Mapping Protein Conformational Landscapes under Strongly Native Conditions with Hydrogen Exchange Mass Spectrometry

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Protein thermodynamic stability and kinetic barriers are crucial for function and disease.
• Traditional methods use denaturants, limiting analysis to non-native conditions and small proteins.
• Hydrogen exchange (HX) is ideal for native-state analysis, but NMR detection is limited.

Purpose of the Study:

• To develop an improved Hydrogen Exchange Mass Spectrometry (HX-MS) approach for analyzing protein conformational dynamics.
• To address experimental and theoretical challenges in quantitative HX-MS analysis.
• To enable the study of larger proteins and lower concentrations under native conditions.

Main Methods:

• Proposed a novel HX-MS approach using standards to correct for experimental artifacts.
• Developed a theoretical framework based on the Linderstrøm-Lang formalism for HX behavior.
• Applied the method to native-state HX of a globular protein.

Main Results:

• Successfully eliminated systematic experimental artifacts in HX-MS profiles using standards.
• Established a comprehensive theoretical framework for describing HX behavior across all regimes.
• Demonstrated proof of principle for native-state HX analysis of a globular protein.

Conclusions:

• The developed HX-MS framework and tools advance the extraction of thermodynamic and kinetic conformational parameters.
• This method significantly enhances the capability to study protein dynamics under native-like conditions.
• Facilitates a deeper understanding of protein function and conformational diseases.