N Jamin, F. Toma/ Progress in Nuclear Magnetic Resonance Spectroscopy 38(2001)83-114 of the molecular magnetic susceptibility and the The most remarkable changes take place in the loop square of the magnetic field strength. As a result, between helices II and Ill: His29 within this loo the dipolar couplings or the chemical shifts vary contacts the DNA. A large decrease in backbone with the strength of the magnetic field and depend mobility within this loop is detected. The relaxation on the orientation of the bond vector or chemical parameters of mostN-containing side-chains hift tensors relative to the magnetic susceptibility ( GIn18, Arg22, Asn25, GIn26, Asn50, and Arg51) tensor. These small effects were observed for dna have also been measured (Fig. 5). Some of the side- or protein-DNA complexes due to the contributions chains of DNA-contacting residues show a significant of the stacked aromatic groups of the dNa bases to decrease in mobility upon dNa binding while others the magnetic susceptibility tensor. The dipolar are about equally mobile in both the free and the coupling restraints have been incorporated in the bound state. This indicates that interactions with simulated annealing protocol for structure determina- DNA do not necessarily restrict the mobility of the tion of the ce the DN ing domain of side-chain upon binding and that some flexibility GATA-1 with a 20 base pair DNA [37]. When remains at the interface between the protein and the ompared with the structure calculated without DNA. N TI measurements indicate that the side- H-IN andCa-H dipolar couplings, the overall chain of residues Gln18, Arg22 and Asn25 undergo precision of the coordinates increased only slightly intermediate exchange (us to ms time-scale) which but the percentage of residues in the most favorable may indicate that these atoms are changing partners region of the Ramachandran map and the number of in hydrogen bonds bad contacts improved significantly. A large displace The dynamics of the three aminoterminal zinc ment in the short loop connecting strands B3 and B4 fingers of X. laevis TFIlla(zf1-3) bound to a 15- was found. The magnetic field dependentN shifts mer DNA has been studied byN NMR [41]. The correlated well with the structure of the gatal flexibility of the backbone of the linker residues DNA complex refined with H-N and CaH (except Lys41)is significantly reduced upon DNA dipolar coupling constraints [38] binding. This reduction is associated with the forma tion of a defined conformation and close packing 2.8. Dynamic interactions between the side-chains within the linker and with the side-chains of the neighboring finger. Measurements of N spin-lattice and spin-spin Some flexibility has been found for the protein- relaxation rates as well as steady state H-N hetero- DNA interface as indicated by the broadening of reso- nuclear Noes ide information about internal nances or weak connectivities observed for some motions on the pico- to nanosecond time-scale and lysine resonances (Lys26, Lys29, Lys87). In fact, on conformational dynamics on the micro- to nano- analysis of the surface electrostatic potential at the econd time-scales [39]. The three examples given DNA binding site where these side-chains interact below, illustrate the role of dynamics in protei ggests that these fluctuations arise from the DNA recognition. The dynamics studies on lac repres- that these side-chains adopt different isoenergetic sor headpiece (1-56)[40] and on the three amino- conformations with different patterns of hydrogen terminal zinc fingers of X laevis TFIIIA [41] show bonds to DNA bases that the process of recognition is dynamic and not The essential Dna binding domain of the ADRI undergoes a disorder-to-order transition NTI, Tlo, and [H-N] NOE experiments were it binds to a 14 base-pair DNA duplex containing the performed on uniformly N-labeled free and DNA UASI binding site [13] as evidenced by Relaxation bound lac repressor headpiece(1-56)[40]. For the measurements. The free dNa binding domain of free lac repressor headpiece(1-56), the backbone of ADRI is composed of three distinct motional regions the three a-helices and of the turn of the hTh motif is and behaves like two beads linked by a flexible strin rather rigid, whereas the backbone of the loop Upon binding, most of this domain tumbles like a between helices I and ml is more mobile. Upon bind- single domain with reduced picosecond time-scale ing to the DNA, several changes in the mobility occur. motions compared to the free formof the molecular magnetic susceptibility and the