Conformational Changes in the Plasma Protein Fibrinogen upon

Citation data is made available by participants in Crossref's Cited-by Linking service. .... Fibrinogen Adsorption on Mica Studied by AFM and in Situ ...
7 downloads 4 Views 219KB Size
Langmuir 2003, 19, 9883-9888

9883

Conformational Changes in the Plasma Protein Fibrinogen upon Adsorption to Graphite and Mica Investigated by Atomic Force Microscopy Katherine L. Marchin and Cindy L. Berrie* Department of Chemistry, University of Kansas, Lawrence, Kansas 66045-7582 Received June 25, 2003. In Final Form: September 2, 2003 Atomic force microscopy has been used to investigate the adsorption of the plasma protein fibrinogen on graphite and mica substrates. These substrates serve as model hydrophobic and hydrophilic surfaces, respectively. The overall structure of submonolayer coverage films is dramatically different on the mica and graphite substrates when imaged under ambient conditions after being dried under a nitrogen stream. The molecules show a tendency to aggregate on the graphite substrate but adsorb as isolated single molecules in the case of mica. On the mica substrate, individual fibrinogen molecules appear globular in structure whereas, on graphite, the trinodular structure is most commonly observed. The average height of the fibrinogen molecules as measured by tapping mode AFM in air is 1.71 ( 0.65 nm, and the average height on the graphite substrate is 1.05 ( 0.13 nm. The average lengths and widths of the molecules on these two substrates vary as well, the average length being 31 ( 7 nm on mica and 63 ( 10 nm on graphite. These differences are consistent with a change in conformation of the protein upon adsorption of these two surfaces due to the differences in surface chemistry of the substrates, which suggests a change in mechanism of adsorption between the two substrates.

Introduction The interaction of proteins with surfaces is an important topic in areas ranging from biocompatibility of medical implants to biosensor development.1,2 The conformation and amount of protein adsorbed on the surface depend strongly on the surface chemistry, but the details of the differences in binding are not well understood. This study uses atomic force microscopy to investigate the adsorption of the plasma protein fibrinogen on two well-defined surfaces, graphite and mica. The observed shapes are consistent with a model presented for conformational changes of the molecule on the different surfaces upon adsorption. The plasma protein fibrinogen is a large (∼340 kD) protein involved in thrombosis. It plays two roles in this process: fibrinogen is cleaved and converted to fibrin, which is polymerized and is involved in platelet aggregation.3,4 Being one of the most abundant plasma proteins, fibrinogen adsorbs strongly to a variety of surfaces, with the amount of adsorbed protein varying less with surface chemistry than is the case for other proteins.4 The conformation of surface-bound fibrinogen has been shown to play an important role in platelet adhesion and thrombus formation,5-8 but the details of the conformational changes fibrinogen undergoes are not well understood. The native structure of fibrinogen has been described as trinodular4,9 with three hydrophobic domains connected * To whom correspondence should be addressed. E-mail: [email protected]. (1) Brash, J. L., Horbett, T. A., Eds. Proteins at Interfaces. Physicochemical and Biochemical Studies. ACS Symp. Ser. 1987, 343, 1987. (2) Horbett, T. A., Brash, J. L., Eds. Proteins at Interfaces II: Fundamentals and Applications (Developed from a symposium sponsored by the Division of Colloid and Surface Science at the 207th National Meeting of the American Chemical Society, San Diego, CA, March 1317, 1994). ACS Symp. Ser. 1995, 602, 1995. (3) McManama, G.; Lindon, J. N.; Kloczewiak, M.; Smith, M. A.; Ware, J. A.; Hawiger, J.; Merrill, E. W.; Salzman, E. W. Blood 1986, 68, 363-371. (4) Feng, L.; Andrade, J. D. ACS Symp. Ser. 1995, 602, 66-79.

