Analytical Ultracentrifuge Studies of a Cytokine, Soluble Receptor Interaction: Comparison of Simulations and Experiments
| Autor: | John J. Correia, Walter F. Stafford, Peter J. Sherwood, Robert Wright |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Chemistry Monte Carlo method Complex system Biophysics Experimental data Analytical Ultracentrifugation 03 medical and health sciences 030104 developmental biology 0302 clinical medicine 030220 oncology & carcinogenesis Sedimentation equilibrium Data analysis Ultracentrifuge Biological system Equilibrium constant |
| Zdroj: | Biophysical Journal. 110(3) |
| ISSN: | 0006-3495 |
| DOI: | 10.1016/j.bpj.2015.11.2078 |
| Popis: | There is a long tradition in analytical ultracentrifugation (AUC) to perform simulation studies to investigate the utility of new data analysis methods or to verify the reliability of established approaches to complex systems. Here we compare the use of sedimentation velocity and sedimentation equilibrium methods for a heterogeneous associating system involving a trimeric cytokine complex (TNFα) and the stepwise binding of three soluble receptors (TNFR1). We will outline the methods for simulating velocity and equilibrium experimental data using the AUC analysis program SEDANAL corresponding to a titration of the ligand to a fixed concentration of trimer. We then outline the methods for analyzing the velocity and equilibrium data and compare the ability to extract the equilibrium constants for the system. The results suggest that complex hetero-associating systems can be successfully analyzed by sedimentation velocity approaches and that velocity analysis may be preferable to sedimentation equilibrium approaches for some complex hetero-reaction mechanisms. A detailed discussion of error analysis by Bootstrap with replacement, Monte Carlo and Fstat is presented in order to establish criteria for comparing and selecting velocity or equilibrium approaches. Experimental data are also compared to simulated data to present the additional challenge of experimental complexity, including sample purity, sample aggregation, and the presence of additional reaction modes. (Funded by UMC AUC Facility.) |
| Databáze: | OpenAIRE |
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