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Direct Observation of Antifreeze Glycoprotein-Fraction 8 on Hydrophobic and Hydrophilic Interfaces Using Atomic Force Microscopy David M. Sarno,‡ Anastasia V. Murphy,† Evan S. DiVirgilio,† Wayne E. Jones, Jr.,*,‡ and Robert N. Ben*,† Department of Chemistry and Department of Chemistry/Institute of Materials Research, State University of New York at Binghamton, Binghamton, New York 13902 Received December 20, 2002. In Final Form: March 5, 2003 The adsorption of antifreeze glycoprotein-fraction 8 (AFGP 8) was studied via a drop deposition method onto hydrophobic (HOPG) and hydrophilic (mica) surfaces. On HOPG, preferential adsorption occurred at the hydrophilic step edges with homogeneous nucleation on hydrophobic planes. Individual particle sizes are consistent with aggregates, and the uniform size of these particles suggests aggregation is a preadsorption event. The adsorption of AFGP 8 onto hydrophilic mica was also consistent with aggregates of individual molecules. Adsorption studies of other glycosylated and nonglycosylated proteins suggest that not only is the adsorption event onto different surfaces influenced by glycosylation but also secondary and tertiary protein structure may play a critical role.
Introduction Antifreeze glycoproteins (AFGPs) are biological antifreezes found in several species of Atlantic and Antarctic teleost fish. These novel compounds have the ability to inhibit the growth of ice and protect organisms from cryoinjury and death.1 AFGPs are complex glycopolymers ranging in molecular weight from 2.4 to 34 kDa. The core repeating unit consists of an L-alanyl-L-alanyl-L-threonyl tripeptide where the secondary hydroxyl group of Lthreonine is glycosylated with β-D-galactosyl-(1,3)-R-DN-acetylgalactosamine (Figure 1). Unlike colligatively acting substances, AFGPs affect a noncolligative depression of the freezing point below that of the melting point; this phenomenon is referred to as thermal hysteresis (TH). The macromolecular mechanism has been described as an adsorption-inhibition process in which the AFGP adsorbs irreversibly onto the surface of an ice crystal.2 While the ice front continues to grow in areas adjacent to individual AFGP molecules, these surfaces possess a high radius of curvature (ice pitting). As the radius of curvature increases, it becomes energetically unfavorable to incorporate water molecules into the ice lattice and a localized freezing point depression is observed; this is referred to as the Kelvin effect.3 While this mechanism sufficiently rationalizes observed changes in the ice surface, it does not explain why different biological antifreezes do not bind to the same prism faces of an ice crystal.1b Nor does it elucidate the key structural features necessary for ice binding. Central to these issues is the role of hydrophilic and/or hydrophobic interactions between AFGP and the ice surface. For instance, AFGP contains one dissacharide for every three amino acid residues and each disaccharide possesses six hydrophilic hydroxyl groups. However, these glycoproteins possess a significant hydrophobic compo† ‡
Department of Chemistry. Department of Chemistry/Institute of Materials Research.
(1) For recent reviews of antifreeze proteins and antifreeze glycoproteins, see: (a) Davies, P. L.; Sykes, B. D. Curr. Opin. Stuct. Biol. 1997, 7, 828. (b) Ewart, K. V.; Lin Q.; Hew, C. L. Cell. Mol. Life Sci. 1999, 55, 271. (c) Ben, R. N. ChemBioChem 2001, 2, 161. (2) Knight, C. A. Nature 2000, 406, 249. (3) Wilson, P. W. Cryo-Lett. 1993, 14, 31.
Figure 1. Typical antifreeze glycoprotein.
