Bn. Green et al., A HIERARCHY OF DISULFIDE-BONDED SUBUNITS - THE QUATERNARY STRUCTURE OF EUDISTYLIA CHLOROCRUORIN, Biochemistry, 37(18), 1998, pp. 6598-6605
The quaternary structure of the cysteine-rich, similar to 3500-kDa chl
orocruorin (Chl) from the marine polychaete Eudistylia vancouverii was
investigated using maximum entropy deconvolution of the electrospray
ionization mass spectra (ESIMS). The native Chl provided two groups of
peaks, at similar to 25 and similar to 33 kDa, and one peak at simila
r to 66 kDa. ESIMS of the reduced and reduced and carbamidomethylated
Chl and of its subunits obtained by HPLC provided the complete subunit
composition of the Chl. Two groups of nonglobin linker chains were ob
served: L1a-f(25 000.4, 25 017.9, 25 039.6, 25 057.0, 25 074.4 and 25
096.8 Da) and L2a-d (25 402.7, 25 446.0, 25 461.6 and 25 478.3 Da) (+/
- 2.5 Da), with relative intensities L1:L2 = 5:2. Six globin chains we
re found, a1, a2, and b1-4, with reduced masses of 16 051.5, 16 172.4,
16 853.5, 17 088.9, 17 161.2 and 17 103.6 (+/- 1.0 Da) and relative i
ntensities of 8:4:1:4:2: 1, respectively. Disulfide-bonded dimers and
a tetramer of globin chains were identified: D1 = a1 + b3 at 33 207.1;
D2 at 33 374.1, which had a cysteinylated Cys (a2 +/- b2 + Cys); and
D3 = a1 + b4 at 33 149.4 Da (+/- 3.0 Da), with relative intensities D1
:D2:D3 = 5:4:1 and T = a1 + a2 + b1 + b2 at 66 154.8 +/- 4.0 Da. A 206
-kDa dodecamer subunit obtained by dissociation of the Chl in 4 M urea
[Qabar, A. N., et al. (1991) J. Mol. Biol. 222, 1109-1129], was found
to consist only of tetramers T. A model was proposed for the Chl, bas
ed on a dimer:tetramer ratio of 2.1. four 206-kDa dodecamers (trimer o
f tetramers) and 48 dimers tethered to a framework of 30 L1 and 12 L2
linker chains. The 144 globin chains (2480 kDa) and 42 linker chains (
1059 kDa) provide a total mass of 3539 kDa, in good agreement with the
3480 +/- 225 kDa determined previously by STEM mass mapping. The hier
archy of disulfide-bonded globin subunits observed for Eudistylia Chl
provides a built-in heterogeneity of hexagonal bilayer structures.