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TABLE 11.1
destabilization of Triple-Helix Δ
T
m
Caused by Gly Substitutions in Various Collagens and Synthetic Peptide Models, with
Triple-Helix domains of different lengths
Sample
T
m
Δ
T
m
Method
Reference
Collagen OI fibroblasts
(Gly-Xaa-Yaa)
338
42°C
0-4.6°C
DSC
36
Collagen OI fibroblasts
(Gly-Xaa-Yaa)
338
-
0.5-20°C
Enzyme digestion
34
Recombinant type I fragment
(Gly-Xaa-Yaa)
31
40°C
G901S: 10°C
G913S biphasic: ~10°C and ~20°C
CD
37
Recombinant bacterial collagen
(Gly-Xaa-Yaa)
79
37°C
1.5-2°C
CD
39
Homotrimer peptides
(Gly-Pro-Hyp)
8
(Gly-Pro-Hyp)
10
44.5°C
60°C
35°C
30-35°C for G→A,S >45°C for
G→larger residues
CD
6
40
7
Heterotrimer
synthetic peptides
(Gly-Xaa-Yaa)
13
A·A·B 60°C
A·A·B 44.5°C
A A B 42.5°C
A A B 36.5°C
1st chain: 15.5°C
2nd chain: 17.5°C
3rd chain: 23.5°C
CD
21
to the mutation site,
26
and such asymmetric disrup-
tion of hydrogen bonds was experimentally observed
in NMR studies of a peptide with a Gly→Ser mutation
(
Figure 11.2
).
25
It is still not clear whether the presence of one
mutant chain per triple helix is more or less destabiliz-
ing than having two or even three mutant chains in the
triple helix. As mentioned in “Effect of Gly Missense
Mutations on the Collagen Triple-helix Structure,”
above, OI molecules with two chains having Gly→Cys
mutations are more enzyme resistant than molecules
with one mutant chain,
19
but it is possible that this is
related to the Cys residue and its formation of disulfide
bonds. For many OI collagens, which are mixtures of
different molecular species, DSC thermograms showed
a control peak together with an additional peak of
decreased stability, suggesting that the molecular spe-
cies with one and two mutant chains may overlap. For
a small number of OI collagens, two distinct DSC peaks
of decreased stability were seen, likely reflecting a dif-
ference in stability between the molecules containing
one and two mutant chains.
36
The homotrimer bacte-
rial collagen proteins with three mutant chains show
T
m
decreases in the same range as OI heterotrimers,
so the number of mutant chains does not necessarily
determine the degree of destabilization. Again, peptides
can magnify local perturbations and stability effects,
and studies of heterotrimeric triple-helical peptides
designed by the Hartgerink laboratory indicated the
introduction of the first chain with a Gly→Ser substitu-
tion has the most destabilizing effect, while the intro-
duction of the second chain is much less, and the third
even less.
21
GLY MISSENSE MUTATIONS AND
F
OLDING OF OI COLLAGEN
S
In a 2001 review article, Byers wrote “Misfolding of
collagen is the major outcome of most mutations in col-
lagen genes”
8
and evidence has accumulated that defec-
tive folding is a key step in OI pathology. It has been
suggested that OI may fall into the category of protein
folding diseases, which includes Alzheimer's disease
and the most common type of cystic fibrosis. Early
observations of unusually high levels of hydroxylysine
(Hyl) and glycosylated Hyl in OI type I collagen impli-
cated folding defects, since these post-translational
modifications can only occur on unfolded chains.
44,45
Collagen folding starts from the C-terminus, and care-
ful analysis indicated overmodification to be present
only N-terminal to the mutation site,
1,46
consistent with
the mutation leading to delayed folding. This indirect
but strong evidence was complemented by the study of
Raghunath et al.
47
on folding on five OI collagens with
Gly→Cys mutations at different sites along the triple-
helix domain of the α1(I) chain. Folding, monitored by
measuring the appearance of enzyme-resistant disulfide
linked dimers, was delayed for all mutant collagens
compared with controls, with the delay length varying
from 5 to 60 minutes for different mutations. Although
there was no simple correlation with distance from the
C-terminus, the mutation near the C-terminus clearly
showed the slowest folding. As discussed in Raghunath
et al.,
47
the results did not define the molecular basis
for the observed delays, but were consistent with some
pause in triple-helix propagation at the mutation site
which could be followed by either normal folding or
