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Potassium Ion Mediated Collagen Microfibril Assembly on Mica Richard W. Loo* and M. Cynthia Goh* Department of Chemistry, UniVersity of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6 Canada ReceiVed September 16, 2008. ReVised Manuscript ReceiVed October 10, 2008 Potassium ion can critically effect the interaction between collagen microfibrils and mica leading to different ordered structures that vary dramatically with changing ion concentration. AFM images of the structures formed at different ion concentrations appear to be intermediate stages in the progression from disordered to ordered film. At 200 mM potassium ion concentration, a nanometer-thick array of aligned and bundled microfibrils covering large areas can be created easily and reproducibly on mica.
The formation of collagen fibrils presents a fascinating case for the study of hierarchical structure and assembly. Collagen in its monomeric form is approximately 300 nm long and ∼1.5 nm in diameter and is made up of three polypeptide chains wrapped around each other in a triple helix. Higher-order structures result from the aggregation of the monomers: it is generally believed that five monomers combine by overlapping and intertwining with each other to form microfibrils,1 which in turn can aggregate both laterally and longitudinally with other microfibrils to form a native collagen fibril.2 The self-assembly process from monomer to fibril has been reproduced in Vitro.3 When other, typically anionic, species are added to the fibril forming solution, a number of other structurally different fibrils are formed.4 Once formed, atomic force microscopy (AFM) has been used extensively to study all these different collagen macrostructures on mica substrates. The AFM images of these collagen fibrils often show a background film of disordered monomer and/or microfibril that have been largely ignored until recently when Mu¨ller and co-workers discovered conditions for forming arrays of aligned collagen microfibrils.5 Originally, the direction the aligned microfibrils take was attributed to the flow of the collagen solution over the mica substrate. However, Merschrod and coworkers have since proven that the direction of alignment is actually due to interactions between collagen and the mica lattice.6 In doing so, they highlighted the importance of the mica surface for templating the assembly process. We have found that the presence of potassium ions dramatically influences that interaction between collagen and mica. When a solution of collagen microfibrils in citrate buffer (0.4 mM, pH 4.3) without any added KCl is deposited on a mica substrate, the microfibrils appear as a disordered mesh (Figure 1a). However, upon addition of KCl to the initial collagen solution, the morphology of the deposited microfibril film changes, and ordering in the film can be observed. The film continues to change as the order progressively increases with increasing amounts of added KCl. *
[email protected],
[email protected]. (1) Hulmes, D. J. S. J. Struct. Biol. 2002, 137, 2. (2) Kadler, K. E.; Holmes, D. F.; Trotter, J. A.; Chapman, J. A. Biochem. J. 1996, 316, 1. (3) Gale, M.; Pollanen, M. S.; Markiewicz, P.; Goh, M. C. Biophys. J. 1995, 68, 2124. (4) Paige, M. F.; Rainey, J. K.; Goh, M. C. Micron 2001, 32, 341. (5) Jiang, F.; Ho¨rber, H.; Howard, J.; Mu¨ller, D. J. J. Struct. Biol. 2004, 148, 268. (6) Sun, M.; Stetco, A.; Merschrod S., E. F. Langmuir, 2008, 24, 5418.
With 50 mM added KCl (Figure 1b), there is no clear ordering yet, but one can see that parts of some of the microfibrils align with neighboring microfibrils. Overall, there is still a mesh-like appearance with many junction points where the microfibrils cross over each other. At 100 mM KCl (Figure 1c), the local alignment of microfibrils is enhanced, while the crossover points have decreased. The microfibrils pack into circular, swirling patterns. At 150 mM KCl (Figure 1d), the microfibrils appear to have straightened out and begin to show alignment in one of three different directions, each of which is separated by an angle of ∼120°. However, there is predominate alignment in two of the directions, as indicated by the arrows (see also Supporting Information Figure S1). At 200 mM KCl (Figure 1e), the microfibrils appear to intertwine around each other in sheet-like bundles. The bundles of microfibrils are aligned in just two different directions now and still separated by an angle of ∼120°. By 300 mM KCl (Figure 1f), the microfibril bundles appear more tightly packed, but are still aligned in two different directions separated by an angle of ∼120°. At higher concentrations of KCl, there are noticeably fewer collagen microfibrils found on the mica surface. In a 400 mM solution of KCl, the density of microfibrils has diminished quite substantially, but they still form bundles that are aligned in the two directions (Figure 2d). As the concentration of KCl is further increased, fewer and fewer microfibrils are adsorbed, and by 1 M KCl, microfibril bundles are no longer found. Larger area AFM scans are presented in Figure 2 to highlight the long-range order that can be found in samples prepared at the higher KCl concentrations. At low concentrations (100 mM and lower), there appears to be no long-range order. Figure 2a shows a 4 × 4 µm2 image of a collagen microfibril film made in 100 mM KCl. Note that there is local alignment of microfibrils, but at the 4 µm scale, this order is clearly not persistent. However, at 200 mM and higher KCl concentrations, the microfibrils appear to straighten out, and long-range order between fibril bundles can be clearly observed (Figure 2b-d). The microfibrils continue to be present as bundles oriented along two directions that are 120° apart, but interestingly, it appears that microfibril bundle orientation begins to predominate in a single direction. In addition, as noted earlier, fewer microfibrils are adsorbed with increasing KCl concentrations. The observed order appears to persist for distances much larger than what our AFM can image in a single scan. Scans of different regions millimeters apart typically show the same orientation of microfibril bundles. Potentially, the effect of the added KCl could be from the K+ ion, the Cl- ion, or a change in ionic strength. We ran three
10.1021/la803041v CCC: $40.75 2008 American Chemical Society Published on Web 10/31/2008
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Figure 1. AFM images of the different structures formed on mica by collagen in the presence of added KCl. Collagen (12 µg/mL) in citrate (0.4 mM, pH 4.3) with (a) 0 mM, (b) 50 mM, (c) 100 mM, (d) 150 mM, (e) 200 mM, and (f) 300 mM added KCl was applied to mica, left for ∼10 min, and then rinsed with water and dried. The tapping mode AFM images shown are of the dried samples. All the images are 1.25 × 1.25 µm2 with a vertical scale of 5 nm.
