of maltose binding protein (MBP) at high structural resolution.
• To identify intermediate states and their role in the folding process of large proteins.

Main Methods:

• Utilized advanced hydrogen-exchange pulse-labeling mass spectrometry (HX MS) for high-resolution kinetic studies.
• Employed small-angle X-ray scattering (SAXS) to analyze protein conformation during folding.

Main Results:

• MBP undergoes rapid molecular collapse (<20 ms) into a heterogeneous polyglobular state.
• A distinct helix-plus-sheet folding intermediate forms with a 7-s time constant, persisting throughout folding.
• This intermediate contains sites of destabilizing mutations, indicating it's an obligatory step in MBP folding.

Conclusions:

• Large proteins like MBP can fold through distinct, native-like intermediates.
• The identified intermediate is a critical, obligatory step in the MBP folding pathway.
• This study adds MBP to the growing list of large proteins with complex, multi-step folding pathways involving intermediates.