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

2. Outline Aims of the paper Amide Proton Exchange Methods Results
Conclusion

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

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

5. 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

6. 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

7. 15N-H HSQC spectrum

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

9. Protection factor and opening rates

10. 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

11. Dissociation pathway

12. 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

13. 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

14. Thank you !!