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Thermodynamic vs. kinetic stability of collagen triple helices
Matrix Biology 20 Ž2001. 267᎐269
Response to letter to the editor
Thermodynamic vs. kinetic stability of collagen triple helices a,U Hans Peter Bachinger , Jurgen Engel b ¨ ¨ a
Research Center, Department of Biochemistry and Molecular Biology, Shriners Hospital for Children Portland, OR, USA b Abteiling fur ¨ Biophysikalische Chemie, Biozentrum der Uni¨ ersitat ¨ Basel, Basel, Switzerland Received 5 April 2001; accepted 18 April 2001
Miles and Bailey correctly state that the precise denaturation temperature of collagen triple helices depends on the rate of heating. They performed isothermal measurements of the rate of unfolding of type I collagen in tendon tissue and found that the rate is extremely slow at low temperatures, but increases dramatically if the temperature is raised. Based on these observations, they conclude that this is the source of collagen’s extremely sharp transition curve, and that collagen is only kinetically stable. There is ample evidence, however, that collagen triple helices are thermodynamically stable. Piez and Sherman Ž1970. showed that the unfolding of ␣1-CB2 is fully reversible and can be described by a concentration-dependent monomer᎐trimer equilibrium. For collagen-like peptides the studies by Kobayashi et al. Ž1970., Go and Suezaki Ž1973. and the more recent work of the group of Barbara Brodsky ŽBrodsky and Ramshaw, 1997; Ackerman et al., 1999. also indicate complete reversibility by equilibrium and kinetic measurements. The one quarter fragment of type III collagen also shows a completely reversible transition curve ŽDavis and Bachinger, 1993.. ¨ There is no reason to believe that the thermodynamic model does not hold for intact collagens. The large intact collagens, however, show a hysteresis loop with a difference of a few degrees between transition
U
Corresponding author. Tel.: q1-503-221-3433; fax: q1-503221-3451. .. E-mail address: [email protected] ŽH.P. Bachinger ¨
curves obtained by thermal unfolding and refolding. Fig. 1 shows the thermal denaturation curves of pN type III collagen at different rates of increasing the temperature, a plot of the endpoints of the refolding kinetics and the unfolding kinetics. Endpoints were determined by extrapolating the time course at constant temperature for 24 h and, as an example, the results at 33.2 and 34.9⬚C are shown ŽFig. 2.. In contrast to Miles and Bailey who found first order kinetics and complete unfolding, we obtained biphasic kinetics whose extrapolated endpoints indicated equilibrium values and not completely unfolded molecules as predicted by the model of Miles and Bailey. The extrapolated endpoints match the endpoints of the refolding kinetics ŽFig. 1., and the resulting temperature profile is the true thermodynamic equilibrium transition curve of pN type III collagen. We think that the discrepancies between our results and those of Miles and Bailey arise from the difficulty of accurate measurements at very low heating rates. Determination of the final value of unfolding is open to artifacts originating from instrumental stability, chemical modifications like deamidation, and the presence of proteases. The collagen triple helix is normally resistant to cleavage by proteases, but large numbers of cleavage sites become available during unfolding. This is especially true for measurements made in tissue including the tendon suspensions and, to a lesser degree, for measurements with purified type III collagen. The rest of the criticism by Miles and Bailey concerns details, which are not directly linked with
0945-053Xr01r$ - see front matter 䊚 2001 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved. PII: S 0 9 4 5 - 0 5 3 X Ž 0 1 . 0 0 1 3 8 - X
268
Letter to the editor r Matrix Biology 20 (2001) 267᎐269
Fig. 1. Thermal transition profile of pN type III collagen measured at different rates of temperature increase and red s 0.5⬚Crh. and by the endpoints of the unfolding Žsquares. and refolding Žspheres. kinetics. The calculated by measuring the optical rotation ␣ at 365 nm from F s Ž ␣ y ␣ d .rŽ ␣ n y ␣ d ., in which the indices native and denatured type III collagen.
