NMR experiments were performed on Bruker spectrometers.

1H/15N HSQC spectrum

were acquired at 283K or 298K on a Bruker DRX500 spectrometer using a conventional
1H/13C/15N triple resonance probe (Bruker). All data were processed using NMRPipe
(Delaglio et al. (1995)) and analysed using NMRView (Johnson (2004)). Figures were
generated using Sparky (Goddard & Kneller).

The essential new ingredient in two-dimensional spectroscopy is a second time variable t1

called the evolution time. A two dimensional experiment has the form

Preparation → evolution during t1 → mixing → detection during t2

and consists of the repetition of a basic pulse sequence n times, t1 being incremented each
time. A fid is acquired for each new value of t1, so at the end we will have a two dimensional
data array S(t1,t2), which after two stages of Fourier transformation yields a two-dimensional
spectrum. An important idea is that of correlation.

The standard protocol consist of running a series of 2D-experiments, COSY, TOCSY

(correlations are obtained for one given proton, all other protons scalarly coupled to it, and all
protons coupled to the latter), and NOESY. The first two experiments are used to assign the
resonances of the different residues in the protein, what additionally provides an indication
about the type of residue (Fig. 7). The latter experiment is used in a first step to establish
connections between adjacent residues. By deriving string of residues of known type and by
comparing those strings with the known sequence, one can assign resonances to particular
segments of the protein. This stage is known as resonance assignment, and constitutes the
most intricate and painful part of the whole process.

Once all resonances have been duly assigned, a further and thorough analysis of the NOESY
spectra provides a set of internuclear distance constraints derived from NOE intensities
(normally ~1500 interproton distance constraints for a protein of 100 residues). Usually, a set
of torsion angle restraints derived from vicinal coupling values is also introduced as an
additional input. A computing algorithm is then used to calculate the structure or structures
compatible with those constraints. The starting structure consists of the protein amino acid
sequence in a randomly generated conformation, in which the bond angles and distances are
assumed to be unchanged along the computational process. The computing algorithm is based
on distance geometry methods combined with restrained molecular dynamics.

Crucial to the solution of the structure was the use of perdeuteration of all of the side chains
with reverse-labeling techniques of selective reintroduction of protonated side chains (11).
Perdeuterated samples provide high-quality spectra but lose the structural information of
the side chains. Therefore, we reintroduced proton nuclei in several side chains including
those of Ile, Val and Leu (12), methyl groups, aromatic Tyr and Phe (13), and Met and Arg
residues. The gain in sensitivity afforded high-quality 2D (Fig. 1) and 3D data (Fig. 2) that
were combined with the analysis of partly deuterated samples to get many restraints (Table
1). The spectra indicated some conformational heterogeneity by the absence of several
amide signals from the 1H-15N-heteronuclear single quantum correlation (HSQC) spectrum.
Line broadening was still detectable in perdeuterated samples around Lys-120, whose signal
was missing from the spectra, and for Met-246, which appeared to occupy two
conformations.

The core domain of p53 aggregates at temperatures of ≈37°C (21). The transverse relaxation times of the amide
protons indicated the presence of species of high M r in fast exchange with the monomers at concentrations >400
μM (pH 7.2; 150 mM ionic strength). Excellent spectra were obtained at 20°C by using 200–400 μM samples.
Triple resonance experiments were performed on a triple-labeled (13C,15N,2H) sample to confirm and complete
our previous backbone assignments of p53 core domain (K. B. Wong, S.M., and V.F., unpublished data). HNCA,
HNCO, HN(CO)CA, CBCA(CO)HN, HN(CO)CC (8 and 12 ms for carbon spin lock), and HNCACB
experiments were modified from the standard (22) to include deuterium decoupling. Side-chain assignments
were completed with various HCCH-COSY in double (13C,15N) or triple (13C,15N,2H 50%) labeled samples.
Distance constraints were obtained from a collection of NOESY spectra with mixing times ranging from 100 ms
(protonated samples) to 200 ms (deuterated and reverse-labeled samples). The following series was used in the
order (sample number, labeling regime, and spectra): 1, unlabeled, 2D NOESY water, and D2O (80 ms); 2, 2H
(50%), 13C, 15N, 2D NOESY and various 3D NOESY-HSQC; 3, 2H (100%), 13C, 15N, 2D NOESY (100–600 ms
mixing), 3D NOESY-HSQC; 4, as 3 plus reverse 13CH3 Ile,Leu,Val, 2D NOESY, 3D NOESY-13C-HSQC,
3D 13C-HSQC-NOESY-13C-HSQC; 5, as 4 plus reverse 12C/1H Tyr, 2D NOESY, 3D NOESY-13C-HSQC; 6, as 3
plus reverse 12C/1H Phe, Met, 2D NOESY (200 ms); and 7, as 3 plus reverse 12C/1H Phe, Arg, 2D NOESY (200
ms). For the perdeuterated sample, several 2D NOESY with 100, 200, 400, and 600 ms were recorded to check
the absence of spin-diffusion. In general, all of the NOESY-like experiments were recorded for samples between
300 and 400 μM in the optimal buffer conditions (25 mM d11-Tris·HCl, pH 7.1/150 mM NaCl/10 mM d10-DTT)
at 298 K in 600- (with cryoprobe) and 800-MHz spectrometers. For the Tyr reverse-labeled sample, an
additional set of 2D NOESY experiments was recorded at various temperatures to analyze the changes in
chemical exchange regime of the ring resonances as a function of temperature.