Probing the Residual Structure of the Low Populated
Denatured State of ADA2h under Folding Conditions by Relaxation Dispersion
Nuclear Magnetic Resonance Spectroscopy
- Publication date
- Publisher
Abstract
The structural characterization of
low populated states of proteins
with accuracy comparable to that achievable for native states is important
for understanding the mechanisms of protein folding and function,
as well as misfolding and aggregation. Because of the transient nature
of these low populated states, they are seldom detected directly under
conditions that favor folding. The activation domain of human procarboxypeptidase
A2 (ADA2h) is an α/β-protein that forms amyloid fibrils
at low pH, presumably initiated from a denatured state with a considerable
amount of residual structure. Here we used Carr–Parcell–Meiboom–Gill
relaxation dispersion (CPMG RD) nuclear magnetic resonance (NMR) spectroscopy
to characterize the structure of the denatured state of the ADA2h
I71V mutant under conditions that favor folding. Under these conditions,
the lifetime of the denatured state of I71V ADA2h is on the order
of milliseconds and its population is approximately several percent,
which makes this mutant amenable to studies by CPMG RD methods. The
nearly complete set of CPMG RD-derived backbone <sup>15</sup>N, <sup>13</sup>C, and <sup>1</sup>H NMR chemical shifts in the I71V ADA2h
denatured state reveals that it retains a significant fraction (up
to 50–60%) of nativelike α-helical structure, while the
regions encompassing native β-strands are structured to a much
lesser extent. The nativelike α-helical structure of the denatured
state can bring together hydrophobic residues on the same sides of
α-helices, making them available for intra- or intermolecular
interactions. CPMG RD data analysis thus allowed a detailed structural
characterization of the ADA2h denatured state under folding conditions
not previously achieved for this protein