Mixed osmolytes: the degree to which one osmolyte affects the protein stabilizing ability of another.
Mixtures of organic osmolytes occur in cells of many organisms, raising the question of whether their actions on protein stability are independent or synergistic. To investigate this question it is desirable to develop a system that permits evaluation of the effect of one osmolyte on the efficacy of another to either force-fold or denature a protein. A means of evaluating the efficacy of an osmolyte is provided by its m-value, an experimental quantity that measures the ability of the osmolyte to force a protein to unfold or fold. An experimental system is presented that enables evaluations of the m-values of osmolytes in the presence and absence of a second osmolyte. The experimental system involves use of a marginally stable protein in 10 mM buffer (pH 7, 200 mM salt, and 34 degrees C) that is at the midpoint of its native to denatured transition. These conditions enable determination of m-values for protecting and denaturing osmolytes in the presence and absence of a second osmolyte, permitting assessment of the extent to which the two osmolytes affect each other's efficacy. The two osmolytes investigated in this work are the denaturing osmolyte, urea, and the protecting osmolyte, sarcosine. Results show unequivocally that neither osmolyte alters the efficacy of the other in forcing the protein to fold or unfold-the osmolytes act independently on the protein despite their combined concentrations being in the multi-molar range. These osmolytes avoid altering one another's efficacy at these high concentrations because the number of osmolyte interaction sites on the protein is large and the binding constants are quite small. Consequently, the site occupancies are low enough in number that the two osmolytes neither compete nor cooperate in interacting with the protein.
Holthauzen
Luis Marcelo F
LM
Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, TX 77555, USA.
Bolen
D Wayne
DW
D009994:Osmolar Concentration; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D012521:Sarcosine / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
D011506:Proteins; D012521:Sarcosine