by R-helical coiled coil domains.10 The length of an individual fibrinogen molecule is 45-50 nm.4,9,11 Several AFM12-19 experiments have been conducted on fibrinogen, including an early investigation of the formation of fibers,20 as well as a number of electron microscopy11,21-24 studies to investigate the structure of surface-adsorbed fibrinogen. The trinodular structure of this molecule has been observed in both AFM and EM studies on a variety of different substrates including silicon dioxide and hydro(5) Pitt, W. G.; Park, K.; Cooper, S. L. J. Colloid Interface Sci. 1986, 111, 343-362. (6) Lindon, J. N.; McManama, G.; Kushner, L.; Merrill, E. W.; Salzman, E. W. Blood 1986, 68, 355-362. (7) Pekala, R. W.; Merrill, E. W.; Lindon, J.; Kushner, L.; Salzman, E. W. Biomaterials 1986, 7, 379-385. (8) Balasubramanian, V.; Grusin, N. K.; Bucher, R. W.; Turitto, V. T.; Slack, S. M. J. Biomed. Mater. Res. 1999, 44, 253-260. (9) Weisel, J. W.; Stauffacher, C. V.; Bullitt, E.; Cohen, C. Science 1985, 1388-1391. (10) Ta, T. C.; Sykes, M. T.; McDermott, M. T. Langmuir 1998, 14, 2435-2443. (11) Gorman, R. R.; Stoner, G. E.; Catlin, A. J. Phys. Chem. 1971, 75, 2103-2107. (12) Ortega-Vinuesa, J. L.; Tengvall, P.; Lundstrom, I. Thin Solid Films 1998, 324, 257-273. (13) Sit, P. S.; Marchant, R. E. Surf. Sci. 2001, 491, 421-432. (14) Wigren, R.; Elwing, H.; Erlandsson, R.; Welin, S.; Lundstrom, I. FEBS Lett. 1991, 280, 225-228. (15) Cacciafesta, P.; Humphris, A. D. L.; Jandt, K. D.; Miles, M. J. Langmuir 2000, 16, 8167-8175. (16) Sit, P. S.; Marchant, R. E. Thromb. Haemostasis 1999, 82, 10531060. (17) Marchant, R. E.; Barb, M. D.; Shainoff, J. R.; Eppell, S. J.; Wilson, D. L.; Siedlecki, C. A. Thromb. Haemostasis 1997, 77, 1048-1051. (18) Jandt, K. D.; Finke, M.; Cacciafesta, P. Biomaterials 2000, 19, 301. (19) Taatjes, D. J.; Quinn, A. S.; Jenny, R. J.; Hale, P.; Bovill, E. G.; McDonagh, J. Cell Biol. Int. 1997, 21, 715-726. (20) Drake, B.; Prater, C. B.; Weisenhorn, A. L.; Gould, S. A. C.; Albrecht, T. R.; Quate, C. F.; Cannell, D. S.; Hansma, H. G.; Hansma, P. K. Science 1989, 243, 1586-1589. (21) Nygren, H.; Stenberg, M.; Karlsson, C. J. Biomed. Mater. Res. 1992, 26, 77-91. (22) Nygren, H.; Stenberg, M. J. Biomed. Mater. Res. 1988, 22, 1-11. (23) Fowler, W. E.; Erickson, H. P. J. Mol. Biol. 1979, 134, 241-249. (24) Stoner, G. E.; Srinivasan, S.; Gileadi, E. J. Phys. Chem. 1971, 75, 2107-2111.

10.1021/la035127r CCC: $25.00 © 2003 American Chemical Society Published on Web 10/16/2003