Žgreen s 10⬚Crh; blue s 2⬚Crh; fraction of helix formation was d and n designate the values for
Fig. 2. Unfolding kinetics of pN type III collagen at 33.2 Žblue curve. and 34.9⬚C Žblack curve. with the corresponding biphasic fits Žred..
the main argument of kinetic vs. thermodynamic stability. However, the integration of our equation 3 is correct for measurements made at constant temperatures and the kinetic model must hold for such measurements as well. In conclusion, the experimental evidence clearly points to a thermodynamic stability model for the collagen triple helix and an explanation of the sharp transition by a high co-operativity of the equilibrium steps, shown in detail in our initial publication ŽEngel and Bachinger, 2000.. Nevertheless, a ¨ slow kinetic process, which we hypothetically relate to
a slow annealing step, is involved in the triple helix coil transition of type III collagen and leads to a hysteresis. References Ackerman, M.S., Bhate, M., Shenoy, N., Beck, K., Ramshaw, J.A., Brodsky, B., 1999. Sequence dependence of the folding of collagen-like peptides. Single amino acids affect the rate of triple-helix nucleation. J. Biol. Chem. 274, 7668᎐7673. Brodsky, B., Ramshaw, J.A., 1997. The collagen triple-helix structure. Matrix Biol. 15, 545᎐554.
Letter to the editor r Matrix Biology 20 (2001) 267᎐269 Davis, J.M., Bachinger, H.P., 1993. Hysteresis in the triple helix-coil ¨ transition of type III collagen. J. Biol. Chem. 268, 25965᎐25972. Engel, J., Bachinger, H.P., 2000. Co-operative equilibrium transi¨ tions coupled with a slow annealing step explain the sharpness and hysteresis of collagen folding. Matrix Biol. 19, 235᎐244. Go, N., Suezaki, Y., 1973. Letter: analysis of the helix-coil transition in ŽPro᎐Pro᎐Glyn. n by the all-or-none model. Biopolymers 12, 1927᎐1930.
269
Kobayashi, Y., Sakai, R., Kakiuchi, K., Isemura, T., 1970. Physicochemical analysis of ŽPro᎐Pro᎐Gly. n with defined molecular weight᎐temperature dependence of molecular weight in aqueous solution. Biopolymers 9, 415᎐425. Piez, K.A., Sherman, M.R., 1970. Equilibrium and kinetic studies of the helix-coil transition in ␣1-CB2, a small peptide from collagen. Biochemistry 9, 4134᎐4140.
Response to letter to the editor
Thermodynamic vs. kinetic stability of collagen triple helices a,U Hans Peter Bachinger , Jurgen Engel b ¨ ¨ a
Research Center, Department of Biochemistry and Molecular Biology, Shriners Hospital for Children Portland, OR, USA b Abteiling fur ¨ Biophysikalische Chemie, Biozentrum der Uni¨ ersitat ¨ Basel, Basel, Switzerland Received 5 April 2001; accepted 18 April 2001
Miles and Bailey correctly state that the precise denaturation temperature of collagen triple helices depends on the rate of heating. They performed isothermal measurements of the rate of unfolding of type I collagen in tendon tissue and found that the rate is extremely slow at low temperatures, but increases dramatically if the temperature is raised. Based on these observations, they conclude that this is the source of collagen’s extremely sharp transition curve, and that collagen is only kinetically stable. There is ample evidence, however, that collagen triple helices are thermodynamically stable. Piez and Sherman Ž1970. showed that the unfolding of ␣1-CB2 is fully reversible and can be described by a concentration-dependent monomer᎐trimer equilibrium. For collagen-like peptides the studies by Kobayashi et al. Ž1970., Go and Suezaki Ž1973. and the more recent work of the group of Barbara Brodsky ŽBrodsky and Ramshaw, 1997; Ackerman et al., 1999. also indicate complete reversibility by equilibrium and kinetic measurements. The one quarter fragment of type III collagen also shows a completely reversible transition curve ŽDavis and Bachinger, 1993.. ¨ There is no reason to believe that the thermodynamic model does not hold for intact collagens. The large intact collagens, however, show a hysteresis loop with a difference of a few degrees between transition
U
Corresponding author. Tel.: q1-503-221-3433; fax: q1-503221-3451. .. E-mail address: [email protected] ŽH.P. Bachinger ¨
