Mapping transient partial unfolding by protein engineering and native-state proteolysis.

Area of Science:

• Structural Biology
• Protein Biochemistry
• Biophysics

Background:

• Transient partial unfolding of proteins under native conditions impacts their properties.
• Native-state proteolysis can probe accessible unfolded protein forms, but determining their structure is challenging.
• Many proteins do not accumulate detectable intermediates during proteolysis, hindering structural analysis.

Purpose of the Study:

• To develop a systematic approach for determining the structures of transiently cleavable protein forms.
• To elucidate the structure of the cleavable form of Escherichia coli maltose-binding protein (MBP) using a novel method.

Main Methods:

• Protein engineering by mutating buried residues to alanine.
• Devising and applying phi(c) analysis, analogous to conventional phi analysis.
• Assessing the effects of mutations on protein global stability and proteolytic susceptibility.

Main Results:

• The phi(c) analysis successfully determined the structure of the cleavable form of MBP, even without accumulated intermediates.
• The results indicate that two C-terminal helices of MBP are unfolded in its cleavable form.
• Ligand binding and C-terminal deletion mutations corroborated the proposed structure of the cleavable form.

Conclusions:

• The developed phi(c) analysis provides a robust method for structural determination of transiently unfolded protein forms.
• This methodology is broadly applicable to understanding proteolysis mechanisms and other processes involving protein partial unfolding, such as misfolding and aggregation.