9884

Langmuir, Vol. 19, No. 23, 2003

phobic and hydrophilic self-assembled monolayers terminating in methyl or amine groups on silicon dioxide,14,16,17 titanium oxide,15,18,25 PMMA,25 graphite and mica,13,16,17 and polylysine coated mica.19 In some experiments, two types of molecules termed “globular” and “trinodular” were observed.14 In addition, there have been investigations of the adhesion force between fibrinogen and a variety of surfaces.26,27 While there have been a number of measurements of fibrinogen absorption on different surfaces, the conclusions from such studies are sometimes contradictory and there is still not a clear understanding of how surface chemistry influences conformation. In addition, in a study involving the time evolution of the protein film structure, differences were observed in the overall structure of the fibrinogen film on graphite and mica substrates.10 While the conformations of individual molecules were not investigated in this experiment, it was speculated that the differences in the structure were a result of differences in the conformation of the individual molecules. One goal of this work is to probe the conformations of the individual molecules in detail. In addition to the microscopy investigations, there have been a number of other methods used to investigate fibrinogen adsorption at surfaces including infrared spectroscopy,28,29 ellipsometry,21,22,30-32 total internal reflection fluorescence (TIRF),33,34 and elution measurements.35-39 The elution studies suggest several conformations of adsorbed protein which have different affinity for the surface. Infrared spectroscopy allows the investigation of average changes in the secondary structure of the protein. Ellipsometry allows kinetic information to be obtained by monitoring the amount of adsorbed protein as a function of time. The concentration and flow rate of the solution above the sample have been shown to have an impact on the distribution of molecules bound on the surface through TIRF measurements.33,34 The orientation and degree of unfolding of the molecules are affected by the rate at which new molecules arrive at the surface and the free surface area available around the molecule. In addition, these measurements have shown that the average footprint of a molecule is larger on a hydrophobic surface than a hydrophilic one.33,34 While these techniques provide a great deal of valuable information, they measure the average properties of the film and not the structure of individual molecules. In this paper, we report detailed AFM investigations of the conformation of the fibrinogen molecules adsorbed on graphite and mica substrates. The results demonstrate (25) Jandt, K. D. Surf. Sci. 2001, 491, 303-332. (26) Hemmerle, J.; Altmann, S. M.; Maaloum, M.; Horber, J. K. H.; Heinrich, L.; Voegel, J. C.; Schaaf, P. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6705-6710. (27) Kidoaki, S.; Matsuda, T. Langmuir 1999, 15, 7639-7646. (28) Lu, D. R.; Park, K. J. Colloid Interface Sci. 1991, 144, 271-281. (29) Lenk, T. J.; Horbett, T. A.; Ratner, B. D.; Chittur, K. K. Langmuir 1991, 7, 1755-1764. (30) Lassen, B.; Malmsten, M. J. Colloid Interface Sci. 1996, 179, 470-477. (31) Malmsten, M.; Lassen, B. ACS Symp. Ser. 1995, 602, 228-238. (32) Elwing, H. Biomaterials 1998, 19, 397-406. (33) Wertz, C. F.; Santore, M. M. Langmuir 2001, 17, 3006-3016. (34) Wertz, C. F.; Santore, M. M. Langmuir 2002, 18, 706-715. (35) Poumier, F.; Schaaf, P.; Voegel, J. C. Langmuir 1999, 15, 62996303. (36) Slack, S. M.; Horbett, T. A. J. Colloid Interface Sci. 1989, 133, 148-165. (37) Chinn, J. A.; Posso, S. E.; Horbett, T. A.; Ratner, B. D. J. Biomed. Mater. Res. 1991, 25, 535-555. (38) Chinn, J. A.; Posso, S. E.; Horbett, T. A.; Ratner, B. D. J. Biomed. Mater. Res. 1992, 26, 757-778. (39) Retzinger, G. S.; Cook, B. C.; DeAnglis, A. P. J. Colloid Interface Sci. 1994, 168, 514-521.

Marchin and Berrie

dramatic differences in the conformation of the molecules on these two surfaces. Statistically significant differences were observed in the average heights and lengths of the molecules on the different surfaces, consistent with a change in conformation of the molecule upon binding. These changes in conformation could have an influence on the biocompatibility of surfaces. Experimental Section Materials. Bovine fibrinogen was obtained from Sigma and used without further purification. The fibrinogen solutions were made using a 0.05 M pH 7.0 phosphate buffer. The concentration of protein solution was varied from 0.05 to 10 µg/mL. HOPG graphite (Digital Instruments) and ruby muscovite mica (Lawrence Mica Company) were used as substrates, and both were freshly cleaved immediately prior to use. AFM Measurements. AFM experiments were conducted with a Digital Instruments Nanoscope IIIA Multimode system in tapping mode with typical scan rates of 1-3 Hz. The measurements were performed in tapping mode using standard silicon cantilevers with resonance frequencies from 300 to 400 kHz (Digital Instruments). The protein solution was allowed to adsorb on the surface for 10 min and then removed and rinsed with Millipure water (resistivity > 18 MΩ) and dried under a nitrogen stream before imaging with the AFM. The images are unfiltered and have been smoothed with a plane fit when needed. The measurements have not been corrected for the convolution with the tip shape that can significantly affect the absolute values for the lengths and widths measured; however, the trends in the relative values of these measurements should not be affected. In addition, the smaller the dimension, the larger the effect of the tip shape on the measured size; that is, the widths would likely be more affected than the lengths. The volumes will also tend to be overestimated.

