Structure and flexibility adaptation between nonspecific and specific protein- DNA complexes

Text S1: Expression and preparation of the dimeric lac HP62-V52C protein and its complexes with the nonspecific DNA fragment (NOD) and its natural operators O1and O3. The HP62-V52C mutant was amplified from the corresponding Lac I genes by the polymerase chain reaction and expressed in E. coli using T7 polymerase based system. The protein was purified as described earlier (1). Uniformly C/Nand N-labeled proteins were grown in BIOEXPRESS-CN 5000 (CIL) media. The oligonucleotides were purchased at Carl Roth GmbH (Germany) and further purified on a Q-Sepharose (Pharmacia) column. For complex formation the protein was mixed with an equimolar amount of the operator and dissolved in 0.06 M KPi buffer (pH 6.0) containing 0.4 M KCl for the nonspecific complex and 0.01 KPi and 0.02 KCl for the specific ones. All samples were concentrated using Centricon concentrators (Amicon) and dissolved in 95% H2O / 5% D2O. Trace amounts of NaN3 were added as a preservative.