nent, as their composition is two-thirds alanine. Recent experiments with type I AFP mutants, close analogues of AFGP, have suggested that the hydrophobic alanine residues are extremely important for antifreeze activity.4 Consequently, researchers are divided in opinion as to which is the dominant force for adsorption of AFGP to ice5 and these factors have made the rational design and de novo synthesis of chemically and biologically stable AFGP analogues suitable for many medical, industrial, and commercial applications6 a formidable challenge. In recent years, various analytical techniques have assisted in the study of complex biomolecules. Through the application of atomic force microscopy (AFM), scientists have been able to directly characterize molecular structure at the nanometer scale, effectively probing and quantifying the affinity with which proteins adsorb at solid-liquid interfaces.7 Given this precedent, we sought (4) Haymet, A. D. J.; Ward, L. G.; Harding, M. M. J. Am. Chem. Soc. 1999, 121, 941. (5) (a) Wierzbicki, A.; Taylor, M. S.; Knight, C. A.; Madura, J. D.; Harrington, J. P.; Sikes, C. S. Biophys. J. 1996, 71, 8. (b) Chao, H.; Houston, M. E., Jr.; Hodges, R. S.; Kay, C. M.; Sykes, B. D.; Loewen, M. C.; Davies, P. L.; Sonnichsen, F. D. Biochemistry 1997, 36, 14652. (6) (a) Griffith, M.; Ewart, K. V. Biotechnol. Adv. 1995, 13, 375. (b) Hays, L. M.; Feeney, R. E.; Crowe, L. M.; Crowe, J. E.; Oliver, A. E. Proc. Natl. Acad. Sci. USA 1996, 93, 6835. (c) Tablin, F.; Oliver, A. E.; Walker, N. J.; Crowe, L. M.; Crowe, J. H. J. Cell. Physiol. 1996, 165, 305. (d) Hansen, T. N.; Smith, K. M.; Brockbank, K. G. M. Transplat. Proc. 1993, 25, 3182. (7) (a) Fritz, M.; Radmacher, M.; Cleveland, J. P.; Allersma, M. W.; Stewart, R. J.; Gieselmann, R.; Janmey, P.; Schmidt, C. F.; Nansma, P. K. Langmuir 1995, 11, 3529. (b) Lea, A. S.; Pungor, A.; Hlady, V.; Andrade, J. D.; Herron, J. N.; Voss, E. W., Jr. Langmuir 1992, 8, 68. (c) Lin, J. N.; Drake, B.; Lea, A. S.; Hansma, P. K.; Andrade, J. D. Langmuir 1990, 6, 509.
10.1021/la027046l CCC: $25.00 © 2003 American Chemical Society Published on Web 04/22/2003
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to examine the adsorption of AFGP 8 (the smallest glycoprotein fraction, with a molecular weight of approximately 2.6 kDa) on hydrophilic and hydrophobic surfaces with the hope of gaining insight into the nature of the protein-surface interface. While ice is the natural substrate for AFGPs, the exact structure of the ice surface has not been well characterized. Conventional twodimensional models of the ice lattice depict the surface as highly ordered, and researchers have attempted to predict glycoprotein-ice interactions by aligning hydroxyl groups of the carbohydrate residues with the ice lattice.8 However, the latest evidence suggests that a 10 Å, “quasi-ordered” dynamic ice layer acts as a boundary between the highly ordered ice surface and the water.9 Given that a functional dynamic model for the ice lattice has not been devised, attempts to predict ice-protein interactions via alignment of carbohydrate hydroxyl groups are not likely to elucidate the molecular mechanism of action. However, it may be possible to discern whether the driving force for adsorption of AFGP onto ice is hydrophilic or hydrophobic by studying the adsorption onto both hydrophilic and hydrophobic substrates. The surfaces of mica and highly order pyrolytic graphite (HOPG) are ideal for such studies, as they have been thoroughly characterized10,11 and represent extremes of hydrophilicity and hydrophobicity. While the adsorption of AFGP 1-5 on mica surfaces has been previously studied,12 we present the first observations of AFGP 8 on HOPG surfaces and describe the relative adsorption mechanism. Experimental Section Proteins. AFGP 8 was generously donated by A/F Protein Inc. as a lyophilized powder after extraction and purification from the rock or Greenland cod (Gadus ogac). Both oxytocin and glycosylated bovine serum albumin (BSA, galactose glycoconjugate with 20 carbohydrate residues per mole of peptide) were purchased from Sigma. Surfaces. Highly ordered pyrolytic graphite (HOPG) and mica were kindly donated by Dr. C. J. Zhong. These substrates were attached to 15 mm diameter steel disks for mounting in the atomic force microscope. Prior to solution deposition, a fresh HOPG surface was obtained by cleavage with adhesive tape. This was repeated until the HOPG surface was visibly smooth. Fresh mica surfaces were cleaved with a stainless steel razor blade. No further cleaning protocols were employed. Sample Preparation. All protein solutions were prepared in doubly distilled deionized water. AFGP 8 was prepared as 1 × 10-5 and 1 × 10-7 g/mL solutions, BSA was prepared as 1 × 10-6 and 1 × 10-12 g/mL solutions, and oxytocin was prepared as 1 × 10-6 and 1 × 10-9 g/mL solutions. One drop (0.1 mL) of solution was deposited via pipet onto a freshly cleaved surface. The sample was dried under a stream