  Hyperpolarized water can be a valuable aid in protein NMR, leading to amide
group 1H polarizations that are orders of magnitude larger than their thermal
counterparts. Suitable procedures can exploit this to deliver 2D 1H-15N
correlations with good resolution and enhanced sensitivity. These enhancements
depend on the exchange rates between the amides and the water, thereby yielding
diagnostic information about solvent accessibility. This study applied this
HyperW method to four proteins exhibiting a gamut of exchange behaviors: PhoA4,
an unfolded 122-residue fragment; barstar, a fully folded ribonuclease
inhibitor; R17, a 13.3 kDa system possessing folded and unfolded forms under
slow interconversion; and drkN SH3, a protein domain whose folded and unfolded
forms interchange rapidly and with temperature-dependent population ratios. For
PhoA4 HyperW sensitivity enhancements were larger than 300-fold, as expected
for an unfolded protein sequence. Though fully folded barstar also exhibited
substantial enhancements; these, however, were not uniform, and according to
CLEANEX experiments reflected the solvent-exposed residues. R17 showed the
expected superposition of 100-fold enhancements for its unfolded form,
coexisting with more modest folded counterparts. Unexpected, however, was the
behavior of drkN SH3, for which HyperW enhanced the unfolded but even more
certain folded protein sites. These preferential enhancements were repeatedly
and reproducibly observed; a number of explanations including three-site
exchange magnetization transfers between water, unfolded and folded states;
cross-correlated relaxation processes from hyperpolarized structural waters and
labile sidechain protons; and the possibility that faster solvent exchange
rates characterize certain folded sites over their unfolded counterparts are
considered to account for them.