Results The tapping mode images of the fibrinogen films show a dramatic dependence on the surface chemistry. Figure 1 (substrate: A ) graphite, B ) mica) shows images of the fibrinogen film taken in tapping mode as described above. The lines crossing the image in the case of the graphite sample are individual atomic steps on the graphite substrate. There are several features of interest in these images. First, the structures of the films are very different. The molecules appear in clusters on the graphite substrate whereas they appear as isolated single molecules in the case of the mica substrate. The molecules are more uniformly distributed on the mica substrate than on the graphite substrate with large bare areas visible on the graphite substrate. This is consistent with earlier timeresolved work that showed that the gross structures of the films formed on these two substrates were different. In addition, the features are higher on average on the mica substrate than on the graphite substrate. These images are stable, and the molecules can be imaged again with no notable change in structure under the imaging conditions employed in these experiments. By zooming in on a smaller length scale, one can observe some of the internal structure of the individual fibrinogen molecules. This has been demonstrated before in both EM11,21-24 and AFM12-19 investigations of fibrinogen adsorption. Figure 2A shows an individual fibrinogen molecule adsorbed on the graphite substrate. The trinodular structure is clearly evident in this image. A crosssectional cut through this molecule is shown in Figure 2B, where the height and width of the molecule as well as the three nodes of the molecules are clearly observed. The height of this molecule is 1.18 nm with a length of 58 nm. This is consistent with previous AFM measurements that provide estimates of 40-67 nm for the length of a fibrinogen molecule on a variety of substrates including

Fibrinogen Adsorption

Langmuir, Vol. 19, No. 23, 2003 9885

Figure 2. AFM image of a single fibrinogen molecule adsorbed on a graphite substrate. This molecule is ∼1.1 nm high. The trinodular structure is clearly evident in the cross-sectional profile.

Figure 1. 2 µm × 2 µm AFM images of fibrinogen adsorbed on (A) graphite and (B) mica. The images were taken in tapping mode. Aggregation is observed on the graphite substrate, while individual molecules are predominant on the mica substrate.

silicon dioxide, PMMA, OTS, and ATPES SAM films, mica, and titanium dioxide.15 It should be noted that the height measured in these images is significantly less than that expected on the basis of the known structure. This is likely a result of the force of the AFM causing a perturbation to the molecules during the imaging process. While many of the molecules are contained within clusters, the majority of the isolated molecules that can be measured do demonstrate this trinodular structure. We occasionally observe a molecule that has a bent structure instead of the linear trinodular structure that is most often observed. In contrast, on the mica substrate, the trinodular structure is much less frequently observed. The majority of the molecules appear much more globular in structure than is the case on the graphite substrate. An example is shown in Figure 3. A cross-sectional cut through this molecule shows a single peak with a height of approximately 1.7 nm. While the vast majority of the molecules on this substrate appear globular in structure, there is a significantly broader distribution of molecules than that on the graphite substrate. As shown in Figure 4, within some images containing many globular molecules, there will be molecules in which the trinodular structure can be resolved. These experiments have been repeated more than five times with a large number of images collected during each

Figure 3. AFM image of a single fibrinogen molecule on a mica surface. The cross-sectional profile is shown below for the line marked in the image. This molecule is very globular in shape and has a height of ∼1.7 nm.

experiment. The results are quite reproducible under similar conditions, even with the use of different tips and different substrates on different days. Statistical analysis of the images is possible and allows us to determine the average height, width, and length of molecules on the surface. A height histogram of an image of fibrinogen on graphite (shown in Figure 5A) is shown in Figure 5B. This histogram is for an individual image and, therefore, is from a single measurement. This height histogram

9886

Langmuir, Vol. 19, No. 23, 2003

Marchin and Berrie

Figure 4. AFM image of fibrinogen on a mica substrate. This image shows a mixture of globular molecules as well as more extended molecules, some of which exhibit trinodular structure.

