S 1 Supporting Online Material Structure and flexibility adaptation between nonspecific and specific protein- DNA complexes Charalampos G. Kalodimos, Nikolaos Biris, Alexandre M.J.J. Bonvin, Marc M. Levandoski, Marc Guennuegues, Rolf Boelens, Robert Kaptein Materials and methods 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 13 C/ 15 N- and 15 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% H 2 O / 5% D 2 O. Trace amounts of NaN 3 were added as a preservative. Text S2: NMR assignment. All NMR spectra were recorded at 300 K for the free protein and the nonspecific complex and at 300 and 315 K for the specific complexes on Bruker DRX750 and DRX600 spectrometers equipped with triple resonance gradient probes. Sequential assignment of the 1 H, 13 C, and 15 N protein chemical shifts was achieved by means of through- bond heteronuclear scalar correlations along the backbone and the side chains using conventional 3D pulse sequences (2). DNA assignments were obtained from 2D NOE and simultaneous 13 C- 15 N double-half filter NOE experiments recorded on a sample containing a 1:1 complex of 13 C- 15 N-dimeric HP62-V52C protein and unlabeled operator, using conventional sequential assignment methodology for nucleic acids (3). Interproton distance restraints within the protein were derived from 3D 13 C- and 15 N-separated NOE experiments. Protein-DNA interactions were assigned from 2D simultaneous 13 C- 15 N double-half filter, 2D-NOE and 3D-NOESY-HSQC