A significant focus in the reporting period was the further development of fluorescence-detected sedimentation velocity (FDS-SV). Previously we have created data analysis and experimental techniques that can take into account the specific data structure of FDS-SV and thereby provide a highly quantitative analysis of the sedimentation process of fluorescent macromolecules, and sufficient sensitivity for the detection of EGFP at low pM concentrations. We have now studied in more detail the temporal aspect of the fluorescent signal in FDS-SV, in view of photophysical effects induced by the detector, as a function of laser power. For standard fluorophores, such as GFP and fluorescein-related compounds, we found no evidence of photobleaching. This finding justifies the use of the higher laser power and concomitantly increased signal-to-noise ratio. For photoswitchable molecules, however, significant photobleaching or photoactivation could be observed. This could be modelled well by the introduction of a new time-domain for overall signal intensity changes. This allows the seamless study of proteins fused to photoswitchable fluorescent protein tags, as used commonly in super-resolution microscopy. With the goal to develop multi-wavelength fluorescence detection capabilities, we have embarked on the modification of one our our analytical ultracentrifuges that is equipped with the commercial single-wavelength fluorescence detector. We have designed and implemented fiber optical and electrical feedthroughs that will allow supplying different wavelengths of light and control detector components. We have also continued the development of calibration procedures in analytical ultracentrifugation after recognizing their crucial importance when using current instrumentation, in order to avoid up to 10 % or higher systematic errors discovered in individual instruments. In order to make the external calibration of the radial dimension easier for other laboratories without mechanical fabrication capabilities, we have engaged in a collaboration with the National Institute of Stndards and Technology (Dr. Fagan and Dr. LeBrun) to create a sapphire cell assembly window with a lithographically deposited patterned mask. The dimensions of the mask will be validated by NIST and the windows be made available for public distribution. We have also developed analysis software for extracting calibration parameters from the scan of such windows. For better temperature calibration, we have developed a modified rotor handle that allows iButton integrated circuits with temperature logging capability to be run at high speed and to record in real-time the rotor temperature during the sedimentation experiments. In parallel, we have also developed an alterante procedure that eliminates the need for custom fabrication of a holder for the temperature probe, based on the measurement of the temperature of the resting rotor. Although not providing real-time data, we found the latter approach to be more practical for many laboratories. Finally, we have concluded the data acquisition phase of our multi-laboratory benchmark study of analytical ultracentrifuge calibration, with identical samples measured in >100 instruments by 80 laboratories worldwide. Although the rigorous statistical data analysis is still on-going, from an initial inspection, the results seem to confirm our previous observation based on a small number of intstruments: the simple external calibration procedures developed by us can improve the accuracy of sedimentation coefficients on average by fivefold, and reduce the range of values obtained by a factor of ten. We believe this will be critically important both for the use of sedimentation velocity for aggregate detection in the pharmaceutical industry, as well as for the interpretation of sedimentation coefficients in the study of protein conformation through hydrodynamic modelling.
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