Abiotic Stress Adaptation: Protein Folding Stability and Dynamics - Abiotic Stress: Plant Responses and Applications in Agriculture - page 13

Figure 7. Comparison of N-H order parameters (S 2 ) of cold shock protein A (CspA) (red). Addition of 0.4M myo -Inositol
(black) showed an overall increase in protein compactness by rigidification of former flexible parts of the protein (S 2 =0
flexible, S 2 =1 rigid)
Order parameter (S 2 ) for cold shock protein A (CspA) showed an overall increase in the
presence of the model osmolyte myo -Inositol. Residues in very flexible parts of the protein
that have low motional restrictions tend to become more rigid and motional restricted upon
the addition of the myo -Inositol. The overall protein compactness increases in the presence
of the osmolyte, most profoundly observed in protein regions with high locally structural
fluctuations.
(CPMG)-type NMR relaxation dispersion experiments
NMR relaxation dispersion methods have been introduced enabling studies on protein fold‐
ing under native conditions without the need for disturbing the equilibrium. Studying pro‐
tein folding and unfolding requires a thoroughly view of all states including the native state,
folding intermediates and the unfolded state [12] as it is increasingly recognized that even
small proteins fold via intermediates. Because these intermediates are low populated and
short-lived (in the order of ms), their structural characterization has been a difficult task. In
NMR relaxation dispersion experiments conformational exchange between a native ground
state and low populated partially folded states can be characterized even if states are not
visible in NMR spectra [50].
CPMG (Carr-Purcell-Meiboom-Gill)-type NMR relaxation dispersion techniques have been
employed to investigate the site-specific conformational exchange processes of proteins on a
microsecond-to-millisecond time scale that is highly sensitive to solvent and co-solvent con‐
ditions. These experiments are particular useful for simple two state exchange processes,
providing information about the kinetics of the exchange process, the relative populations
and structural features of invisible states in terms of NMR chemical shifts [51, 52]. Residues
that undergo conformational exchange on the μs-ms time scale contribute to the effective
transverse 15 N relaxation rates (R 2.eff ). By measuring the increased contribution, R ex , to the ef‐
fective transverse relaxation rate as a function of CPMG pulse spacing relaxation, typical
non-flat dispersion profiles are obtained (Figure 8).