A residue specific view of the association and dissociation Pathway in protein-DNA recognition Nature structural Biology, vol. 9 number 3, march 2002

Outline Aims of the paper Amide Proton Exchange Methods Results Conclusion

Lac repressor-DNA complex 357 residues long Tetrameric protein binds to DNA Regulates lactose transport Prototype for transcription regulation Unknown association and dissociation pathways

Lac repressor Structure DNA binding domain ( 1-49) Core domain (63-357) Hinge region ( 50-62) Disordered in uncomplexed state , α–helix in complex

Hydrogen exchange Amide protons exchange with Deuterium in D2O Scheme Kobs = Kop x Kint / ( Kcl + Kint ) Mechanisms: EX2: Kcl >> Kint Kobs = ( Kop / Kcl ) Kint EX1: Kint >> Kcl Kobs = Kop

Determination of exchange rates from NMR measurement H-atom – spin ½ D-atom – spin 1 nuclei 15N- H HSQC spectrum shows all amide protons Start with measuring in H2O then a series in D2O with time intervals Signal decays and vanishes for deuterated residues Rate of decay is the rate of exchange Kobs

15N-H HSQC spectrum

Determination of opening and closing rates Kint is function of pH. Plot Kobs vs Kint EX2 to EX1 transition at Kcl =Kint

Protection factor and opening rates

Opening rates mapped on complex kop = 0.20 h-1 is red; kop = 0.11 h-1, orange; kop = 0.04 h-1, yellow; and kop = 0.02 h-1 Violet

Dissociation pathway

Association pathway Follow protected protons only in bound state The recognition helix is first to fit to DNA Hinge helix is last to bind to DNA

Conclusions DNA is an allosteric activator of the Lac repressor Lac repressor is Highly flexible in free state Order of the events is important for function Amide exchange studies using NMR is a powerful technique to study pathways of complex formation

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