curves obtained by thermal unfolding and refolding. Fig. 1 shows the thermal denaturation curves of pN type III collagen at different rates of increasing the temperature, a plot of the endpoints of the refolding kinetics and the unfolding kinetics. Endpoints were determined by extrapolating the time course at constant temperature for 24 h and, as an example, the results at 33.2 and 34.9⬚C are shown ŽFig. 2.. In contrast to Miles and Bailey who found first order kinetics and complete unfolding, we obtained biphasic kinetics whose extrapolated endpoints indicated equilibrium values and not completely unfolded molecules as predicted by the model of Miles and Bailey. The extrapolated endpoints match the endpoints of the refolding kinetics ŽFig. 1., and the resulting temperature profile is the true thermodynamic equilibrium transition curve of pN type III collagen. We think that the discrepancies between our results and those of Miles and Bailey arise from the difficulty of accurate measurements at very low heating rates. Determination of the final value of unfolding is open to artifacts originating from instrumental stability, chemical modifications like deamidation, and the presence of proteases. The collagen triple helix is normally resistant to cleavage by proteases, but large numbers of cleavage sites become available during unfolding. This is especially true for measurements made in tissue including the tendon suspensions and, to a lesser degree, for measurements with purified type III collagen. The rest of the criticism by Miles and Bailey concerns details, which are not directly linked with
0945-053Xr01r$ - see front matter 䊚 2001 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved. PII: S 0 9 4 5 - 0 5 3 X Ž 0 1 . 0 0 1 3 8 - X
268
Letter to the editor r Matrix Biology 20 (2001) 267᎐269
Fig. 1. Thermal transition profile of pN type III collagen measured at different rates of temperature increase and red s 0.5⬚Crh. and by the endpoints of the unfolding Žsquares. and refolding Žspheres. kinetics. The calculated by measuring the optical rotation ␣ at 365 nm from F s Ž ␣ y ␣ d .rŽ ␣ n y ␣ d ., in which the indices native and denatured type III collagen.
Žgreen s 10⬚Crh; blue s 2⬚Crh; fraction of helix formation was d and n designate the values for
Fig. 2. Unfolding kinetics of pN type III collagen at 33.2 Žblue curve. and 34.9⬚C Žblack curve. with the corresponding biphasic fits Žred..
the main argument of kinetic vs. thermodynamic stability. However, the integration of our equation 3 is correct for measurements made at constant temperatures and the kinetic model must hold for such measurements as well. In conclusion, the experimental evidence clearly points to a thermodynamic stability model for the collagen triple helix and an explanation of the sharp transition by a high co-operativity of the equilibrium steps, shown in detail in our initial publication ŽEngel and Bachinger, 2000.. Nevertheless, a ¨ slow kinetic process, which we hypothetically relate to
a slow annealing step, is involved in the triple helix coil transition of type III collagen and leads to a hysteresis. References Ackerman, M.S., Bhate, M., Shenoy, N., Beck, K., Ramshaw, J.A., Brodsky, B., 1999. Sequence dependence of the folding of collagen-like peptides. Single amino acids affect the rate of triple-helix nucleation. J. Biol. Chem. 274, 7668᎐7673. Brodsky, B., Ramshaw, J.A., 1997. The collagen triple-helix structure. Matrix Biol. 15, 545᎐554.
Letter to the editor r Matrix Biology 20 (2001) 267᎐269 Davis, J.M., Bachinger, H.P., 1993. Hysteresis in the triple helix-coil ¨ transition of type III collagen. J. Biol. Chem. 268, 25965᎐25972. Engel, J., Bachinger, H.P., 2000. Co-operative equilibrium transi¨ tions coupled with a slow annealing step explain the sharpness and hysteresis of collagen folding. Matrix Biol. 19, 235᎐244. Go, N., Suezaki, Y., 1973. Letter: analysis of the helix-coil transition in ŽPro᎐Pro᎐Glyn. n by the all-or-none model. Biopolymers 12, 1927᎐1930.
269
Kobayashi, Y., Sakai, R., Kakiuchi, K., Isemura, T., 1970. Physicochemical analysis of ŽPro᎐Pro᎐Gly. n with defined molecular weight᎐temperature dependence of molecular weight in aqueous solution. Biopolymers 9, 415᎐425. Piez, K.A., Sherman, M.R., 1970. Equilibrium and kinetic studies of the helix-coil transition in ␣1-CB2, a small peptide from collagen. Biochemistry 9, 4134᎐4140.