of nitrogen gas for 3 h and then dried under vacuum for approximately 10 h prior to imaging. All experiments were performed at room temperature (25 °C). Instrumentation. Atomic force microscopy was performed with a Digital Instruments NanoScope IIIa (Santa Barbra, CA). Images were obtained in tapping mode using silicon cantilevers with resonance frequencies ranging from approximately 260 to 320 kHz in air. The scan rates were adjusted to optimize the image quality for each sample and are included in the figure captions. All scans were taken at a resolution of 512 pixels per line. Images were simultaneously collected in height and (8) Knight, C. A.; Driggers, E.; DeVries, A. L. Biophys. J. 1993, 64, 252. (9) Karim, O. A.; Haymet, A. D. J. J. Chem. Phys. 1988, 89, 6889. (10) For characterization of the mica surface, see: Hunter, J. Foundations of Colloid Science, Vol. 1; Clarendon Press: Oxford, U.K., 1987. (11) For characterization of the HOPG surface, see: Zhong, C. J.; Han, L.; Maye, M. M.; Luo, J.; Jones, W. E., Jr. J. Chem. Educ. 2003, 80, 194. (12) Lavalle, Ph.; DeVries, A. L.; Cheng, C.-C. C.; Scheuring, S.; Ramsden, J. J. Langmuir 2000, 16, 5785.
Langmuir, Vol. 19, No. 11, 2003 4741 amplitude modes to help identify instrumental artifacts that could have been mistaken for surface features. Image Processing. Image processing and analysis was carried out using the NanoScope IIIa software. All images were brought through a standard flattening protocol. Average feature heights and widths were obtained from cross-sectional measurements at several locations on each image. Molecular Dynamics. The AFGP 8 structure was constructed with the Biopolymer module/Insight II version 98 (BIOSYM/ Molecular Simulations Inc.) operating on a Silicon Graphics O2 workstation. All calculations were carried out using the AMBER force field interfaced with the molecular mechanics package DISCOVER (BIOSYM/Molecular Simulations Inc.). Atom potential types and charges were set according to Homans potential types for carbohydrates in the AMBER force field.13 The initial structures were solvated in a 5 Å layer of water and minimized through 10 000 iterations using conjugate gradients. The amino and carboxylic termini were treated as charged, and no counterion was introduced. After energy minimization, a constrained molecular dynamics simulation was initiated. The temperature was raised 25 K in 1 ps steps from 100 to 600 K, followed by cooling to 300 K for 500 ps. A final minimization was performed until a derivative of less than 0.001 kcal/Å was achieved.
Results and Discussion Initial attempts at studying the adsorption of AFGP 8 from solution onto HOPG as a function of time were conducted using a standard AFM flow cell. Unfortunately, images of adsorbed AFGP 8 could not be obtained even after the extended time 6 h. Two possible reasons exist for this. First, the rate of adsorption of AFGP onto HOPG might be very slow, implying that AFGP does not bind well to a hydrophobic surface. Alternatively, if the surface interactions of adsorbed AFGP are very weak, the AFM tip might sweep the AFGP off the surface. In this instance, AFGP 8 might favor binding to the AFM tip.14 To circumvent these issues, we employed a slightly modified deposition procedure based upon work by Droz et al.15 In this procedure, a 100 µL drop of aqueous AFGP 8 was placed on a fresh HOPG surface. The sample was dried under a stream of nitrogen gas for 3 h, followed by vacuumdrying overnight and subsequent imaging. Figure 2 shows AFM images obtained after the deposition from a 1.0 × 10-5 g/mL solution of AFGP 8 on HOPG. The fact that the AFGP 8 remains on the surface during scanning suggests that interactions between the cantilever tip and AFGP 8 are minimal and that the solution cell experiments failed because the rate of adsorption onto the HOPG surface was very low. One of the most striking features in Figure 2 is that the protein is clearly visible as irregularly shaped particles oriented along straight lines. These lines represent the nanometer-sized step edges and planes typical of the freshly cleaved HOPG surface. The heights of individual globules range from 4 to 7 nm and the widths are between 80 and 100 nm. While AFGP 8 appears to have been deposited in an even or homogeneous fashion over the entire surface, the step edges are almost completely covered by the glycoprotein (Figure 2b). When the experiment is repeated at 100-fold dilution (1.0 × 10-7 g/mL), the distribution of glycoprotein on the flat hydrophobic HOPG planes is significantly reduced, while the step edges remain evenly coated (Figure 3). The observed affinity of AFGP 8 for the step edges of the HOPG may be explained by the fact that these edges are readily oxidized under ambient conditions and, (13) Homans, S. W. Biochemistry 1990, 29, 9110. (14) Cullen, D. C.; Lowe, C. R. J. Colloid Interface Sci. 1994, 166, 102. (15) Droz, E.; Taborelli, M.; Descouts, P.; Wells, T. N. C. Biophys. J. 1994, 67, 1316.
