At. Alexandrescu et al., STRUCTURE AND DYNAMICS OF A DENATURED 131-RESIDUE FRAGMENT OF STAPHYLOCOCCAL NUCLEASE - A HETERONUCLEAR NMR-STUDY, Biochemistry, 33(5), 1994, pp. 1063-1072
A partially folded form of staphylococcal nuclease has been obtained b
y deleting residues 4-12 and 141-149 of the 149-residue wild-type prot
ein. Sequence-specific NMR resonance assignments have been obtained fo
r 106 of the 131 residues in this protein fragment by using multi-dime
nsional triple resonance NMR of samples enriched with C-13 and N-15. R
esidues corresponding to helix 2 (residues 98-106) and helix 1 (residu
es 54-68) of the native state give chemical shifts and NOE effects cha
racteristic of helical structure. These same residues, however, give c
oupling constants and NOE effects indicative of fast conformational av
eraging between helical and extended conformations. The residual helix
structure observed in the nuclease fragment is thus considerably less
persistent than the corresponding structure in the native state. Base
d on H alpha chemical shifts, we estimate the fractional population of
helical conformers to be 30% for helix 2 and 10% for helix 1. Two seg
ments, 83-86 and 94-97, show NOE effects, coupling constants, and lowe
red amide temperature coefficients consistent with a native-like rever
se-turn structure. The C-terminal alpha-helix as well as the fourth an
d fifth strands of the 5-strand beta-barrel show little evidence for o
rdered structure. The first three strands of the beta-sheet, part of t
he catalytic loop, and the first turn of helix 3 give significantly po
orer NMR data than the rest of the protein, possibly as a result of ex
change broadening, and could not be characterized in detail. That the
most persistent elements of structure in the fragment are native-like
suggests that nuclease may fold by a hierarchical mechanism.