Quantifying Protection in Disordered Proteins Using Millisecond Hydrogen Exchange-Mass Spectrometry and Peptic...

Area of Science:

• Biochemistry
• Structural Biology
• Protein Dynamics

Background:

• Intrinsically disordered proteins (IDPs) exhibit transient structures crucial for their function.
• Millisecond amide hydrogen exchange (HX) coupled with mass spectrometry (HX-MS) can probe these structures.
• Accurately determining the chemical HX rate is essential for quantifying transient structuring in IDPs.

Purpose of the Study:

• To evaluate methods for accurately modeling the chemical HX rate for millisecond HX-MS analysis of IDPs.
• To compare the Englander method, denaturant-based HX measurements, and reference peptide HX measurements.
• To identify the most reliable approach for quantifying transient structure in disordered proteins.

Main Methods:

• Utilized model peptides and disordered protein domains (e.g., thyroid receptor, CREB binding protein).
• Assessed the Englander method for chemical HX rate calculations.
• Measured HX rates in high concentrations of denaturants (urea, guanidinium chloride).
• Employed HX-MS on reference peptides derived from the target IDPs.

Main Results:

• The Englander method slightly underestimated chemical HX rates.
• High denaturant concentrations significantly altered HX rates in model peptides.
• HX measurements using reference peptides provided the most accurate chemical HX rates.
• This method allows for precise quantification of transient structure in IDPs.

Conclusions:

• Accurate chemical HX rate determination is critical for millisecond HX-MS studies of IDPs.
• Reference peptide HX measurements offer a robust method for this determination.
• This approach enhances the understanding of IDP conformational ensembles and function.