Case 40
A Collection of Collagen Cases
Focus concept
Factors important in the stability of collagen are examined.
Prerequisites
∙
∙
∙
Amino acid structures and properties
Primary and secondary structure
Basic collagen principles
Case 1
Several investigators have sought to explain why hydroxyproline-containing collagen molecules have
increased stability. Some suggested that the hydroxyproline on the pyrrolidine ring of Hyp participates
in a network of hydrogen bonds with “bridging” water molecules. However, this has been called into
question by others who have noted that (Pro-Pro-Gly)10 and (Pro-Hyp-Gly)10 are stable in methanol. To
investigate the question of stability further, one group of investigators synthesized a synthetic collagen
that contains 4-fluoroproline (Flp), which resembles hydroxyproline except that fluorine substitutes for
the hydroxyl group. The melting points of three synthetic collagen peptides were measured and are
shown in the table.
Table 40-1: Stability of collagen-like peptides
Synthetic peptide
Forms a triple helix?
Tm (°C)
(Pro-Pro-Gly)10
yes
41
(Pro-Hyp-Gly)10
yes
60
(Pro-Flp-Gly)10
yes
91
(a) Compare the structure of hydroxyproline and fluoroproline. Why do you suppose the investigators
chose fluorine?
(b) Compare the melting points of the three synthetic collagens. What factor is responsible for the
increased stability of the synthetic collagen? What factor is not responsible?
1
2
Case 2
A group of investigators synthesized a series of collagen-like peptides each containing 30 amino acid
residues in order to study the important interactions among the three chains in the triple helix. The
following peptides were synthesized: (1) (Pro-Hyp-Gly)10, (2) (Pro-Hyp-Gly)4-Glu-Lys-Gly-(Pro-HypGly)5 and (3) Gly-Lys-Hyp-Gly-Glu-Hyp-Gly-Pro-Lys-Gly-Asp-Ala-(Gly-Ala-Hyp)2-(Gly-ProHyp)4GV. These peptides are abbreviated (1) (POG)10, (2) EK-containing peptide and (3) T3-487 in the
table below. (Note that “O” is the one-letter abbreviation for hydroxyproline.)
Table 40-2:Stability of three collagen-like peptides.
Peptide
Forms
trimers?
Imino acid
content
Tm, °C
(1) (POG)10
yes
67%
pH = 1
pH = 7
pH = 13
61
58
60
(2) EK-containing peptide
yes
60%
pH = 1
pH = 7
pH = 13
44
46
49
(3) T3-487
yes
41%
pH = 1
pH = 7
pH = 13
18
26
19
(a) Rank the stability of the three collagen-like peptides (POG)10, EK-containing peptide and T3-487 at
pH = 7. What is the reason for the observed stability?
(b) Compare the Tm values of T3-487 at the various pH values. At what pH does T3-487 have a
maximum Tm value? What interactions are primarily responsible for this?
(c) Compare the Tm values of (POG)10 at the various pH values. Why is there less of a difference of Tm
values at different pH values for (POG)10 than for T3-487?
(d) Consider the answers to the above questions. Which factor is more important in stabilizing the
structure of the collagen-like peptides?
3
Case 3
The deep sea hydrothermal vent worm Riftia pachyptila resides under extreme conditions of high
temperature, low oxygen content and drastic temperature changes. The worm has a thick collagencontaining cuticle that protects it from its harsh environment. Recently the structure of this collagen was
investigated. Sequence analyses indicated that the collagen had the customary -Gly-X-Y- triplet but that
hydroxyproline occurred only in the X position and that Y was often a glycosylated threonine (a
galactose sugar residue was covalently attached via a condensation reaction between a hydroxyl group
on the galactose and the hydroxyl group of the threonine side chain). Experiments were carried out using
synthetic peptides in order to evaluate the stability of each. Results are shown in the table.
Table 40-3: Stability of synthetic collagen-like peptides
Synthetic peptide
Forms a triple helix?
Tm (°C)
(Pro-Pro-Gly)10
yes
41
(Pro-Hyp-Gly)10
yes
60
(Gly-Pro-Thr)10
no
N/A
(Gly-Pro-Thr(Gal))10
yes
41
OH OH
(a) Compare the melting temperatures of (Pro-Pro-Gly)10 and
(Pro-Hyp-Gly)10. What is the structural basis for the
difference?
(b) Compare the melting temperatures of (Pro-Pro-Gly)10 and
(Gly-Pro-Thr(Gal))10 and provide an explanation.
(c) Why was (Gly-Pro-Thr)10 included by the investigators?
O
H
O
+H3N
CH
O
CH
C
H
HO
H
H
OH
H
CH3
Gal-Thr
Figure 40-1: Structure of Thr(Gal)
References
Case 1
Holmgren, S., Taylor, K., Bretscher, L. E., and Raines, R. (1998) Nature 392, 666-667.
Case 2
Venugopal, M. G., Ramshaw, J. A. M., Braswell, E., Zhu, D., and Brodsky, B. (1994) Biochemistry 33,
7948-7956.
Case 3
Bann, J. G., Peyton, D. H., and Bächinger, H. P. (2000) FEBS Lett. 473, 237-240.
4
O-