118 INSTRUMENTAL METHODS
Horse heart cytochrome c solutions were prepared at concentrations of
∼ 4 – 6 mM in 30% CD 3 CN (deuterated acetonitrile) and 70% D 2 O or H 2 O solu-
tions containing 50 mM phosphate buffer (pH 6.9). The external standard (^1 H,
0.00 ppm) used was 2,2 - dimethyl - 2 - silapentane - 5 - sulfonate sodium salt (DSS),
a commonly used standard in biological and pharmacological NMR experi-
ments. Data were collected on one of two 600 - MHz NMR instruments, and
then they were exported and processed in Felix 98, offl ine data processing,
visualization, and analysis software from Molecular Simulations (now Accel-
rys; see http://www.accelrys.com/ ). The ferrocytochrome c NOESY spectra
were recorded with mixing times of 100 ms with 64 scans per FID using a
spectral width of 20 ppm. A total of 2048 points were collected in the F2 dimen-
sion and 256 points in theF1 dimension. TOCSY spectra were recorded with
mixing times of 20, 50, and 75 ms with the same acquisition parameters as the
NOESY spectra. TOCSY, total correlation spectroscopy, is a COSY - like
method that shows different^1 H connectivities at different mix times, providing
more detailed structural information and thus reducing the number of possible
structures. DQF - COSY spectra were recorded with 128 scans per FID with
2048 × 256 ( F2 × F1 ) points acquired. DQF - COSY, double quantum fi ltered
COSY, uses a pulsed - fi eld gradient to assist in the separations of overlapping
spectral peaks and thus to reduce the number of possible structures. In this
case, DQF - COSY produced^3 J Hα HN coupling constants (coupling constants
between the proton attached to the aaα - carbon atom and the proton attached
to the peptide main chain amide nitrogen) for 73 of the ferrocytochrome c ’ s
104 amino acid residues.
The next calculations establish distance restraints using cross - peak volumes
generated from the 100 - ms 2D NOESY spectra. The fi rst step involves
manual assignment of cross peaks due to intra - residue and sequential proton –
proton interaction ( ∼ 500 NOESY peaks assigned). The second step is an
automated NOESY cross - peak assignment using the NOAH module in the
DYANA structure calculation program. The DYANA software program
suite for structural analysis of NMR data results from the work of Kurt W ü t-
hrich and fellow researchers.^25 Professor W ü thrich shared the 2002 Nobel Prize
in Chemistry “ for his development of nuclear magnetic resonance spectros-
copy for determining the three - dimensional structure of biological macromol-
ecules in solution. ” Researchers Sivakolundu and Mabrouk performed 30
cycles until no further changes in the resonance assignments of cross peaks
were observed in NOAH. This process resulted in assignment of 2232 NOESY
cross peaks.
Next, the researchers performed structural calculations using the DYANA
program suite. First, the ferrocytochrome c heme was constructed and desig-
nated as HES, using the heme and his18 coordinates from the cytochrome c
solution structure deposited as PDB: 1GIW.^26 The HES residue is added so
that the heme moiety and his18 are free to move independently during sub-
sequent structural calculations. Because DYANA does not recognize the iron
atom, it was represented by a dummy atom. The NOESY cross - peak volumes