Non-ideality by sedimentation velocity of halophilic malate dehydrogenase in complex solvents.
We have investigated the potential of sedimentation velocity analytical ultracentrifugation for the measurement of the second virial coefficients of proteins, with the goal of developing a method that allows efficient screening of different solvent conditions. This may be useful for the study of protein crystallization. Macromolecular concentration distributions were modeled using the Lamm equation with the approximation of linear concentration dependencies of the diffusion constant, D = D(o) (1 + k(D)c), and the reciprocal sedimentation coefficient s = s(o)/(1 + k(s)c). We have studied model distributions for their information content with respect to the particle and its non-ideal behavior, developed a strategy for their analysis by direct boundary modeling, and applied it to data from sedimentation velocity experiments on halophilic malate dehydrogenase in complex aqueous solvents containing sodium chloride and 2-methyl-2,4-pentanediol, including conditions near phase separation. Using global modeling for three sets of data obtained at three different protein concentrations, very good estimates for k(s) and s degrees and also for D degrees and the buoyant molar mass were obtained. It was also possible to obtain good estimates for k(D) and the second virial coefficients. Modeling of sedimentation velocity profiles with the non-ideal Lamm equation appears as a good technique to investigate weak inter-particle interactions in complex solvents and also to extrapolate the ideal behavior of the particle.
Solovyova
A
A
Laboratoire de Biophysique Moléculaire, Institut de Biologie Structurale J. P. Ebel, F-38027 Grenoble, France.
Schuck
P
P
Costenaro
L
L
Ebel
C
C
D004058:Diffusion; D019614:Haloarcula marismortui / Q000201:enzymology; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000302:isolation & purification*; D008956:Models, Chemical; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D012965:Sodium Chloride / Q000737:chemistry; D012997:Solvents / Q000737:chemistry*; D013816:Thermodynamics; D014461:Ultracentrifugation; D014867:Water / Q000737:chemistry
D011506:Proteins; D012997:Solvents; D014867:Water; D012965:Sodium Chloride; D008291:Malate Dehydrogenase