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Figure 2. AFM images of AFGP 8 deposited from aqueous solution (1.0 × 10-5 g/mL) onto a freshly cleaved HOPG surface: (a) scan rate, 1.20 Hz; (b) scan rate, 1.65 Hz.
Figure 3. AFM images of AFGP 8 deposited from aqueous solution (1.0 × 10-7 g/mL) onto a freshly cleaved HOPG surface: (a) scan rate, 2.35 Hz; (b) scan rate, 1.80 Hz.
consequently, are hydrophilic relative to the planar surface regions of HOPG.16 To ascertain whether AFGP 8 was actually adsorbed to the surface, a rinsing experiment was performed. In this experiment, several drops of aqueous AFGP 8 (1.0 × 10-5 g/mL) were deposited on the HOPG surface and then immediately washed three times using distilled water from a syringe. The sample was then dried under a stream of nitrogen gas for 4 h and vacuum-dried overnight prior to being imaged. The images looked nearly identical to those in Figure 2 with homogeneous nucleation of AFGP 8 at both hydrophilic and hydrophobic surfaces. This result suggests not only that AFGP 8 physically binds to the HOPG surface but also that it is resistant to desorption processes. To rule out the possibility of electrostatic attraction between the protein and the HOPG, several samples were prepared in which the steel sample disks were electrically grounded during the deposition process. The images that were obtained were identical to those in Figure 2, suggesting that AFGP 8 was actually adsorbed or at least closely associated with the HOPG surface. Another interesting observation is that the dimensions of individual particles at both the step edges and planes do
not correspond to single AFGP 8 molecules. We have performed molecular dynamics simulations of solvated AFGP 8, which estimate that an individual molecule is approximately 2.4 nm in length by 1.3 nm in height with an overall ellipsoidal geometry (Figure 4). Individual particles in Figures 2 and 3 range from 80 to 100 nm, which is consistent with aggregates of AFGP 8. In contrast, experiments with longer chain glycopolymers (AFGP 1-5) show the adsorption as individual molecules, not aggregates.12 Our observation may be especially relevant to the molecular mechanism of action for lower molecular weight AFGPs. The relatively narrow range of particle size is consistent with aggregation prior to adsorption (i.e., aggregation after adsorption should produce heterogeneously sized particles). Since aggregates are observed, it is also not possible to determine from these data whether AFGP 8 undergoes a conformational change during the adsorption event. In an effort to better understand the nature of the AFGP interaction with hydrophilic surfaces, the adsorption of AFGP 1-5 on amorphous silica-titania and muscovite mica has been studied using flow cell techniques.12 The images obtained on each surface were initially very similar.
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Figure 4. Molecular dynamics simulation of solvated AFGP 8 performed using MSI Insight II.
Figure 5. AFM image of AFGP 8 deposited from aqueous solution (1.0 × 10-5 g/mL) onto a freshly cleaved mica surface: scan rate, 1.80 Hz.
