Use of urea and glycine betaine to quantify coupled folding and probe the burial of DNA phosphates in Lac repressor - Lac operator bindings

Thermodynamic analysis of urea-biopolymer interactions and effects of urea on folding of proteins and alpha-helical peptides shows that urea interacts primarily with polar amide surface. Urea is therefore predicted to be a quantitative probe of coupled folding, remodeling, and other large-scale changes in the amount of water-accessible polar amide surface in protein processes. A parallel analysis indicates that glycine betaine [N,N,N-trimethylglycine (GB)] can be used to detect burial or exposure of anionic (carboxylate, phosphate) biopolymer surface. To test these predictions, we have investigated the effects of these solutes (0-3 m) on the formation of 1:1 complexes between lac repressor (LacI) and its symmetric operator site (SymL) at a constant KCl molality. Urea reduces the binding constant KTO [initial slope dInK(TO)/dm(urea) = -1.7 +/- 0.2], and GB increases K-TO [initial slope dlnK(TO)/dm(GB) = 2.1 +/- 0.2]. For both solutes, this derivative decreases with an increase in solute concentration. Analysis of these initial slopes predicts that (1.5 +/- 0.3) x 10(3) angstrom(2) of polar amide surface and (4.5 +/- 1.0) x 10(2) angstrom(2) of anionic surface are buried in the association process. Analysis of published structural data, together with modeling of unfolded regions of free Lacl as extended chains, indicates that 1.5 x 10(3) angstrom(2) of polar amide surface and 6.3 x 10(2) angstrom(2) of anionic surface are buried in complexation. Quantitative agreement between structural and thermodynamic results is obtained for amide surface (urea); for anionic surface (GB), the experimental value is similar to 70% of the structural value. For LacI-SymL binding, two-thirds of the structurally predicted change in amide surface (1.0 x 10(3) angstrom(2)) occurs outside the protein-DNA interface in protein-protein interfaces formed by folding of the hinge helices and interactions of the DNA binding domain (DBD) with the core of the repressor. Since urea interacts principally with amide surface, it is particularly well-suited to detect and quantify the extent of coupled folding and other large-scale remodeling events in the steps of protein-nucleic acid interactions and other protein associations.