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In Depth Tutorials and Information
problem, and no simple correlation was seen between
the presence of a kink and a processing defect.
16,17
An obstacle to elucidating the precise nature of the
structural perturbation at a mutation site is that long rod-
like collagen molecules are not amenable to high resolu-
tion structural approaches, such as X-ray crystallography
or multidimensional NMR studies. In general, spectro-
scopic techniques are not sensitive enough to detect very
small local structural perturbations, but the Leikin labo-
ratory has shown the usefulness of enzyme susceptibil-
ity as a probe.
19
Similar to normal collagens, OI collagens
are resistant to trypsin and other enzymes at low tem-
peratures, suggesting there is no substantial looping out
or unfolded region. However, increased non-specific pro-
teolytic degradation at 37°C was reported for collagen
molecules which contain one mutant chain (Gly→Cys),
obtained from the Brtl mouse model.
19
Interestingly,
Forlino et al.
19
found the disulfide bonded molecules
with two Gly→Cys mutant chains showed resistance to
proteolysis similar to wild-type molecules, suggesting
that trimers with two mutant chains may be less dis-
torted than trimers with one mutant chain. It would be
useful to confirm whether a similar pattern is seen when
Gly is replaced by residues other than Cys.
Studies on collagens obtained from OI fibroblasts
have been complemented by investigations of model
peptides and by computational approaches. Peptides
of defined amino acid sequence can form a stable
triple helix if sufficient Gly-Pro-Hyp or Gly-Pro-Pro
triplets are included, and such triple-helical peptides
have proved amenable to high-resolution structural
techniques.
20
One limitation of peptide models is their
homotrimeric nature, with a mutation in all three
chains, in contrast with the heterotrimers containing
one or two mutant chains found in OI collagens. Recent
approaches by Hartgerink's laboratory have indicated
the feasibility of creating heterotrimer peptides with a
mutation in one or two chains,
21
and computational
studies with mutations in one or two chains have been
reported.
22-24
High-resolution structures have been determined
for homotrimeric peptides where one Gly has been
replaced by a larger residue, and the results can guide
thinking about the effect of mutations on collagen struc-
ture. The first insight came from the high-resolution
(1.9 Å) crystal structure for a peptide in which a single
Gly→Ala mutation is introduced into a (Pro-Hyp-Gly)
10
sequence: (Pro-Hyp-Gly)
4
-Pro-Hyp-Ala-(Pro-Hyp-Gly)
5
(PDB ID 1CAG).
11
The helix was surprisingly straight,
with no “looping out” features, and the perturbations
were highly localized. The regular pattern of direct
interchain Gly NH…CO (Xaa) hydrogen bonds was
interrupted at the mutation site, where direct hydro-
gen bonds were replaced by water-mediated hydrogen
bonds involving the Ala NH (
Figure 11.3C
). Several
φ,ψ angles near the mutation site fell outside the stan-
dard collagen range in the Ramachandran plot. But,
as predicted by Traub and Steinmann,
15
the structure
regained a standard triple helix within one triplet away
from the mutation site. One long range effect was seen:
the perturbation at the mutation site resulted in a local
untwisting of the triple helix, which put the superhelix
at the N-terminal end of the chain out of phase with the
C-terminal end (
Figure 11.3C
).
11
NMR studies on a peptide with a Gly→Ser mutation
indicated an asymmetric disruption of the backbone
hydrogen bonding, with more perturbation C-terminal
to the mutation site.
25
This is consistent with recent
suggestions relating increased clinical severity with
the presence of destabilizing residues C-terminal vs.
N-terminal to a mutation site.
26
The NMR studies sup-
port the potential of hydrogen bonding of the Ser side
chain when it is in a Gly-position, a concept raised in
earlier computational studies.
27
A conformational perturbation of the triple helix
could contribute to OI pathology at a number of levels.
As discussed in other chapters, it could trigger quality
control problems in the endoplasmic reticulum, leading
to degradation,
28
or interfere with binding at the fibril-
lar level to other matrix molecules or receptors.
10
If the
loss of axial registration seen for the Gly→Ala peptide
structure is present in OI collagen, this could disrupt
the packing of adjacent type I collagen molecules into
D periodic fibrils. The increased hydroxylation and gly-
cosylation of Lys in OI collagens, which results from
delayed folding (see “Gly Missense Mutations and
Folding of OI Collagens,” below), could affect collagen
fibril formation and crosslinking, features that would
impact collagen function.
COLLAGEN TRIPLE-HELIX STABILITY
AN
D GLY MISSENSE MUTATIO
NS
Protein destabilization has been suggested as an
important property of the pathogenic mechanism by
which missense mutations lead to monogenic dis-
eases.
29
The fine tuning of collagen thermal stability
is known to be essential for its biosynthesis, fibril for-
mation, and overall function,
30-32
so the effect of OI
mutations on stability has been an important topic of
investigation. Stability was first assessed for OI col-
lagens expressed in fibroblasts by measuring their
enzyme susceptibility at different temperatures using
a trypsin/chymotrypsin assay.
33
A considerable num-
ber of mutations led to a small decrease in melting tem-
perature (
T
m
), while several showed surprisingly large
decreases and some led to no change at all.
34,35
A more
precise definition of changes in thermal stability was
carried out in the Leikin laboratory using differential
