Gas-Phase Unfolding Reveals Stability Shifts Associated with Substrate Binding in Modular Polyketide Synthases

Area of Science:

• Biochemistry and Structural Biology
• Mass Spectrometry and Biophysical Techniques

Background:

• Native mass spectrometry (MS), ion mobility (IM), and collision-induced unfolding (CIU) are established methods for studying protein-small molecule interactions.
• Challenges persist in applying these techniques to large, multidomain protein complexes, such as polyketide synthases (PKSs), which are crucial for natural product biosynthesis and pharmaceuticals.
• Existing methods struggle to detect subtle binding events in massive protein assemblies due to their complexity and size.

Purpose of the Study:

• To develop advanced CIU methods for analyzing large, multiprotein complexes.
• To assess the quantitative capabilities of CIU for detecting minor binding events in massive protein assemblies.
• To investigate the stability of approximately 280 kDa polyketide synthase (PKS) dimer complexes.

Main Methods:

• Development and application of novel collision-induced unfolding (CIU) methodologies.
• Probing the gas-phase stability of large, multidomain protein complexes, specifically PKS dimers.
• Quantitative analysis of stability shifts associated with substrate binding in intact protein assemblies.

Main Results:

• Successfully adapted CIU methods to extract meaningful data from large multiprotein complexes.
• Demonstrated the ability of quantitative CIU to detect subtle stability changes upon substrate binding.
• Observed stability shifts corresponding to substrate binding, which constituted less than 0.1% of the total mass of the PKS dimer complex.

Conclusions:

• The developed CIU methods significantly advance the study of large protein complexes.
• Quantitative CIU is capable of detecting low-abundance binding events in massive protein assemblies.
• This work expands the application of MS-based techniques to complex biological systems like PKSs.