Backbone dynamics for the wild type and a double H52R/T56W mutant of the vnd/NK-2 homeodomain from Drosophila melanogaster.
The (15)N relaxation behavior and heteronuclear Overhauser effect data for the wild type and an H52R/T56W double mutant protein that encompasses the vnd/NK-2 homeodomain from Drosophila melanogaster were used to characterize and describe the protein backbone dynamics. This investigation, which includes a description of a model structure for the H52R/T56W double mutant vnd/NK-2 homeodomain, was carried out for the two proteins in both the free and DNA-bound states. The double residue replacement at positions 52 and 56 within the DNA recognition helix of vnd/NK-2 has been shown to lead to a significant secondary structural modification resulting in an increase in the length of the recognition helix for the unbound protein. These structural changes are accompanied by corresponding changes in the T(1) and T(1)(rho) relaxation times as well as in the heteronuclear Overhauser effect (XNOE) values that show that the structural stability of the protein is enhanced by the two residue replacements. The values of the rotational anisotropy, D(parallel)/D(perpendicular), derived from analysis of the (15)N T(1) and T(1)(rho) relaxation values are small (1.189 for the unbound homeodomain and 1.110 for the bound homeodomain; both analyzed as prolate ellipsoids of revolution). A comparison of the T(2) values of the wild type and double mutant homeodomain reveals the presence of a low-frequency exchange contribution for the wild type analogue. These relaxation studies show that the motional behavior of the protein primarily reflects the tertiary structure and stability of the homeodomain backbone as well as the respective changes induced upon site-directed residue replacement or DNA binding.
Fausti
S
S
Istituto di Studi Chimico-Fisici di Macromolecole Sintetiche e Naturali (IMAG), National Research Council, Via De Marini, 6, 16149 Genova, Italy.
Weiler
S
S
Cuniberti
C
C
Hwang
K J
KJ
No
K T
KT
Gruschus
J M
JM
Perico
A
A
Nirenberg
M
M
Ferretti
J A
JA
D000818:Animals; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D013816:Thermodynamics; D014157:Transcription Factors
D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D014157:Transcription Factors; C094797:vnd protein, Drosophila