One of the techniques with major promise for the study of multi-protein complexes is multi-signal sedimentation velocity, and we have continued the development of this approach. We have devised a novel mass conservation constraint to be used as a form of Bayesian regularization. We have mathematically shown that it can significantly aid in the spectral discrimination of components. In collaboration with Dr. Chad Brautigam, the performance of this approach is now being tested with simulated and experimental data sets. A fundamental quantity derived from sedimentation velocity is the hydrodynamic frictional coefficient of translation, which reports on the protein solution conformation. In particular, this coefficieint is very informative in conjunction with structural predictions. In a collaboration with Drs. Ghirlando, Brautigam, and Aragon, we have set out to compare results from the new high-precision boundary element methods for structure-based prediction of friction coeffcients with experimental data from sedimentation velocity. From initial collection of data, we have identified key experimental factors. All ultracentrifugation experiments are subject to macromolecular buoyancy as a dominating factor of sedimentation behaviour. To complement existing compositional and densimetry approaches for the determination of macromolecular partial-specific volume, we have developed a density contrast sedimentation velocity method. This was successfully tested on several model proteins. Finally, we have updated our SEDFIT software with new tools for sedimentation equilibrium and sedimentation velocity studies of intrinsically polydisperse macromolecules, such as carbohydrates.

Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2011
Total Cost
$63,780
Indirect Cost
Name
National Institute of Biomedical Imaging and Bioengineering
Department
Type
DUNS #
City
State
Country
Zip Code
Chaturvedi, Sumit K; Ma, Jia; Zhao, Huaying et al. (2017) Use of fluorescence-detected sedimentation velocity to study high-affinity protein interactions. Nat Protoc 12:1777-1791
Chaturvedi, Sumit K; Zhao, Huaying; Schuck, Peter (2017) Sedimentation of Reversibly Interacting Macromolecules with Changes in Fluorescence Quantum Yield. Biophys J 112:1374-1382
Ma, Jia; Zhao, Huaying; Sandmaier, Julia et al. (2016) Variable Field Analytical Ultracentrifugation: II. Gravitational Sweep Sedimentation Velocity. Biophys J 110:103-12
Desai, Abhiksha; Krynitsky, Jonathan; Pohida, Thomas J et al. (2016) 3D-Printing for Analytical Ultracentrifugation. PLoS One 11:e0155201
Zhao, Huaying; Fu, Yan; Glasser, Carla et al. (2016) Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions. Elife 5:
Schuck, Peter (2016) Sedimentation coefficient distributions of large particles. Analyst 141:4400-9
Ma, Jia; Metrick, Michael; Ghirlando, Rodolfo et al. (2015) Variable-Field Analytical Ultracentrifugation: I. Time-Optimized Sedimentation Equilibrium. Biophys J 109:827-37
Ma, Jia; Zhao, Huaying; Schuck, Peter (2015) A histogram approach to the quality of fit in sedimentation velocity analyses. Anal Biochem 483:1-3
Zhao, Huaying; Ghirlando, Rodolfo; Alfonso, Carlos et al. (2015) A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation. PLoS One 10:e0126420
Zhao, Huaying; Balbo, Andrea; Metger, Howard et al. (2014) Improved measurement of the rotor temperature in analytical ultracentrifugation. Anal Biochem 451:69-75

Showing the most recent 10 out of 39 publications