Studies of interacting protein systems using multi-wavelength AUC
Challenge
Studying multiple components simultaneously is
essential, necessitating methods that can identify and characterize them concurrently.
Traditional single-wavelength sedimentation
velocity experiments might not offer the depth required for comprehensive analysis,
especially when components share similar hydrodynamic properties.
Solution
Introduction of MW-AUC (Multi-wavelength AUC):
This technique delves deeper than single
wavelength
experiments by utilizing both
hydrodynamic and optical properties.
It capitalizes on the excitation spectra of
fluorescent proteins, such as Ultramarine
(blue), mTFP1 (green), and mPapaya (red). Although all fluorescent proteins have a
common peak at 280 nm due to tryptophan and tyrosine absorbance, their excitation
spectra in the visible region are significantly different.
This differentiation enables MW-AUC to
distinguish
between spectra in a multi-wavelength
AUC experiment, even when the analytes have similar hydrodynamic properties.
Optical Deconvolution with UltraScan:
The process involves comparing the spectral
angle θ
between two distinct spectra (u and
v).
Effective deconvolution is possible when the
angle θ
is greater than zero, with the
larger the angle indicating more definitive separation.
This separation is further enhanced by
selecting
wavelength regions with minimal overlap
Conclusion
Mixtures of three fluorescent proteins (Ultramarine,
mTFP1, and mPapaya) were sedimented and analyzed. Relative ratios of these mixed proteins
could be resolved even within the margin of pipetting errors.
Complete resolution of proteins was achieved in all
instances.
Unlike single-wavelength methods, MW-AUC was able to
differentiate mPapaya and mTFP1 due to their distinct spectral properties, even though they
had identical sedimentation coefficients.
This led to accurate characterization and determination
of molar ratios for multiple analytes, even in the presence of pipetting errors.
MW-AUC also accurately determined
interactions and molar ratios of associating proteins when tagged to different fluorescent
proteins, all validated by molar extinction coefficients.