Figure 6. (A) Image of fibrinogen on mica and (B) height histogram for the image in part A. The histogram shows two features, the separation of which is related to the average height of the molecules. Table 1. Average Fibrinogen Dimensions Obtained from Cross-sectional Measurements of Individual Molecules, Such as Those Shown in Figures 2 and 3

Figure 5. (A) Image of fibrinogen on graphite and (B) height histogram for the image in part A. The histogram shows two peaks, the separation of which is related to the average height of the molecule on the surface.

shows two peaks, one at the height corresponding to the substrate and one at the height related to the average height of the molecules. From the difference between these two peaks we can estimate an average height for the molecules on the surface. Figure 6 shows a similar histogram for the mica substrate. In this case, the second peak is not as high and is significantly broadened, but it is still present. An average of the results, calculated from

substrate

length (nm)

width (nm)

height (nm)

mica graphite

31 ( 7 62 ( 9

26 ( 5 28 ( 7

1.71 ( 0.65 1.05 ( 0.13

the histograms for several images, shows a height of 1.3 ( 0.2 nm on the mica substrate and 0.92 ( 0.04 nm on the graphite substrate. In addition, by taking crosssectional measurements on individual molecules, the average length, width, and height of the molecules is estimated. Table 1 summarizes the measurements of the molecules obtained from these cross-sectional measurements. The number of molecules analyzed for the mica (75) is significantly larger than the number of molecules on the graphite (30), since the graphite molecules are often contained in clusters from which it is difficult to obtain accurate measurements. The molecules, on average, appear higher and more globular in structure on the mica substrate and flatter and more trinodular on the graphite substrate. Crude calculations of the volumes based on a rectangular box with the average dimensions for the molecules presented in Table 1 suggest that the volume is consistent with a single molecule in both cases. We have also conducted experiments where the imaging is done under buffer, and the results show very similar trends; this will be the subject of a future publication. The molecules still appear more clustered on the graphite substrate than on the mica substrate, but the molecules on both substrates were still stably imaged under buffer.

Fibrinogen Adsorption

Figure 7. Schematic diagram of one possible conformational change induced by the changes in surface chemistry. On the hydrophobic surface, the molecule interacts through the globular hydrophobic domains and lies flat while, on the hydrophilic substrate, the molecule interacts through the hydrophilic “arms” and the molecule appears globular in shape. This type of conformational change is consistent with the AFM measurements presented above.

Discussion The fact that the molecules tend to cluster together on the graphite substrate and adsorb as isolated single molecules on mica had previously been observed, and conformational changes of the individual molecules were invoked as a possible explanation, although the conformational changes could not be observed directly.10 A schematic drawing of the structure of fibrinogen is shown in Figure 7. The three hydrophobic regions (domains D and E) are connected by helical coiled coil domains with a section of relatively hydrophilic composition of amino acid residues sticking out from each end (R chain “arms”). Interaction with the hydrophobic graphite substrate is likely to occur through the hydrophobic regions of these molecules, leaving the “arms” free in solution while interaction with the hydrophilic mica surface is likely to involve the “arms” containing the more hydrophilic residues. In this case, with the arms tethered to the surface, the “arms” are no longer free to interact with neighboring molecules as they adsorb to the surface, so adsorption occurs as isolated single molecules. On the other hand, if adsorption on the graphite substrate occurs through the hydrophobic domains, the “arms” would remain free in solution to interact with other molecules. This is a possible explanation for the aggregation observed on the graphite substrate and the absence of aggregation observed on the mica substrate. Protein-protein interactions then are obviously important in the adsorption of fibrinogen to graphite, but they are less important in the mechanism of fibrinogen adsorption on mica. Analysis of the images is consistent with this picture. The average height of the molecules is larger on the mica substrate than on the graphite substrate. One possible way that the hydrophilic arms could interact with the substrate on the mica surface would be for those arms to fold up under the molecule, as depicted in the schematic drawing in Figure 7, making the molecule protrude farther from the surface and appear higher than in the case of the graphite substrate, where interaction occurs directly with the hydrophobic domains. Not only is the difference in height consistent with this idea, but also the measurements of the length of the molecules can be explained in this way. The longer, more trinodular structure observed

