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scanning calorimetry (DSC).
36
Collagens secreted by
fibroblasts from heterozygous patients represent-
ing 41 distinct Gly missense mutations along the α1(I)
and α2(I) chains were examined and most of the DSC
thermograms had a normal peak together with one or
two peaks of lower stability, corresponding to trimers
containing one or two mutant chains. The thermal sta-
bility of the peaks assigned to molecules containing
mutations were 1.5-4°C lower than normal, indicating
a small but significant effect on global collagen stabil-
ity. A detailed analysis of the energetics suggested that
the degree of destabilization reflects the stability of the
region or folding domain containing the mutation site,
rather than local stability or the identity of the residue
replacing Gly.
36
As discussed in Chapter 8 by Leikin
and Makareeva, the degree of destabilization caused by
a mutation is not related in any simple manner to clini-
cal severity, and the folding itself may be a more mean-
ingful step in the pathology (see below).
The variable effect of different OI mutations on col-
lagen stability has been approached through recombi-
nant collagen systems. The effect of Gly→Ser mutations
at two different sites within a 63 residue recombinant
fragment of type I collagen with no Hyp was studied
in a bacterial expression system by Xu laboratory.
37
C-terminal terminal foldon domain for trimerization
and short flanking (Gly-Pro-Pro)
n
sequences were used
for stability.
37
The original α1(I) sequence was modi-
ied by site-directed mutagenesis to model Gly901Ser,
known to cause a mild OI case, and Gly913Ser, known
to cause perinatal lethal type II OI. Circular dichroism
spectroscopy and enzymatic susceptibility indicated
that the Gly901Ser mutation decreased the thermal sta-
bility of the triple helix by ~10°C. The Gly913Ser muta-
tion led to destabilization, a biphasic melting curve
and additional disruptive effects, including unfold-
ing at low temperatures (15°C) of a 24 residue Pro-
free zone immediately C-terminal to the mutation site.
Experiments showed that the greater disruptive effect
of Gly913Ser is mediated by a nearby KGE sequence,
previously shown to provide a major electrostatic con-
tribution to triple-helix stability.
37,38
Stability studies were also carried out on a recom-
binant bacterial collagen-like protein, where Gly
missense mutations were introduced at variable loca-
tions.
39
A small but reproducible decrease of 1.5-2°C
in the
T
m
value was observed as a result of a Gly→Ser
or Gly→Arg replacement in the (Gly-Xaa-Yaa)
79
colla-
gen-like domain. The small drop in
T
m
values for this
homotrimeric recombinant bacterial collagen mole-
cule with mutations in all three chains is similar to the
Δ
T
m
= 1.5-5°C values reported by Makareeva et al.
36
for OI type I heterotrimers. A Gly→Arg mutation very
close to the N-terminal resulted in the same decrease
in thermal stability, but introduced a new nearby
trypsin-sensitive site within the triple helix, highlight-
ing the presence of locally destabilized regions which
have limited effect on the overall
T
m
value.
The effect of triple-helix length is seen for 24-36 resi-
due peptides, where a perturbation to local stability
affects the stability of the entire peptide, translates into
a large change in the global
T
m
value of the peptide,
and often leads to the loss of triple-helical conforma-
tion. In host-guest peptides of the form (Gly-Pro-Hyp)
8
,
replacement of a central Gly by another residue leads to
a >35°C drop in the
T
m
value, and the degree of desta-
bilization depends on the identity of the residue replac-
ing Gly.
6
The replacement residues could be ranked
from the least to the most damaging as follows: Gly
<Ala~Ser<Cys<Arg<Va l<Glu<Asp<Trp.
6
The good
correlation between the severity of the OI clinical out-
come and residue ranking of the degree of triple-helix
destabilization in model peptides suggesting local
destabilization is an important factor in OI etiology.
The introduction of a Gly→Ala or Gly→Ser mutation
in (Pro-Hyp-Gly)
10
leads to a drop in
T
m
similar to that
seen for (Gly-Pro-Hyp)
8
, but the decrease is smaller in
peptides with lower amino acid contents (
Table 11.1
).
7,40
A local destabilization due to a mutation, as reflected
in the peptide studies, may result in only a small drop
in the collagen global
T
m
value because of the pres-
ence of multiple domains within type I collagen (
Table
11.1
).
36,41
The recombinant protein studies described
above suggest a >10°C decrease in
T
m
is seen when
a Gly mutation is introduced into a (Gly-Xaa-Yaa)
21
sequence,
37
while a smaller 1.5-2°C decrease is seen
when the mutation is introduced into a longer (Gly-
Xaa-Yaa)
79
bacterial collagen-like sequence.
39
The length
of the bacterial collagen-like protein may reach a value
similar to that of independently melting domains in full
length type I collagen chains.
Computational approaches have been used to inves-
tigate how Gly missense mutations interfere with inter-
actions stabilizing the triple helix, including hydrogen
bonding and hydration. Molecular mechanics calcula-
tions demonstrated that disruption of hydrogen bond-
ing network and triple-helix hydration depend strongly
on the identity of the amino acid replacement for Gly
in clinically observed EDS-IV-associated type III colla-
gen mutations.
42
Applying a high throughput molecu-
lar dynamics approach, the Klein group showed the
degree of perturbation depended on the residue replac-
ing Gly and found lethal mutations had fewer back-
bone hydrogen bonds than non-lethal mutations.
27
A relationship was also reported between the side chain
volume and hydropathy of the residue replacing Gly and
the computed effective mechanical properties (Young's
modulus).
43
A recent machine-tool-learning approach
suggested a relationship between OI clinical sever-
ity and the presence of low stability triplets C-terminal
