N Jamin, F. Toma/ Progress in Nuclear Magnetic Resonance Spectroscopy 38 (2001)83-114 i.e. the dissociation constants Kd are less than 10M GR [R2R3] and detailed information can be obtained on the IMIM,I complexes because of the slow exchange regim between free and bound states (lifetimes greater than I s) at the chemical shift time-scale. The rate of exchange is much less than the difference in the chemical shift between the two states and. at a mole ratio less than the stoichiometric ratio, two sets of resonances are observed corresponding to the free and bound states. Therefore. the resonances of the complex have to be assigned using NMR techniques employed for large molecules and/or edited/filtered Fig. 2 shows the imino region of the H obtained upon addition of different amounts of ESapeolra tion of R2R3 dna binding domain of c-Myb to a solution of mim12 oligonucleotide [12]. On addition of the protein, new resonance lines corresponding to the bound mim12 dodecamer appear. Some of these lines are split into two signals which indicate the enc forms. The lifetimes of these two forms are longer than the inverse of the frequency difference between the free and bound state 14.0 12.0 Chemical shifts are very sensitive probes of the Fig.2. Imino region of the H-NMR 600 MHz spectra obtained local environments of the nucleus but unfortunately upon addition of different amour ding domain of c-Myb to a sol olton of the rzR DNA it is not possible to predict their values from the 20°C. conformation of the complex or conversely to deduce the conformation from their values. Nevertheless they are useful parameters to gain insight into the desired DNA sequence in E. coli with N andc parts of the molecules influenced by the interaction nutrients. These methods have been applied to the Schmiedeskamp and coworkers [13] have shown by synthesis of fully or partiallyC,N orN-labeled analysis of H and Ca chemical shifts that little double strand oligonucleotides of 10-21 base pairs. a change in the structure of the zinc-finger domain 32 base DNA oligonucleotide that folds to form an from the yeast transcription factor ADRI occurs intramolecular quadruplex as well as a 12 base oligo- upon binding to a 14mer DNA containing the UAS nucleotide that dimerizes and folds to form a quadru- half site. A correlation between the protein-DNA plex uniformly C, N-doubly labeled have also been interface mapped by chemical shift changes and that produced for NMR studies. by muta ts was found Both these methods require a high level of exper However, the identification of the dna binding site tise Site specific labeling is more easily attained with using DNA induced chemical shift changes should be the chemical method and is therefore the method of done with care. This approach is not feasible for hoice for the synthesis of site specific labeled DNA. numerous protein-DNA complexes where proteins undergo conformational transitions and dynamics 2. 2. Chemical shift changes changes upon binding that will affect the chemical shifts. This has been recently demonstrated by Foster Interactions of protein with DNA fragment contain- and coworkers [14]. These authors analyzed the corre ing specific binding sites are tight binding interactions lation between the chemical shift changes upondesired DNA sequence in E. coli with 15N and 13C nutrients. These methods have been applied to the synthesis of fully or partially 13C,15N or 15N-labeled double strand oligonucleotides of 10±21 base pairs. A 32 base DNA oligonucleotide that folds to form an intramolecular quadruplex as well as a 12 base oligo￾nucleotide that dimerizes and folds to form a quadru￾plex uniformly 13C,15N-doubly labeled have also been produced for NMR studies. Both these methods require a high level of exper￾tise. Site speci®c labeling is more easily attained with the chemical method and is therefore the method of choice for the synthesis of site speci®c labeled DNA. 2.2. Chemical shift changes Interactions of protein with DNA fragment contain￾ing speci®c binding sites are tight binding interactions i.e. the dissociation constants Kd are less than 1028 M and detailed information can be obtained on the complexes because of the slow exchange regime between free and bound states (lifetimes greater than 1 s) at the chemical shift time-scale. The rate of exchange is much less than the difference in the chemical shift between the two states and, at a mole ratio less than the stoichiometric ratio, two sets of resonances are observed corresponding to the free and bound states. Therefore, the resonances of the complex have to be assigned using NMR techniques employed for large molecules and/or edited/®ltered techniques. Fig. 2 shows the imino region of the 1 H spectra obtained upon addition of different amounts of a solu￾tion of R2R3 DNA binding domain of c-Myb to a solution of mim12 oligonucleotide [12]. On addition of the protein, new resonance lines corresponding to the bound mim12 dodecamer appear. Some of these lines are split into two signals which indicate the simultaneous presence of two forms. The lifetimes of these two forms are longer than the inverse of the frequency difference between the free and bound state resonances. Chemical shifts are very sensitive probes of the local environments of the nucleus but unfortunately it is not possible to predict their values from the conformation of the complex or conversely to deduce the conformation from their values. Nevertheless, they are useful parameters to gain insight into the parts of the molecules in¯uenced by the interaction. Schmiedeskamp and coworkers [13] have shown by analysis of 1 H and 13Ca chemical shifts that little change in the structure of the zinc-®nger domain from the yeast transcription factor ADR1 occurs upon binding to a 14mer DNA containing the UAS half site. A correlation between the protein±DNA interface mapped by chemical shift changes and that mapped by mutagenesis experiments was found. However, the identi®cation of the DNA binding site using DNA induced chemical shift changes should be done with care. This approach is not feasible for numerous protein±DNA complexes where proteins undergo conformational transitions and dynamics changes upon binding that will affect the chemical shifts. This has been recently demonstrated by Foster and coworkers [14]. These authors analyzed the corre￾lation between the chemical shift changes upon 86 N. Jamin, F. Toma / Progress in Nuclear Magnetic Resonance Spectroscopy 38 (2001) 83±114 Fig. 2. Imino region of the 1 H-NMR 600 MHz spectra obtained upon addition of different amount of a solution of the R2R3 DNA binding domain of c-Myb to a solution of mim12 oligonucleotide at 208C