Langmuir, Vol. 19, No. 23, 2003 9887

on the graphite substrate is an extended structure with interaction occurring through each of the nodes of the molecule. The more globular, compact structure observed on the mica substrate is consistent with the molecule folding over on itself and becoming more compact. As stated previously, this type of conformational change in fibrinogen on these two surfaces had been hypothesized as a possible explanation for the gross structure of the protein films observed,10 but the conformation of the molecules was not directly observed. In this experiment, we have provided direct evidence of the proposed conformational changes. The deposition on both the mica and graphite substrates was done from the same solution for the same amount of time at the same temperature, so these differences cannot be attributed to any difference in preparation conditions. The remarkable reproducibility of the results over many runs also provides confidence that the differences observed on these two substrates are a true result of the differences in interactions of the molecules with the surfaces. Recent results indicate the distribution of molecules on the mica surface may possibly be affected by changes in humidity, ionic strength, concentration, or temperature. As mentioned above, graphite is a hydrophobic surface while mica is hydrophilic. In addition, the mica surface as cleaved in air is negatively charged while the graphite surface is neutral. At pH 7.0, as in these experiments, the fibrinogen should have a net negative charge.4 The “arms” that extend from the terminal globular D domain have a +2 charge, thus making this a very favorable interaction site for the molecule with the negatively charged mica.4 Therefore, it is possible that not only the hydrophilicity but also the surface charge are important in determining the distributions of conformations observed on the surface. These results are consistent with recent TIRF measurements in which it was observed that the average footprint of fibrinogen is larger on a hydrophobic surface than on a hydrophilic one.33,34 We observe a larger footprint on the hydrophobic graphite than on the hydrophilic mica. In addition to the differences in shape observed on the two substrates, there also seems to be a larger variation on the mica substrate than on the graphite. This could be due to differences in the kinetics of equilibration on the surface with the graphite and mica or it could be a result of more similar energetics of adsorption in the different conformations on the mica than on the graphite substrate. This requires further investigation. In addition, the molecules on graphite seem to accumulate at the step edges and leave other areas on the terraces relatively free from adsorption. This is also observed in our solution images and is a topic to be investigated further in future studies. The distribution of molecules on the terraces and step edges does not change with continued imaging, suggesting that the probe tip is not responsible for the molecules being swept to the step edges during imaging. This may suggest that the molecules are initially fairly mobile on the surface and eventually get trapped at the step edge. Previous studies disagree as to the interaction of fibrinogen with a mica surface. In one experiment, the trinodular structure of fibrinogen was observed on mica16 while, in another, fibrinogen was found not to adsorb on mica without precoating with poly-L-lysine.19 Our studies show that the molecules do adsorb on the bare mica substrate and that a primarily globular conformation is adopted, although other conformations are also observed.

9888

Langmuir, Vol. 19, No. 23, 2003

Conclusions Differences in the height and length of individual fibrinogen molecules have been observed using atomic force microscopy, as have dramatic differences in the gross structure of the film. The molecules tend to aggregate on the graphite substrate but not on the mica. In addition, height histograms show that the average height of an individual fibrinogen molecule is larger on the mica substrate than on the graphite. Our results are consistent with a proposed conformational change10 in the molecules and both the size distributions and the aggregation state of the films. These results are also consistent with significant protein-protein interactions during the adsorption on the graphite substrate. The present results demonstrate the power of AFM in resolving conformational

Marchin and Berrie

changes induced in molecules through the interactions with surfaces as well as the ability to discern submolecular structure. Conformational changes in the plasma protein fibrinogen can lead to changes in activity which could dramatically affect biocompatibility; therefore, a detailed understanding of the effect of surface chemistry on conformation and the mechanism of adsorption is critically important. Acknowledgment. This material is based upon work supported by Kansas Technology Enterprise Corporation through a KTEC/NSF First Award and a 3M Nontenured Faculty Award. LA035127R