Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition

Abstract

Sedimentation equilibrium is a powerful tool for the characterization of protein self-association and heterogeneous protein interactions. Frequently, it is applied in a configuration with relatively long solution columns and with equilibrium profiles being acquired sequentially at several rotor speeds.The present study proposes computational tools, implemented in the software SEDPHAT, for the global analysis of equilibrium data at multiple rotor speeds, multiple concentrations, and multiple optical detection methods. The detailed global modeling of such equilibrium data can be a non-trivial computational problem. It was shown previously that mass conservation constraints can significantly improve and extend the analysis of heterogeneous protein interactions. Here, a method for using conservation of mass constraints for the macromolecular redistribution is proposed in which the effective loading concentrations is being calculated from the sedimentation equilibrium profiles. The approach is similar to that described by Roark (Biophys. Chem. 5 (1976) [185][186][187][188][189][190][191][192][193][194][195][196], but its utility is extended by determining the bottom position of the solution columns from the macromolecular redistribution. For analyzing heterogeneous associations at multiple protein concentrations, additional constraints are introduced that relate the effective loading concentrations of the different components or their molar ratio in the global analysis. Equilibrium profiles at multiple rotor speeds also permit the algebraic determination of radial-dependent baseline profiles, which can govern interference optical ultracentrifugation data, but usually also occur, to a smaller extent, in absorbance optical data. Finally, the global analysis of equilibrium profiles at multiple rotor speeds with implicit mass conservation and computation of the bottom of the solution column provides an unbiased scale for determining molar mass distributions of non-interacting species. The properties of these tools are studied with theoretical and experimental data sets.

Mutagenesis, biochemical, and biophysical characterization of Mycoplasma arthritidis-derived mitogen

“…All SV AUC data were recorded with absorbance detection at wavelengths of 280, 294, and 300 nm, for low, moderate, and high concentrations of proteins, respectively. Absorbance fringe displacement profiles were analyzed with the software SEDFIT (http://www.analyticalultracentrifugation.com/) (Vistica et al, 2004), using a model for continuous sedimentation coefficient distributions c(s) .…”

CED-4 forms a 2 : 2 heterotetrameric complex with CED-9 until specifically displaced by EGL-1 or CED-13

“…34 Sedimentation velocity data at multiple time points were fitted to either a noninteracting discrete species model of up to three components or a continuous sizedistribution model 35 using the program SEDFIT, which is available at www.analyticalultracentrifugation.com. Sedimentation equilibrium data at multiple speeds were globally fitted to single discrete species model by employing the program SEDPHAT, 36 which is similarly available.…”

Loss of iron–sulfur clusters from biotin synthase as a result of catalysis promotes unfolding and degradation

“…Each protein was subjected to centrifugation at 10000, 15000, and 20000 rpm for 2-4 hrs, achieving an equilibrium radial concentration distribution that depends on the native molecular weight and centrifugation speed. Using the program Sedphat [24], the data at three speeds were globally fit to a nonequilibrating mixture of monomer, dimer, and tetramer. The centrifugation data for 2Fe-BioB are fit best to a mixture of 86 % dimer (MW = 92.8 kD and S = 4.97) and 14 % tetramer (MW = 188.2 kD and S = 6.86).…”

“…Data sets collected after 2 and 4 hrs were indistinguishable indicating that equilibrium was achieved and that the samples were stable over several hours. Absorbance data collected at 3 speeds were globally fit using the program Sedphat [24] to a mixture of non-equilibrating species. Initial estimated values of Abs total = 0.3, dimer MW = 80000 and s = 5, tetramer MW = 160000 and s = 7, were provided by prior velocity centrifugation experiments (data not shown).…”

“…Five radial scans at 280 nm were averaged for each data set. Data are globally fit using Sedphat [24] to a mixture of nonequilibrating BioB dimer and tetramer. Circular dichroism spectra of apoBioB, 2Fe-BioB, and 2Fe4Fe-BioB.…”