On silica-titania, proteins were initially deposited as individual molecules that underwent a conformational change and that later aggregated into conical structures. In contrast, adsorption onto mica resulted in a random distribution of proteins with dimensions consistent with that of individual molecules. Figure 5 represents a typical image obtained after the adsorption of AFGP 8 onto mica using our modified drop deposition method. The image is consistent with that reported by Ramsden and co-workers12 in that we see a random distribution of protein evenly coating the surface. However, the dimensions of the AFGP 8 particles are again consistent with aggregates, as we observed on HOPG. The observation of protein aggregation on both hydrophilic and hydrophobic surfaces suggests that this phenomenon may be unique to AFGP 8, and not AFGP 1-5. The preferred binding of AFGP 8 to the hydrophilic step edges of HOPG at low protein concentrations is intriguing, since the peptide backbone is two-thirds alanine and hydrophobic in nature. To determine if this property was unique to AFGPs, we investigated the adsorption of two very different proteins onto HOPG using our experimental conditions. The first was a glycosylated bovine serum albumin (galactose-BSA conjugate), which is a globular protein with a molecular weight of approximately 100 kDa. In this glycoconjugate the galactose
is conjugated to BSA through an amide bond and a flexible organic spacer. This particular glycoconjugate was selected because it has been previously utilized as a negative control in recrystallization-inhibition assays to assess antifreeze protein-specific activity.17 At the concentration 1.0 × 10-6 g/mL, the resulting surface was too rough and irregular to obtain any useful images. However, Figure 6 depicts the AFM image obtained after deposition from a 1.0 × 10-12 g/mL solution. From this image it is immediately apparent that the adsorption of glycosylated BSA onto HOPG is very different from that of AFGP 8, despite the fact that both are glycoproteins. The pattern of adsorption with glycosylated BSA is consistent with the previously reported time-resolved adsorption of glycosylated proteins onto HOPG.14 In these reports, hydrophilic proteins nucleate at the hydrophilic step edge and then spread out from the edges to give an image very similar to that shown in Figure 6. Consequently, the surface coverage is heaviest at the steps and lowest at regions furthest from the edges. As a result, some areas of the hydrophobic HOPG planes contain no bound protein. Upon the basis of the irregular dimensions of the individual nucleation sites, it seems likely that aggregation is a postadsorption event. Using the same procedures, we have also investigated the adsorption of oxytocin, a nonglycosylated protein. Figure 7 shows the AFM image after adsorption from a 1 × 10-6 g/mL aqueous solution onto HOPG. Again, the image is consistent with that of the time-resolved adsorption of other proteins onto HOPG. Adsorption of oxytocin onto HOPG results in a more even surface distribution compared to that of glycosylated BSA or AFGP 8. In the absence of time-resolved experiments, it is difficult to speculate how this occurs or whether the secondary and/ or tertiary structure of the protein has an effect. At lower concentrations, oxytocin binds preferentially to the hydrophilic steps of HOPG like AFGP 8. Conclusions The drop deposition method is an effective way to adsorb proteins onto hydrophilic and hydrophobic surfaces. Our results are comparable to solution cell methods in that proteins are distributed in a random fashion at high concentration. The fact that AFGP 8 remains bound to HOPG after rinsing is consistent with the notion that many proteins adsorb more strongly to hydrophobic than (16) Rice, R. J.; McCreery, R. L. Anal. Chem. 1989, 61, 1637. (17) Eniade, A.; Purushotham, M.; Wang, J. B.; Horwath, K.; Ben, R. N. Cell Biochem. Biophys., in press.
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Figure 6. AFM images of adsorbed glycosylated BSA on HOPG deposited from aqueous solution (1.0 × 10-12 g/mL): (a) scan rate, 1.49 Hz; (b) scan rate, 2.18 Hz.
Unlike the higher molecular weight analogues, it is likely that AFGP 8 aggregation is a preadsorption event. This aggregation may be suggestive of a difference in the mode of action of AFGP 8 relative to the larger, more potent glycopolymer fractions AFGP 1-5. Time-revolved adsorption studies as well as dynamic light scattering experiments are also being considered to support this observation. Finally, it appears that while AFGP 8 may be regarded as an amphipathic glycoprotein (both hydrophilic and hydrophobic), our studies indicate that when presented with both hydrophilic and hydrophobic binding sites (i.e. step edges or planes of HOPG) AFGP 8 binds preferentially to the hydrophilic step edges. This result suggests that the binding mechanism may not be universal to all types of biological antifreezes.
Figure 7. AFM height image of oxytocin adsorbed from aqueous solution (1.0 × 10-6 g/mL) on HOPG (scan rate, 2.18 Hz).
hydrophilic surfaces. However, at lower concentrations, AFGP 8 appears to bind preferentially to hydrophilic step edges of HOPG. It is clear that the surface adsorption and distribution of proteins and glycoproteins is a complex phenomenon in which primary, secondary, and tertiary structures all contribute in some way. The adsorption of AFGP 8 onto HOPG and mica results in homogeneous particle sizes.
Acknowledgment. R.N.B. acknowledges the National Institutes of Health (RO1GM60319), the Petroleum Research Fund, administered by the American Chemical Society (PRF 35280-G1), and A/F Protein for financial support. W.E.J. acknowledges the National Institutes of Health (R15ES10106) and the National Science Foundation (DUE9952628 and DMR9976713). In addition, Professor C. J. Zhong of Binghamton University is thanked for supplying the HOPG and mica samples, and Professor John DiNardo of Drexel University is thanked for helpful discussions. LA027046L
