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We developed new collision-induced unfolding (CIU) methods to study large protein complexes like polyketide synthases (PKSs). This technique reveals subtle stability changes from small molecule binding in massive enzyme assemblies.

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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.