N Jamin, F. Toma/ Progress in Nuclear Magnetic Resonance Spectroscopy 38(2001)83-114 po Wild-type Holo Wild-type 02030405060708090100 102030405060708090100 LAHBH CHDHEHF [AHBHCHDHE HF S100Apo AV77 0orooxu 20 102030405060708090100 Residue Number Residue Numb Fig3. Amide proton exchange rate(s )versus residue number for the wild-type and AV77 apo- and holorepressor, pH 7.6 at 45C(Fi rom Ref [191). Reprinted with the permission of O. Jardetzky and of Cambridge University Press(O 1996) binding of the three aminoterminal zinc fingers of X. In the case of fast exchange between free and laevis TFIlla(zf1-3)to a 15-mer DNa with the inter bound states, the structure of the complex cannot molecular contacts known from the high-resolution be obtained easily. Titration experiments monitor structure of the complex. They found that the chemi- the variation of chemical shifts upon addition of cal shift changes for protein H, N and C reso- DNA and estimation of binding constants(in the nances upon DNa binding are not well correlated millimolar range) can be extracted from the with DNA contacts observed in the solution structure analysis of the titration curves [15]. The chemical of the complex. In fact the protein resonances are shifts of the bound protein resonances are directly affected not only by dna binding but also by changes obtained from these titration experiments. As in in the dynamics and conformation of the protein upon the case of slow exchange, the variation of binding. The DNA base-protons were found to be chemical shifts can be used to map the binding good markers of the DNa binding sites because the surface. conformation of the dNa is not significantly distorted For intermediate exchange between the free and upon binding bound states or between different bound conformationsbinding of the three aminoterminal zinc ®ngers of X. laevis TFIIIA (zf1-3) to a 15-mer DNA with the inter￾molecular contacts known from the high-resolution structure of the complex. They found that the chemi￾cal shift changes for protein 1 H,15N and 13C reso￾nances upon DNA binding are not well correlated with DNA contacts observed in the solution structure of the complex. In fact the protein resonances are affected not only by DNA binding but also by changes in the dynamics and conformation of the protein upon binding. The DNA base-protons were found to be good markers of the DNA binding sites because the conformation of the DNA is not signi®cantly distorted upon binding. In the case of fast exchange between free and bound states, the structure of the complex cannot be obtained easily. Titration experiments monitor the variation of chemical shifts upon addition of DNA and estimation of binding constants (in the millimolar range) can be extracted from the analysis of the titration curves [15]. The chemical shifts of the bound protein resonances are directly obtained from these titration experiments. As in the case of slow exchange, the variation of chemical shifts can be used to map the binding surface. For intermediate exchange between the free and bound states or between different bound conformations, N. Jamin, F. Toma / Progress in Nuclear Magnetic Resonance Spectroscopy 38 (2001) 83±114 87 Fig. 3. Amide proton exchange rate (s-1) versus residue number for the wild-type and AV77 apo- and holorepressors, pH 7.6 at 458C. (Fig. 1 from Ref. [19]). Reprinted with the permission of O. Jardetzky and of Cambridge University Press (q 1996)