Driving integrative structural modeling with serial capture affinity purification

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Systems Biology

Background:

• Characterizing protein complexes is crucial for understanding cellular functions and networks.
• Existing methods for protein complex analysis can be time-consuming and lack multi-faceted validation.
• Developing streamlined techniques for protein interaction network studies is essential.

Purpose of the Study:

• To introduce a novel method, serial capture affinity purification (SCAP), for streamlined protein complex characterization.
• To demonstrate the utility of SCAP in validating protein interactions in vivo and generating integrative structural models.
• To provide a comprehensive approach combining multiple techniques for detailed protein complex analysis.

Main Methods:

• Serial Capture Affinity Purification (SCAP) using HaloTag and SNAP-tag for multistep enrichment.
• In vivo interaction validation via acceptor photobleaching Förster resonance energy transfer (FRET) and fluorescence cross-correlation spectroscopy (FCCS).
• Coupling SCAP with cross-linking mass spectrometry (XL-MS) for structural modeling.

Main Results:

• SCAP successfully enabled multistep affinity enrichment of specific protein complexes.
• In vivo imaging techniques confirmed protein interactions within live cells.
• Integration of SCAP with XL-MS yielded an integrative structural model of the Spindlin1-SPINDOC complex.
• The structural model revealed SPINDOC binding to the histone H3 interaction interface of SPIN1.

Conclusions:

• SCAP offers a streamlined and versatile approach for characterizing protein complexes.
• The combined methodology allows for in vivo validation and the generation of high-resolution structural models.
• This integrated strategy advances the study of protein interaction networks and complex structures.