Abstract:
:The effect of low molecular-weight compounds on the equilibrium constant K(A) can be used to explore the energetics and molecular mechanism of protein-DNA interactions. Here we use the complex composed of the integrase Tn916 DNA-binding domain and its target DNA duplex to investigate the effects of salt and the nonionic osmolytes glycerol and sorbitol on sequence-specific protein-DNA association. Increasing Na(+) concentration from 0.12 to 0.32 M weakens the binding affinity by a factor of 20. The decrease of affinity is dominated by a large loss of binding enthalpy but only a small loss of binding entropy. This contrasts the concept that the salt-induced weakening of protein-DNA binding is mainly entropic. The large enthalpy loss is discussed in the light of recent views about the nature of the general salt effect. Addition of up to 2.5 M sorbitol and up to 3.3 M glycerol causes a slight increase of the binding affinity. However, both osmolytes lead to a large enthalpy gain and a similarly large entropy loss. This intriguing enthalpy-entropy compensation can be explained in part by an enthalpic chelate effect: The osmolyte tightens the structure of the protein-DNA complex whereby the formation of enthalpically favorable noncovalent interactions is promoted at the entropic cost of a more rigid complex. The results were obtained by isothermal titration calorimetry. They are supported by kinetic experiments showing that the rate of formation of the complex is reduced by salt, but the rate of complex dissociation is not. Glycerol and sorbitol reduce both rates in line with an only small effect on complex stability. This work clarifies the thermodynamic and kinetic response of a novel protein-DNA complex to increased salt and the presence of two common, nonionic osmolytes.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Milev S,Bosshard HR,Jelesarov Idoi
10.1021/bi048907nsubject
Has Abstractpub_date
2005-01-11 00:00:00pages
285-93issue
1eissn
0006-2960issn
1520-4995journal_volume
44pub_type
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