Globular or end-to-end? A two-dimensional Spectrum Analysis for Assessing Modes of Aggregation from
Sedimentation Velocity Experiments
Challenge
Researchers are trying to understand
subunit polymerization details, especially molecular weight and shape distributions in mixed
solutions. A significant challenge is analyzing these when multiple components are present.
The 2-dimensional spectrum analysis, introduced here, examines sedimentation velocity
experiments to decipher aggregation modes. It can distinguish between fibril-type and
globular aggregations. To process efficiently, the method leverages supercomputer
parallelization.
Solution
We use the frictional ratio, k=f/f0, to parameterize the
diffusion coefficient.
A sedimentation coefficient formula is applied.
The solute mixture is described using a model.
The NNLS algorithm compares the model to experimental
data, b, identifying solute coefficients.
s and k=f/f0 values are estimated based on known protein
and DNA molecule ranges.
A 2D mesh for each s, f/f0 pair is developed and
refined.
Further refinement methods include maximum
entropy regularization and genetic algorithm optimization, with the 2D analysis as a
starting point.
Conclusion
The 2-dimensional spectrum analysis reliably describes
systems with varied sedimentation coefficients and shapes, outperforming other methods in
variance.
The method accurately represented a heterogeneous
plasmid digest and two systems, "Mo27Fe30, Keplerate" and "Bindzil 30/360", distinct in
molecular weight but consistent in shape.
Using this analysis, it's possible to
determine both sedimentation and shape properties, helping to identify the aggregation mode
of systems (e.g., fibril or globular).
