what-when-how
In Depth Tutorials and Information
might be important for proper formation and function
of collagen fibers.44,46,47
44,46,47
Human type I collagen triple helix contains 338
uninterrupted Gly-X-Y repeats within each of the three
chains. Pro occupies ~1
/
3 of X-positions, 4-Hyp occupies
~1
/
3 of Y-positions, and Arg occupies ~ 1
/
8 of Y-positions
(~80% of Arg residues have Y-position locations). Gly
substitutions account for over 80% of severe OI cases.
1
Substitutions of X-position Pro, Y-position 4-Hyp, and
Y-position Arg also result in OI symptoms,
48-54
but sub-
stitutions of X-position Arg do not seem to cause OI,
52,54-
57
probably because the latter do not destabilize the triple
helix. Thus, triple-helix stability and folding appear to
play an important role in OI.
Different maps of regions with reduced and increased
local triple-helix stability were constructed for type I
collagen based on the content of Pro and 4-Hyp
residues,
58
stability of triple-helical peptides with
different sequences,
42,59
and stability of the whole col-
lagen molecule with Gly substitutions at different sites.
60
Approximate locations of the largest regions with higher
and lower than average stability based on the latter
approach are shown in
Figure 7.2A
. As discussed in the
next section of this review, local triple-helix stability corre-
lates with regional variations in the severity of OI caused
by Gly substitutions, but this correlation is neither simple
nor straightforward.
60
Overall, type I collagen triple-helix stability is sur-
prisingly low, e.g., the native triple-helical structure
of human type I collagen is more thermodynamically
favorable than unfolded chains (and therefore stable)
only below 34-35°C.
63,64
In a physiological environ-
ment at normal body temperature, the triple helix is
unstable and slowly denatures.
63
It is important to
emphasize that the apparent triple-helix denaturation
temperature (
T
m
) depends on the heating
/
equilibration
rate and is usually 5-7°C higher than the equilibrium
stability of the molecule, because typical measurements
allow only for a relatively short equilibration at each
temperature.
63,65-67
Type I collagen instability appears to have been
deliberately designed by Nature, since the apparent
T
m
value correlates with the normal body temperature for
the species from which the protein has been extracted,
from arctic fish to mammals.
47,68
This instability might
be essential for formation of elastic collagen fibers with
unique biochemical and mechanical properties.
47
Triple-
helix denaturation at body temperature is sufficiently
slow to allow for N- and C-propeptide cleavage and
incorporation of native molecules into fibers,63,64
63,64
where
the triple helices are stabilized and protected from dena-
turation by intermolecular interactions.
69
Yet, the low
triple-helix stability presents a significant challenge for
cells, resulting in a unique folding process that requires
highly specialized chaperone machinery.
PROCOLLAGEN FOLDING
Unlike most proteins, which begin to fold from their
N-terminal end while the rest of the chain is still being
synthesized, procollagen folds from the C-terminal end
only after the synthesis of the chains is complete. The
folding process begins from association of two pro-α1(I)
and one pro-α2(I) chain within the C-propetide region
and C-propeptide folding. The next step is folding of
the central triple helix, which proceeds from the C- to
N-terminal end in a zipper-like manner. N-propeptide
folding completes the process. Hydroxylation of most
Y-position Pro to 4-Hyp, few specific X-position Pro to
3-hydroxyproline (3-Hyp), and some Lys to hydroxyly-
sine (Hyl) as well as glycosylation of some Hyl proceed
simultaneously with the synthesis and folding of procol-
lagen chains and occur only within unfolded regions of
the chains (
Figure 7.1A
).
35-37,70,71
The most unusual step in this process is the triple-helix
folding, as it does not conform to the generally accepted
protein folding paradigm. In a test tube, when not dis-
turbed or helped by other macromolecules, folding or
unfolding of a polypeptide chain always proceeds toward
more thermodynamically favorable conformations
(which have lower free energy at equilibrium).
72-74
In
the ER, folding of most proteins also follows this general
direction. Some non-native interactions between residues
(mostly hydrophobic) may be more thermodynamically
favorable than native ones (have even lower free energy),
resulting in misfolding and
/
or aggregation of polypeptide
chains. To prevent such counterproductive interactions,
cells utilize chaperone molecules.
75-77
ER chaperones like
BiP and GRP94 bind to unfolded and partially folded
chains and block counterproductive interactions, e.g., by
cycling on and off large hydrophobic patches. Release of
these chaperones upon completion of the folding pro-
cess serves as one of the signals that the protein can be
transported out of the ER toward its final destination.
75
Misfolding causes sequestration of BiP and GRP94 on
misfolded chains. The resulting release of these chaper-
ones from their receptors on the ER membrane activates
conventional unfolded protein response (UPR) signal-
ing.
78,79
For most proteins, preferential chaperone binding
makes unfolded and partially folded chain conformations
less thermodynamically unfavorable, but not more favor-
able than the native state.
For procollagen, however, the folding scenario is
drastically different. Cells have to utilize specialized
chaperones that make the folding thermodynamically
favorable by preferentially binding to the native triple
helix rather than unfolded chains.
80
Without such chap-
erones, human procollagen folding is favorable below
34-35°C, but unfavorable above this temperature.
64
At
37-38°C, native procollagen denatures within sev-
eral days and remains unfolded, because its unfolded
