N Jamin, F. Toma/ Progress in Nuclear Magnetic Resonance Spectroscopy 38 (2001)83-114 2.9. Hydration water molecules around the backbone amide proton of Ala30, Tyr34 and Tyr35 which are close to phosphate Water molecules are important contributors in the groups. This suggests that these water molecules process of protein-DNA recognition as they may participate in bridging hydrogen bonds between the have structural and /or functional roles sugar-phosphate backbone and the relevant amide NMR can provide information about the location group and lifetime of the contacts between water and th protein/DNA 3, 42, 43]. The residence times of hydra tion water can be estimated from the measurements of NOEs and rOEs between water protons and protein 3. Selected applications or DNA protons. These measurements distinguis residence times of less than 1 ns from longer ones. Table 1 summarizes the protein sequence Typically residence times shorter than I ns are motifs and DNA sequence of the protein-DNA observed on the surface of protein and in the major complexes discussed below. It also includes a groove ofDNA while residence times longer than I ns summary of the direct interactions between the have been observed for water molecules in the interior amino acid side-chains and the nucleic acid of proteins, in the minor grooves of DNA and in bases protein-DNA interfaces The NMR study of the Antennapedia homeodo- 3.1. The helix-turn-helix motif main-DNA complex reveals that water molecules are present at the protein-DNA interface: contacts The HTH motif consists of two nearly perpendicu between protein and water have been observed for The second helix of this motif called the"recognition lar a-helices separated by a link of variable lengt amino acid residues 43 44. 47. 48. 50. 51. 52 and 54(Fig. 6[44. These water molecules exchange helix" inserts into the major groove of the dna to slowly with the bulk solvent(residence times between make specific contacts. Variations between members ns and 20 ms)[45] similar to water molecules in the of the hTH family include the orientation of the helix nterior of proteins and have multiple preferred loca in the major groove, the position of the residues tions. In addition, two residues at the protein-DNA contacting the DNA and the length of the recogniti interface, Asn51(strictly conserved )and Gln50 (func- helix. This motif first identified in procaryotic gene tionally important), contact several DNA bases with regulatory proteins can be found in a wide variety of transient water mediated hydrogen bonds. The model DNA-binding proteins including eukaryotic homeo- proposed for the interactions between the protein and domains and transcription factors the DNA consists of a fluctuating network of hydro- gen bonds between the polar groups of the protein and 3.. Homeodomain the dNa and water molecules A homeodomain protein is the product of homeo- In contrast to other protein-DNA complexes, the box genes. It is a highly conserved DNA-binding complex between the dna binding domain of domain of about 60 amino acid residues that is chicken GATA-I and a 16 base pair duplex is char- found in transcriptional regulators involved in the acterized by only two hydrogen bonds between the genetic control of development. These regulators protein and the DNA [46]. The specific interactions specify to the embryonic cells the positional informa involve hydrophobic contacts between the methyl tion(where they are relative to their neighbors) and groups of the protein and the dna bases. Clore and the segmental identity(what structure they should coworkers have found water molecules around all generate). They act at various levels of the develop surface exposed methyl groups as well as around ment and in all organisms, from yeast to human methyl groups in the neighborhood of the sugar-phos- Mutations in the homeodomain could result in genetic phate backbone but the water molecules are excluded diseases and developmental abnormalities. Therefore, from the interface between the protein and the dna in order to understand the role of individual amino bases in the major groove [47]. They also observed acid residues in tertiary structure formation and2.9. Hydration Water molecules are important contributors in the process of protein±DNA recognition as they may have structural and /or functional roles. NMR can provide information about the location and lifetime of the contacts between water and the protein/DNA [3,42,43]. The residence times of hydra￾tion water can be estimated from the measurements of NOEs and ROEs between water protons and protein or DNA protons. These measurements distinguish residence times of less than 1 ns from longer ones. Typically residence times shorter than 1 ns are observed on the surface of protein and in the major groove of DNA while residence times longer than 1 ns have been observed for water molecules in the interior of proteins, in the minor grooves of DNA and in protein±DNA interfaces. The NMR study of the Antennapedia homeodo￾main±DNA complex reveals that water molecules are present at the protein±DNA interface: contacts between protein and water have been observed for amino acid residues 43, 44, 47, 48, 50, 51, 52 and 54 (Fig. 6 [44]). These water molecules exchange slowly with the bulk solvent (residence times between 1 ns and 20 ms) [45] similar to water molecules in the interior of proteins and have multiple preferred loca￾tions. In addition, two residues at the protein±DNA interface, Asn51 (strictly conserved) and Gln50 (func￾tionally important), contact several DNA bases with transient water mediated hydrogen bonds. The model proposed for the interactions between the protein and the DNA consists of a ¯uctuating network of hydro￾gen bonds between the polar groups of the protein and the DNA and water molecules. In contrast to other protein±DNA complexes, the complex between the DNA binding domain of chicken GATA-1 and a 16 base pair duplex is char￾acterized by only two hydrogen bonds between the protein and the DNA [46]. The speci®c interactions involve hydrophobic contacts between the methyl groups of the protein and the DNA bases. Clore and coworkers have found water molecules around all surface exposed methyl groups as well as around methyl groups in the neighborhood of the sugar-phos￾phate backbone but the water molecules are excluded from the interface between the protein and the DNA bases in the major groove [47]. They also observed water molecules around the backbone amide proton of Ala30, Tyr34 and Tyr35 which are close to phosphate groups. This suggests that these water molecules participate in bridging hydrogen bonds between the sugar-phosphate backbone and the relevant amide groups. 3. Selected applications Table 1 summarizes the protein sequence motifs and DNA sequence of the protein±DNA complexes discussed below. It also includes a summary of the direct interactions between the amino acid side-chains and the nucleic acid bases. 3.1. The helix-turn-helix motif The HTH motif consists of two nearly perpendicu￾lar a-helices separated by a link of variable length. The second helix of this motif called the ªrecognition helixº inserts into the major groove of the DNA to make speci®c contacts. Variations between members of the HTH family include the orientation of the helix in the major groove, the position of the residues contacting the DNA and the length of the recognition helix. This motif ®rst identi®ed in procaryotic gene￾regulatory proteins can be found in a wide variety of DNA-binding proteins including eukaryotic homeo￾domains and transcription factors. 3.1.1. Homeodomain A homeodomain protein is the product of homeo￾box genes. It is a highly conserved DNA-binding domain of about 60 amino acid residues that is found in transcriptional regulators involved in the genetic control of development. These regulators specify to the embryonic cells the positional informa￾tion (where they are relative to their neighbors) and the segmental identity (what structure they should generate). They act at various levels of the develop￾ment and in all organisms, from yeast to human. Mutations in the homeodomain could result in genetic diseases and developmental abnormalities. Therefore, in order to understand the role of individual amino acid residues in tertiary structure formation and 92 N. Jamin, F. Toma / Progress in Nuclear Magnetic Resonance Spectroscopy 38 (2001) 83±114