The proton resonances of the actin peptide (5 µl final volume containing 1 µl of each stock solution peptide at 1 mg/ml) were assigned using standard 2-D NMR procedures for spin system identification (COSY, 1H-1H RELandand TOCSY) and sequential consecutive determination (NOESY and ROESY).
The nonexchangeable DNA protons were assigned using standard protocols.
The resonances of the nonexchangeable protons were assigned using standard approaches.
The NMR resonances for the oligodeoxynucleotide duplex containing the Tg·G mismatch pair were assigned using standard strategies (43, 44).
The chemical shifts of each of the peptides in this study were assigned using standard TOCSY and NOESY H-based strategies.
To examine this more directly, a N,C labeled TGF-β3H4 sample was prepared, and its backbone resonances were assigned using standard triple-resonance methodology.
NMR resonances were assigned using standard TROSY-based 3D triple-resonance and NOESY-TROSY amide proton amide proton experiments, in combination with residue-specific labeling and site-directed mutagenesis (see Figure 3); 89% of backbone assignments were completed for Q1-VSD.
Affinity scores were assigned using standard log-likelihood ratios [ 40] and a binding site defined as an oligonucleotide with log-likelihood ratio higher than 66% of the maximum score possibly associated to the PWM.
The thermal melting of the 5′-XA-3′, 5′-XC-3′, 5′-XT-3′, and 5′-XY-3′ duplexes was also examined by monitoring NMR spectra of the imino protons as a function of temperature; the imino proton resonances were assigned using standard methods.
For structure determination, N-edited Nuclear Overhauser Enhancement Spectroscopy (NOESY) and C-edited NOESY spectra with 150 ms mixing times were recorded on a Brucker AVANCE 900 spectrometer at 296 K. Sequence-specific backbone chemical shifts were assigned using standard triple-resonance experiments (59).
