Stoichiometry of intrinsically disordered protein complexes

Importance of the Topic

Disordered protein interactions play crucial roles in cellular regulation and cancer pathways. S100 family proteins bind the C-terminal region of p53, influencing its oligomerization and tumor suppressor functions. Detailed stoichiometric insights into these complexes are essential for therapeutic targeting and mechanistic understanding.

Objectives and Study Overview

This work employs size-exclusion chromatography with multi-angle light scattering (SEC-MALS) to quantify binding stoichiometry between S100B and p53 variants. Monomeric (L344P), dimeric (L344A) and tetrameric wild-type p53 forms were mixed at various ratios to determine complex assembly under native conditions.

Methodology and Instrumentation

SEC-MALS combines an HPLC-based SEC column with light scattering and differential refractive index detection to derive absolute molar masses independent of calibration standards. Samples of p53 variants and S100B were first analyzed separately, then incubated in 4:1, 2:1, 1:1, 1:2 and 1:4 stoichiometries. Elution profiles for scattering and dRI signals were processed in ASTRA software to calculate weight-average molar masses across each peak.

Instrumentation Used

• HPLC system equipped with a size-exclusion column
• DAWN multi-angle light scattering detector
• Optilab differential refractive index detector
• ASTRA software for data acquisition and analysis

Results and Discussion

Individual analyses confirmed monomeric L344P (~11.2 kDa), dimeric L344A and tetrameric wild-type p53. Mild curvature in the molar mass profiles indicated concentration-dependent equilibria. Mixing studies showed that one S100B dimer binds a single p53 monomer (~32 kDa complex), regardless of input ratio. No complexes featuring two p53 monomers per S100B dimer were detected. Furthermore, S100B disrupted the p53 dimer and formed stable assemblies with tetrameric p53, comprising four S100B dimers bound to one p53 tetramer.

Benefits and Practical Applications

SEC-MALS delivers accurate stoichiometric and oligomeric characterization of protein complexes, including intrinsically disordered proteins. It is invaluable in structural biology, drug discovery and biopharmaceutical quality control for elucidating binding mechanisms under native conditions.

Future Trends and Applications

Emerging directions include coupling SEC-MALS with complementary techniques (native mass spectrometry, SAXS), high-throughput interaction screening, in vivo validation of binding models and expansion to diverse disordered protein complexes relevant to disease.

Conclusion

The application of SEC-MALS revealed distinct binding modes between S100B and p53 variants, shedding light on regulatory shifts in p53 oligomerization. This methodology stands out as a robust platform for probing protein–protein stoichiometry and dynamics.