Biomedical Engineering Reference
In-Depth Information
5.3.3 Binding Without Folding
Although IDPs were originally detected experimentally by observing an
increase in ordered structure upon interaction with a partner, the disordered
domain does not necessarily end up in what we would call a 'folded' state.
When a protein remains conformationally heterogeneous during interaction
with a partner, the interaction has been termed 'fuzzy'
62
or as undergoing
'cloud contacts'.
63
Multiple weak interactions may occur because the IDP uses
multiple binding sites, while contacting multiple sites of the partner.
5.4 Partly Folded and Molten Globule-Like States
Biophysical data (NMR, CD, fluorescence) are available for both ordered and
disordered states, but data on partly folded states are much harder to obtain
and more difficult to interpret. The 'molten globule' state was originally
proposed as a specific intermediate with a native-like fold but without native
tertiary structure,
64,65
and was originally invoked to explain intermediate states
that could be identified during the folding of globular proteins. Such
intermediate states actually consist of a number of conformational statesin
equilibrium; this is commonly taken to be true of molten globule states in
general. Under normal conditions, the exchange between members of a molten
globule conformational ensemble frequently occurs on the tricky timescale
termed 'intermediate exchange', neither fast nor slow compared to the NMR
chemical shift timescale, resulting in extensive broadening and disappearance
of resonances. Methods of addressing these difficulties have recently been
introduced,
13,14
but NMR examination of these systems remains extremely
difficult. Other techniques such as fluorescence and CD spectroscopy, small-
angle X-ray scattering and H/D exchange are frequently employed to give
structural information.
5.4.1 Ankyrin Repeat Proteins
The ankyrin repeat (AR) is a 33-residue structured motif with a b-turn/loop-
helix-loop-helix fold. Multiple repeats have been observed in a large number
of proteins, primarily as a protein recognition motif. There is a wide variety of
AR amino acid sequences within a general homology that includes a consensus
PLHLA sequence at the N-terminal end of the first short helix. This sequence
variety manifests itself as a wide repertoire of structured and partly structured
forms. The Notch AR domain is apparently well folded,
66
but the p19
INK4d
tumor suppressor undergoes a general loosening upon phosphorylation, which
may be a prelude to ubiquitination and degradation.
67,68
The ankyrin repeats
of IkBa have very different stabilities in the free state, with a partly folded
region (AR 5 and 6) near the C-terminus of the free protein.
59,69
This
conformational instability appears to be required for some functions of IkBa:
if the sequence of AR5 and 6 is mutated to the consensus sequence, the partly
