NMR as a Unique Tool in Assessment and Complex Determination of Weak Protein–Protein Interactions - NMR of Proteins and Small Biomolecules - page 38

be qualitatively deduced from the spectrally perturbed residues. In addition, for
wPPIs with fast exchange, characterized by high k off rates, K d can be estimated
from concentration-dependent titrations [ 13 ] . However, the potential problem
associated with this analysis relates to the low affinity of the complex: an interfacial
residue might not necessarily undergo a significant CSP upon binding, meaning that
interfaces derived from CSP data alone are not always complete [ 6 ]. One way to
avoid this caveat is to increase the ratio of the unlabeled titrant, which can drive the
equilibrium towards the bound form with bigger CSP. However, the ratio cannot be
too high since it may cause some non-specific effects. The weakest PPI analyzed by
CSP demonstrated a K d of 10 2 M for the flavodoxin/flavodoxin reductase complex
[ 14 ]. Both (1) and (2) might occur for strong PPIs, which often undergo significant
conformational rearrangements upon binding, especially if a disorder-order transi-
tion occurs. In such cases, analysis of binding interfaces is more challenging and less
straightforward, and one has to rely on additional techniques, such as incorporation of
a cross-saturation [ 15 ] or inter-molecular Nuclear Overhauser Effects/Enhancements
(NOEs) (described in detail in the next section). To conclude, although for wPPIs the
CSP method provides fast and robust assessment of the residues forming an intermo-
lecular interface, the mutual orientation of the two partners remains elusive. Thus, if
the goal is to generate the structural model of the complex, additional experiments
have to be performed and/or novel computational approaches have to be employed.

1.2 Nuclear Overhauser Effect

NOE, a relaxation mechanism based upon magnetic dipole-dipole interactions of
the nuclei, allows measurement of interproton distances with the basic r 6 distance
proportionality. This provides major distance restraints for structural calculations.
Supplemented with additional data, such as original dihedral angle restraints
obtained from J-couplings or more recent information about the orientation of the
bond vectors connecting magnetically active nuclei with respect to the external
magnetic field, this approach has been the foundation for NMR-based protein
structure determination since its dawn in 1984 [ 11 ].

1.2.1 NOE in the Determination of the Structure of wPPIs

From the perspective of wPPIs structural characterization, two particular applications
of NOE are proven most fruitful.

Transferred NOE Experiments

Transferred NOE Experiments (trNOESY) is a quick two-dimensional 1 HNMR
experiment that allows the observation of intramolecular proton contacts ( 5 ˚ )

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