Figure 2. AFM images of the different structures formed on mica by collagen in the presence of added KCl. Collagen (12 µg/mL) in citrate (0.4 mM, pH 4.3) with (a) 100 mM, (b) 200 mM, (c) 300 mM, and (d) 400 mM KCl was applied to mica, left for ∼10 min, and then rinsed with water and dried. The tapping mode AFM images shown are of the dried samples. Images (a) and (b) are 4 × 4 µm2. Images (c) and (d) are 10 × 10 µm2. The insets in (c) and (d) are 2 × 2 µm2 scans of the same respective samples presented on the same scale as images (a) and (b) for comparison. All images have a vertical scale of 5 nm.
different sets of experiments to separate these three factors and determine what causes the ordered adsorption and aggregation of collagen microfibrils on the mica surface. Thus, we made collagen solutions containing 200 mM NaCl, 100 mM NaCl + 100 mM KCl, and 200 mM KOAc, and deposited them separately onto mica. These three solutions all have the same ionic strength and yet produce very different results. For the solution containing 200 mM NaCl, we see no aligned or ordered microfibrils
(Supporting Information Figure S2). For the second solution, containing 100 mM KCl and 100 mM NaCl, we found locally aligned microfibrils forming circular patterns with no long-range order (Supporting Information Figure S3), which clearly resemble the images obtained with just 100 mM KCl present (Figure 1c). Finally, for the solution containing 200 mM KOAc, ordered and aligned microfibrils form on mica (Supporting Information Figure S4) similar to samples with 200 mM KCl (Figure 1e). From
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these results, we can clearly conclude that the K+ ions are responsible for the ordered adsorption and aggregation of collagen microfibrils on the mica surface. There is literature precedence for K+ ions playing a specific role in the self-assembly of both a charge transfer complex7 and guanosine 5′-monophosphate8 on mica. However, while those small molecules are both known to bind alkali metal ions, it is not apparent how collagen would interact specifically with K+ ions particularly at pH 4.3 when collagen itself will be positively charged.9 In conclusion, we have shown collagen microfibril film assembly on mica as a single domain array of aligned bundles of intertwined microfibrils. These films can be easily and reproducibly created by simply adding 200 mM potassium ion to the initial collagen solution. Importantly, it appears that a single critical component, the potassium cation, is responsible for creating an ordered film from a system that would otherwise
form a disordered film. Also, perhaps most interestingly, the collagen microfibril solutions containing lower potassium ion concentrations appear to provide insight into the intermediate stages of ordering of the microfibrils on mica. The AFM images from those experiments suggest that the effect of potassium ion on the mica-templated microfibril film assembly proceeds in two stages. In the first stage starting with low concentrations of added potassium ion, there is alignment of microfibril to microfibril locally with initially sections of microfibril aligning with sections of other microfibrils and then more complete lengths of microfibrils aligning with each other. In the second stage, the aligned microfibrils straighten and intertwine into bundles that then align in a long-range ordered array.
(7) Akutagawa, T.; Ohta, T.; Hasegawa, T.; Nakamura, T.; Christensen, C. A.; Becher, J. Proc. Natl. Acad. Sci. 2002, 99, 5028. (8) Kunstelj, K.; Federiconi, F.; Spindler, L.; Drevensek-Olenik, I. Colloids Surf. B 2007, 59, 120. (9) Hattori, S.; Adachi, E.; Ebihara, T.; Shirai, T.; Someki, I.; Irie, S. J. Biochem. 1999, 125, 676.
Supporting Information Available: Experimental details and additional AFM images. This material is available free of charge via the Internet at http://pubs.acs.org.
Acknowledgment. The National Science and Engineering Research Council of Canada supported this work.
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