Oligomerization, Function and stability.
Mature BDUN1 has 190 amino acids and by SDS PAGE has an apparent molecular weight of 20,000 Da. Using ultracentrifugation, native BDUN1 by has an apparent molecular weight of 40,000, but in the presence of 12 bp DNA forms a complex of MW~48,000. (Hint see cover page for important information-how much does the DNA weigh?). When it is phosphorylated on Ser16, the apparent molecular mass is 20,000. Free DNA is used in excess and is observed in both phosphorylated and un-phosphorylated experiments.
1) BRIEFLY (1-3 sentences) Describe and rationalize the oligomerization behavior of BDUN1, with and without DNA, and the effect of phosphorylation
Give two reasons why it is advantageous for BDUN1 bind DNA this way (from (1)) (IGNORE THE EFFECT OF PHOSPHORYLATION
2) Compared to free BDUN1, what happens to protein stability upon BDUN1 binding DNA? (increases/decreases)
3) The crystal structure of BDUN-1 with a 12 bp DNA site GAAGTGCACTCC. shows no direct protein-DNA contacts with the central GpC base-step (underlined). Changing this to an TpA basestep, has little or no effect on DNA affinity. Circle the DNA structure below that most likely appears in the BDUN1/DNA crystal structure.