Assignment of 1H, 15N, and 13C resonances, identification of elements of secondary structure and determination of the global fold of the DNA-binding domain of GAL4.
Almost complete assignments of the 1H, 15N, and aliphatic 13C resonances of the 62-residue N-terminal DNA-binding domain of GAL4 [GAL4(62)] have been obtained using a combination of two-dimensional homonuclear and two- and three-dimensional double- and triple-resonance heteronuclear NMR methods. The sequential NOE connectivities, amide proton exchange measurements, and 13C alpha chemical shift data indicate the presence of two short alpha-helices in the N-terminal half of the polypeptide. Residues 1-9 and 41-62 appear to be unstructured and flexible in solution. Analysis of the 13C alpha chemical shifts also revealed a significant downfield shift of approximately +3 ppm, relative to random-coil values, for the four nonbridging Zn(II) ligands, Cys 14, 21, 31, and 38. Interestingly, no such correlation was observed for the two bridging ligands, Cys 11 and 28. Preliminary structure calculations using a subset of distance restraints derived from three-dimensional 1H-15N and 1H-13C NOESY-HSQC spectra are consistent with the recently reported solution structures of Zn(II)2GAL4(7-49) [Kraulis, P., et al. (1992) Nature 356, 448-450] and of Cd(II)2GAL4(65) [Baleja, J. D., et al. (1992) Nature 356, 450-453].
Shirakawa
M
M
Institute for Protein Research, Osaka University, Japan.
Fairbrother
W J
WJ
Serikawa
Y
Y
Ohkubo
T
T
Kyogoku
Y
Y
Wright
P E
PE
D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D001483:Base Sequence; D002247:Carbon Isotopes; D003198:Computer Simulation; D004268:DNA-Binding Proteins / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry*; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D009841:Oligonucleotides; D011487:Protein Conformation; D017510:Protein Folding; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors
D000577:Amides; D002247:Carbon Isotopes; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D009587:Nitrogen Isotopes; D009841:Oligonucleotides; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D006859:Hydrogen