Structural analysis of gelsolin using synchrotron protein footprinting

Area of Science:

• Biochemistry
• Structural Biology
• Proteomics

Background:

• Protein structure and dynamics are crucial for biological function.
• Understanding conformational changes upon ligand binding is key to elucidating protein mechanisms.
• Existing methods may not fully capture dynamic structural information in solution.

Purpose of the Study:

• To investigate the structural and dynamic changes in gelsolin upon calcium (Ca2+) activation using protein footprinting.
• To identify specific residues and regions affected by Ca2+ binding.
• To correlate structural findings with existing crystallographic data.

Main Methods:

• Radiolytic protein footprinting utilizing synchrotron "white" beam irradiation.
• Hydroxyl radical generation to probe protein surface accessibility.
• Protease digestion followed by liquid chromatography-coupled mass spectrometry (LC-MS) and tandem MS (MS/MS) for peptide analysis and quantitation.
• Monitoring oxidation rates of specific side chain residues.

Main Results:

• Analysis of 81 peptides from trypsin-digested gelsolin revealed oxidation rates for 21 peptides.
• Ca2+ activation induced changes in oxidation rates for 7 peptides: 5 increased oxidation, and 2 showed protection.
• Tandem MS identified specific side chain probes responsible for Ca2+-dependent and -insensitive oxidation.
• Findings were consistent with crystallographic data for inactive gelsolin.

Conclusions:

• Radiolytic protein footprinting is a powerful tool for detailed structural analysis of proteins in solution.
• The study provides a detailed model for gelsolin activation, highlighting Ca2+-dependent conformational dynamics.
• This technique offers insights into ligand-induced structural rearrangements and protein dynamics.