square of the magnetic ®eld strength. As a result, the dipolar couplings or the chemical shifts vary with the strength of the magnetic ®eld and depend on the orientation of the bond vector or chemical shift tensors relative to the magnetic susceptibility tensor. These small effects were observed for DNA or protein±DNA complexes due to the contributions of the stacked aromatic groups of the DNA bases to the magnetic susceptibility tensor. The dipolar coupling restraints have been incorporated in the simulated annealing protocol for structure determina￾tion of the complex of the DNA binding domain of GATA-1 with a 20 base pair DNA [37]. When compared with the structure calculated without 1 H±15N and 13Ca ±1 Ha dipolar couplings, the overall precision of the coordinates increased only slightly but the percentage of residues in the most favorable region of the Ramachandran map and the number of bad contacts improved signi®cantly. A large displace￾ment in the short loop connecting strands b3 and b4 was found. The magnetic ®eld dependent 15N shifts correlated well with the structure of the GATA1± DNA complex re®ned with 1 H±15N and 13Ca ±1 Ha dipolar coupling constraints [38]. 2.8. Dynamics Measurements of 15N spin±lattice and spin±spin relaxation rates as well as steady state 1 H±15N hetero￾nuclear NOEs provide information about internal motions on the pico- to nanosecond time-scale and on conformational dynamics on the micro- to nano￾second time-scales [39]. The three examples given below, illustrate the role of dynamics in protein± DNA recognition. The dynamics studies on lac repres￾sor headpiece (1±56) [40] and on the three amino￾terminal zinc ®ngers of X. laevis TFIIIA [41] show that the process of recognition is dynamic and not static. 15N T1, T1r, and [1 H±15N] NOE experiments were performed on uniformly 15N-labeled free and DNA bound lac repressor headpiece (1±56) [40]. For the free lac repressor headpiece (1±56), the backbone of the three a-helices and of the turn of the HTH motif is rather rigid, whereas the backbone of the loop between helices II and III is more mobile. Upon bind￾ing to the DNA, several changes in the mobility occur. The most remarkable changes take place in the loop between helices II and III: His29 within this loop contacts the DNA. A large decrease in backbone mobility within this loop is detected. The relaxation parameters of most 15N-containing side-chains (Gln18, Arg22, Asn25, Gln26, Asn50, and Arg51) have also been measured (Fig. 5). Some of the side￾chains of DNA-contacting residues show a signi®cant decrease in mobility upon DNA binding while others are about equally mobile in both the free and the bound state. This indicates that interactions with DNA do not necessarily restrict the mobility of the side-chain upon binding and that some ¯exibility remains at the interface between the protein and the DNA. 15N T1r measurements indicate that the side￾chain of residues Gln18, Arg22 and Asn25 undergo intermediate exchange (ms to ms time-scale) which may indicate that these atoms are changing partners in hydrogen bonds. The dynamics of the three aminoterminal zinc ®ngers of X. laevis TFIIIA (zf1-3) bound to a 15- mer DNA has been studied by 15N NMR [41]. The ¯exibility of the backbone of the linker residues (except Lys41) is signi®cantly reduced upon DNA binding. This reduction is associated with the forma￾tion of a de®ned conformation and close packing interactions between the side-chains within the linker and with the side-chains of the neighboring ®nger. Some ¯exibility has been found for the protein± DNA interface as indicated by the broadening of reso￾nances or weak connectivities observed for some lysine resonances (Lys26, Lys29, Lys87). In fact, analysis of the surface electrostatic potential at the DNA binding site where these side-chains interact suggests that these ¯uctuations arise from the fact that these side-chains adopt different isoenergetic conformations with different patterns of hydrogen bonds to DNA bases. The essential DNA binding domain of the yeast ADR1 undergoes a disorder-to-order transition when it binds to a 14 base-pair DNA duplex containing the UAS1 binding site [13] as evidenced by 15N relaxation measurements. The free DNA binding domain of ADR1 is composed of three distinct motional regions and behaves like two beads linked by a ¯exible string. Upon binding, most of this domain tumbles like a single domain with reduced picosecond time-scale motions compared to the free form. N. Jamin, F. Toma / Progress in Nuclear Magnetic Resonance Spectroscopy 38 (2001) 83±114 91