GLPNGLLSGDEDFSSIADMDFSALLSQISS

1. Circular dichroism (CD) spectroscopy, far-UV (293 K; quartz cells (1 mm path length))


2. Nuclear magnetic resonance (NMR) (pH: 5.5; 10% D₂O; 90% H₂O; NaCl 50 mM; protein 2 mM; sodium phosphate 50 mM)

1. Schmitz ML, Baeuerle PA. "The p65 subunit is responsible for the strong transcription activating potential of NF-kappa B." Embo J. 1991; 10(12): 3805-3817. PubMed: 1935902
   
   
2. Schmitz ML, dos Santos Silva MA, Altmann H, Czisch M, Holak TA, Baeuerle PA. "Structural and functional analysis of the NF-kappa B p65 C terminus. An acidic and modular transactivation domain with the potential to adopt an alpha-helical conformation." J Biol Chem. 1994; 269(41): 25613-25620. PubMed: 7929265
   
   

CD and NMR spectra revealed that TA1 exists in aqueous solution at physiological pH in a random coil conformation or is unstructured. The acidic activation domians of p65 TA1 are all capable of forming an alpha-helix in hydrophobic solvents. In an aqueous solution, negatively charged amino acids might prevent formation of a secondary structure due to electrostatic repulsion and/or steric hindrance imposed by hydrate shells. A transition of positively charged random coil sequence into an alpha-helical structure has been reported to occur when leucine zipper proteins contact DNA with their basic domains. Domains of the p65 molecule required for initiating transcription were subjected to deletion analysis. Removal of only 30 amino acids from the C-terminus of p65 reduced the transactivating potential of p65, whereas removal of 250 amino acids from the C-terminus completely negated the transactivating potential. A feature of TA1 appears to be the clustering of serine residues on one side of an alleged amphipathic helix, in which the serine residues may be involved in a zipper-like structure serving a role in protein-protein interaction as has been proposed for leucine repeats. Ser529 and Ser536 are important for inhibiting NF-kB activation, and phosphorylation of these residues is essential for NF-kB activation.

LQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAI
TRLVTGAQRPPDPAPAPL

1. Nuclear magnetic resonance (NMR) (pH: 5.5; 10% D₂O; 90% H₂O; NaCl 50 mM; sodium phosphate 50 mM)

1. Schmitz ML, Baeuerle PA. "The p65 subunit is responsible for the strong transcription activating potential of NF-kappa B." Embo J. 1991; 10(12): 3805-3817. PubMed: 1935902
   
   
2. Schmitz ML, dos Santos Silva MA, Altmann H, Czisch M, Holak TA, Baeuerle PA. "Structural and functional analysis of the NF-kappa B p65 C terminus. An acidic and modular transactivation domain with the potential to adopt an alpha-helical conformation." J Biol Chem. 1994; 269(41): 25613-25620. PubMed: 7929265
   
   

NMR spectra revealed that TA2 exists in aqueous solution at physiological pH in a random coil conformation or is unstructured. NMR analysis the C-terminal 123 amino acids showed a random coil conformation, which indicates that the entire transactivating C-terminus is unstructured. C-terminal deletion of the TA2 domain causes a gradual loss in transactivating activity. A region within TA2 homologous to TA1 is referred to as TA'1. TA'1 is necessary for the activity of TA2 because any deletion affecting the integrity of TA'1 leads to a decrease in the ability of TA2 to stimulate transcription. TA2 activity might also be dependent upon a leucine zipper, but this needs to be studied further.

TQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHT
TEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQ
ISS

1. Nuclear magnetic resonance (NMR) (pH: 5.5; 10% D₂O; 90% H₂O; NaCl 50 mM; sodium phosphate 50 mM)

1. Schmitz ML, dos Santos Silva MA, Altmann H, Czisch M, Holak TA, Baeuerle PA. "Structural and functional analysis of the NF-kappa B p65 C terminus. An acidic and modular transactivation domain with the potential to adopt an alpha-helical conformation." J Biol Chem. 1994; 269(41): 25613-25620. PubMed: 7929265
   
   

NMR analysis of a protein corresponding to the C-terminal 123 amino acids showed a random coil conformation, which indicates that the entire transactivating C-terminus is unstructured.

1. Ruben SM, Dillon PJ, Schreck R, Henkel T, Chen CH, Maher M, Baeuerle PA, Rosen CA. "Isolation of a rel-related human cDNA that potentially encodes the 65-kD subunit of NF-kappa B." Science. 1991; 251(5000): 1490-1493. PubMed: 2006423