Using singular value decomposition to characterize protein-protein interactions by in-cell NMR spectroscopy

Area of Science:

• Biochemistry
• Structural Biology
• Cell Biology

Background:

• Cellular environment significantly impacts protein-protein and protein-ligand interactions compared to in vitro conditions.
• In-cell Nuclear Magnetic Resonance (NMR) analyses are challenging due to low signal-to-noise and spectral changes from protein overexpression over time.

Purpose of the Study:

• To develop a method for unambiguously resolving protein binding modes within the complex cellular environment.
• To differentiate concentration-dependent and independent binding events in vivo.

Main Methods:

• Utilized single-value decomposition (SVD) to analyze time-course in-cell NMR spectral data.
• Applied SVD to spectral data from a target protein during the overexpression of its interacting partner.

Main Results:

• SVD successfully differentiated concentration-dependent and concentration-independent events in the cellular milieu.
• Identified the principal binding mode and implicated specific amino acids involved in the interaction.
• The identified amino acids for interaction showed good agreement with crystallographic data, despite differing from previous NMR analyses.

Conclusions:

• Single-value decomposition is an effective tool for analyzing complex in-cell NMR data to determine protein interaction mechanisms.
• This method provides insights into protein interactions within the cellular context, complementing traditional in vitro and NMR studies.