Chemistry Reference
In-Depth Information
Fig. 1 Three representative cases of wPPIs. Case I: a weak protein-protein interaction found in
a locally highly crowded manner. Case II: a weak domain-domain interaction, exemplified by
A-B pair, as part of a tight multi-domain complex. Such weak binary domain-domain interaction
may be undetectable by many conventional methods including deletion mapping, yeast-hybrid
approach, immunoprecipitation, etc., but become apparent when the tertiary structure of the tight
complex is challengingly determined. However, NMR may be able to pick this interaction at early
stage of the characterization. Case III: a weak protein-protein interaction as a part of multi-protein
complex. Similar to (
II
), a weak A-D pair may not be detectable in isolated manner by any
conventional techniques except NMR
and at near physiological conditions [
7
,
8
,
10
,
11
]. In this chapter we present the various
NMR approaches to assess and characterize these three types of wPPIs structurally.
1.1 Chemical Shift Perturbation Mapping
The resonance frequencies, also known as chemical shifts, of individual atoms in
a particular protein strongly depend on the local environment and, because of that,
are often considered as fingerprints of its structure. The chemical shift patterns of
15
N and directly attached amide
1
H are especially sensitive in this respect. Thus
their perturbation, as the result of complex formation, provides a highly sensitive
tool for mapping the binding interface. Binding surface on the target protein is
identified by titrating the unlabeled target into the solution of the
15
N-labeled
protein and monitoring the resultant spectral changes in its
1
H-
15
N HSQC
(heteronuclear single quantum correlation) spectrum or, for the larger proteins, its
TROSY-based (transverse relaxation-optimized spectroscopy) version [
12
]. The
utility and popularity of this experiment are based upon its straightforward nature
and high sensitivity - the spectrum can be recorded in 5-40 min on a typical protein
sample (~0.1-1 mM). The spectral changes, also denoted as chemical shift
perturbations (CSP), are usually associated with a particular set of amino acids
that either (1) directly participate in interaction or are situated very closely to the
binding site or (2) reflect binding-induced conformational rearrangements (e.g.,
a disorder-order transition). The former happens more frequently for wPPIs,
which have characteristic small CSP with little or no conformational change, at
least within the backbone of well folded domains. Hence the binding interface can
