Analysis of thermally induced protein folding/unfolding transitions using free solution capillary electrophoresis.
It is shown that free solution capillary electrophoresis (FSCE) can be used to monitor the temperature-dependent folding/unfolding transitions of proteins. Furthermore, analysis of the data obtained by FSCE can be used to estimate the apparent thermodynamic parameters (enthalpy change (delta HvH), entropy change (delta S), and transition temperature (Tm)) associated with the folding/unfolding transition. In addition to mobility changes associated with the transition, FSCE analysis is unique in its ability to provide access to the population distribution of mobility states. This is demonstrated by the temperature-dependent change in the electrophoretic peak width and by the appearance of multiple peaks for very slow equilibrium or irreversible processes. Moreover, by comparing the mobility of the denatured state to that of unstructured model peptides, it is possible to characterize the relative degree of structure present in the unfolded state of a protein. This methodology has been applied to the analysis of the thermally induced unfolding of lysozyme at low pH. It is shown that the mobility of thermally denatured lysozyme can be described by the same function that describes unstructured, fully solvated peptides. On the contrary, the mobility of the native lysozyme is significantly higher than the value predicted by the same function. The accuracy of the apparent thermodynamic parameters obtained by this methodology compare within error with values obtained by direct calorimetric measurements using differential scanning calorimetry.
Hilser
V J
VJ
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Worosila
G D
GD
Freire
E
E
D000595:Amino Acid Sequence; D000818:Animals; D004586:Electrophoresis / Q000379:methods*; D005069:Evaluation Studies as Topic; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry; D017510:Protein Folding; D012996:Solutions; D013816:Thermodynamics
D009842:Oligopeptides; D012996:Solutions; D009113:Muramidase