Figure 5.

Proteins like H1 with more than one binding domain can bind their substrates in more than one way. To illustrate, a generic protein with circular and square binding domains X and Y (which could represent the H1 globular and C-terminal domains, for example) can bind its substrate in three ways. In (A) each domain targets an independent set of binding sites. In this case the bound/free fraction in the wild-type (XY) is equal to the sum of bound/free fractions measured in mutants with just the X or Y binding domain (X and Y, respectively), as shown in the bar-plot to the right. Alternatively, in (B), the X and Y domains cooperate to target a mutual site where the binding of one domain tethers the other so it subsequently binds with higher affinity than it would if it were untethered and freely diffusing. In this case, the wild-type bound/free fraction is greater than the sum in mutants, as shown by the grey portion (red online) of the bar-plot to the right. (C) The H1⁰ wild-type bound/free fraction measured by FRAP is significantly greater than the sum of bound/free fractions measured for the globular domain by itself (S1S2=GD) and the C-terminal domain (C) by itself. The difference, shown in grey (red online), represents strong cooperation between these two domains. From the bar-plot, the cooperativity factor γ=27±8 was calculated by dividing the grey portion (red online) by the product of the two white portions (Supplementary equation (S.17)). A full-colour version of this figure is available at The EMBO Journal Online.