OtM3E.3.pdf
Optics in the Life Sciences 2015 © OSA 2015
Tuba Altindal, Marjan Shayegan, Evan Kiefl and Nancy R. Forde
Department of Physics, Simon Fraser University, Burnaby BC V5A 1S6, Canada nforde@sfu.ca
Abstract: The local viscoelasticity was compared between full-length and telopeptide-removed collagen solutions using optical-tweezers-based microrheology. An enhanced modulus at higher concentrations in full-length collagen solutions suggests contributions from telopeptide-associated transient crosslinks.
OCIS codes: (160.1435) Biomaterials; (160.5470) Polymers; (350.4855) Optical tweezers or optical manipulation
1. Introduction
Self-assembly of biomolecules into complex functional structures with precise morphological and mechanical properties is a fascinating aspect of life at the cellular scale, and is of importance also to materials design and assembly. These processes that proceed without external guidance rely on specific interactions between constituent molecules. Collagen is one such biomolecule, a protein that is found abundantly in the extracellular matrix in vertebrates, where it is found to form hierarchical structures. These fibrillar structures are constantly remodeled, i.e.
, broken down and re-assembled, conferring a dynamic nature to the extracellular matrix. Identifying and characterizing interactions between collagen molecules is crucial for our understanding of remodeling processes which play important roles in the regulation of cellular processes such as growth, migration and differentiation.
Telopeptides are short non-helical regions at the ends of triple helical collagen molecules that influence the kinetics of fibril formation (Figure 1).[1-3] Although they constitute less than 5% of the total length of each protein, their proteolytic removal results in a delay in the onset of fibril formation, suggesting their importance to the kinetics of interactions between chains.
The types of interactions between polymers like collagen, such as entanglement and transient cross-linking, determine how stress relaxes in these systems, and consequently define their flow properties. Here, we describe our studies using optical-tweezers-based microrheology[4] to investigate how the removal of telopeptides influences the interaction between collagen molecules. The aim of this work is to provide insight into the underlying mechanism of interactions responsible for the self-assembly process into collagen fibrils.
2. Materials and methods
The collagen solutions used in the comparative study described below were prepared using acid-soluble type I collagen from rat tail tendon (Cultrex Invitrogen) and its pepsin-digested derivative obtained in our lab. By removing telopeptides in-house,[5] we obtained control over the relative collagen concentrations in telo- and atelocollagen samples (with and without telopeptide, respectively). The stock collagen was diluted to various concentrations < 5 mg/ml in 20 mM acetic acid at pH 3.5, concentrations and pH values at which fibril formation does not occur.
The thermally induced short-range fluctuations in the position of optically trapped probing particles (2 µm carboxyl-polystyrene beads) were detected using a quadrant photodiode at 100 kHz. The description of the optical tweezers instrument used in our microrheology measurements can be found in Refs. [6, 7]. The power spectrum of particle fluctuations within the trap was used to calculate the frequency-dependent complex shear modulus of the surrounding collagen solution, G* ( f ) = G’ ( f )+ iG” ( f ), where G’ ( f ) represents the frequency-dependent elastic modulus and G” ( f ) the viscous modulus of the system[6, 8].

OtM3E.3.pdf
Optics in the Life Sciences 2015 © OSA 2015
Fig. 1. (A) A cartoon of a bead in an optical trap probing stress relaxation in a collagen (green curves) solution. (B) An illustration of removal of non-helical telopeptides flanking the tightly wound triple helical domain of collagen by the enzyme pepsin (yellow beans).
3. Experimental results
We have confirmed the results of preliminary studies showing that the removal of telopeptides significantly decreases the elastic modulus of collagen solutions in the ~10-200 Hz range.[5] This finding suggests that telopeptides contribute substantial elasticity to collagen solutions, particularly in this intermediate frequency range.
A proposed mechanism by which telopeptides facilitate fibril formation is by forming transient interactions between collagens in solution, thereby promoting alignment and docking of the triple helices to form fibrils.[3] We sought to test whether transient crosslinking occurs in our system, under acidic conditions, by examining the lowfrequency response of collagen solutions. Transient crosslinks between semiflexible chains have previously been found to influence the power-law scaling of the shear modulus at low frequencies, changing it from a singlerelaxation Maxwellian behaviour ( G’ ~ f 2 ; G” ~ f 1 ) to a multiple relaxation behaviour described by a scaling of G ~ f α , with α <1.[9] At timescales shorter than the characteristic dissociation time of the transient interaction, this scaling is predicted to reach α =½.[9]
At low telo- and atelo-collagen concentrations, we observed G” to scale as f 1 (Figure 2.) However, as collagen concentration increases and intermolecular interactions become more likely, this behaviour changes. While both telo- and atelo-collagen solutions exhibit similar power-law scalings at high frequencies, indicating comparable intramolecular relaxation modes, the difference in the low-frequency scaling of G” suggests that telopeptideinduced transient interactions between collagen molecules cause the system to deviate from the single-mode relaxation Maxwellian behaviour ( G” ~ f 1 ). While even at the highest concentration of 4 mg/ml atelo-collagen still exhibits Maxwellian response at low frequencies, the telopeptide-containing collagen undergoes a change in response, showing an increasingly enhanced modulus at low frequencies. This implies that additional long-range modes contribute to the dynamics of relaxation, an observation consistent with the presence of transient crosslinks between collagens in our system. The concentrations of collagen and/or the strength of transient telopeptide-collagen interactions may not be sufficient to bring the response to α =½.[9]
Fig. 2. The frequency-dependent viscous modulus of untreated, full-length (black symbols) and pepsin-digested (red symbols) collagen solutions at the specified concentrations. Error bars indicating the standard error of the mean among N =6-8 independent measurements at each condition are smaller than the symbols.

OtM3E.3.pdf
Optics in the Life Sciences 2015 © OSA 2015
4. Discussion and Conclusion
Our results show that the presence of intact telopeptides on the ends of collagen proteins leads to a significant enhancement of the complex shear modulus. Here, by examining the loss modulus, we have found that increasing the concentration of telopeptide-containing collagen results in a growing non-Maxwellian behaviour at low frequencies. This result suggests the contribution of additional long-range modes of relaxation to the dynamics of collagen, compared with collagen lacking its telopeptides. Because each triple helical protein contains four different telopeptide sequences (N- and C- ends of constituent α
1
and α
2
chains), there is likely a range of unbinding times, which would result in more complex dynamics than in the model system considered in [9]. Other possible mechanisms for the difference in response between these two types of samples, as seen by others,[10] include increased intermolecular crosslinking in the telopeptide-containing sample, as telopeptides are sites at which intermolecular crosslinks are formed in fibrils. Further investigation, for example inhibiting transient interactions,[3] could help to distinguish between these two mechanisms.
5. Acknowledgements
This research was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC). EK acknowledges support from an NSERC Undergraduate Student Research Award (USRA).
6. References
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[9] C. P. Broedersz, M. Depken, N. Y. Yao, M. R. Pollak, D. A. Weitz, and F. C. MacKintosh, "Cross-Link-
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[10] A. M. Oechsle, X. Wittmann, M. Gibis, R. Kohlus, and J. Weiss, "Collagen entanglement influenced by the addition of acids," European Polymer Journal 58 , 144-156 (2014).