11200
Langmuir 2007, 23, 11200-11205
Conformational Changes in Salivary Proline-Rich Protein 1 upon Adsorption to Calcium Phosphate Crystals Satheesh Elangovan,† Henry C. Margolis,‡ Frank G. Oppenheim,*,† and Elia Beniash*,‡,§ Department of Periodontology and Oral Biology, Boston UniVersity Goldman School of Dental Medicine, Suite W201, 700 Albany Street, Boston, Massachusetts 02118-2392, and Department of Biomineralization, Forsyth Institute, 140 Fenway, Boston, Massachusetts 02115 ReceiVed May 14, 2007. In Final Form: July 29, 2007 Conformational analyses of PRP1, a proline-rich acidic salivary protein and major component of the acquired enamel pellicle, have been carried out in solution and upon binding to two enamel prototypes, hydroxyapatite (HA) and carbonated hydroxyapatite (CHA), using Fourier transform infrared spectroscopy (FTIR) in attenuated total reflection (ATR) mode. We have shown for the first time that, in solution, large portions of PRP1 adopt the hydrated polyproline type II (PPII) helical structure in addition to the random coil structure, with the maximum absorbance of the amide I band around 1620 cm-1. Upon binding to HA or CHA, the protein undergoes significant conformational changes, loosing a considerable portion of hydrated PPII and random coil domains with a shift in the maximum absorbance to 1666 cm-1, indicating that a large fraction of the protein is composed of β turns. A small fraction of PPII in a calcium-bound or anhydrous form (∼1642 cm-1) was also observed in the HA- and CHA-bound proteins, which could play a role in protein-mineral interactions. The conformational changes in PRP1 adsorbed on CHA and HA were similar in nature; however, these changes were greater in the protein bound to HA. Interestingly, these results are in agreement with protein adsorption data that show that less protein is adsorbed onto CHA than onto HA. Our results demonstrate that binding to apatitic mineral surfaces leads to major conformational changes in PRP1, which might reflect the expulsion of water and the formation of protein-mineral and/or protein-protein interactions in the adsorbed layer.
Introduction An understanding of the molecular mechanisms of protein adsorption to solid surfaces is extremely important in a variety of fields, including biomineralization. For example, it is widely accepted that specific protein-mineral interactions regulate the nucleation, growth, and shape of mineral crystals during the formation of mineralized tissues.1,2 In addition, protein-mineral interactions play a vital role in mineral homeostasis by forming protective protein layers such as in the periostracum of mollusk shells3 and in the acquired enamel pellicle (AEP) covering mammalian tooth enamel.4 Dental enamel is the hardest tissue in the human body, containing 96% carbonated hydroxyapatite (CHA) mineral by weight and small amounts of water and residual enamel matrix protein. The carbonate content of enamel mineral is 2 to 3 wt %.5 CHA is characterized by reduced crystallinity6 and decreased resistance to acids compared to hydroxyapatite (HA)7. Within the oral cavity, the AEP forms by the selective adsorption of * Corresponding authors. (F.G.O.) E-mail:
[email protected]. Tel: (617) 638 4727. Fax: (617) 638 4924. (E.B.) E-mail:
[email protected]. Tel: (412) 648-0108. Fax: (412) 634 6685. † Boston University Goldman School of Dental Medicine. ‡ Forsyth Institute. § Current address: University of Pittsburgh School of Dental Medicine, Department of Oral Biology, 693A Salk Hall, 3501 Terrace Street, Pittsburgh, Pennsylvania 15261. (1) Addadi, L.; Weiner, S. Angew. Chem., Int. Ed. Engl. 1992, 31, 153-169. (2) Weiner, S.; Addadi, L. J. Mater. Chem. 1997, 7, 689-702. (3) Lowenstam, H. A.; Weiner, S. On Biomineralization; Oxford University Press: New York, 1989. (4) Lendenmann, U.; Grogan, J.; Oppenheim, F. G. AdV. Dent. Res. 2000, 14, 22-28. (5) Sonju Clasen, S.; Ruyter, I. AdV. Dent. Res. 1997, 11, 523-527. (6) LeGeros, R. Z.; LeGeros, J. P. Carbonate Apatite: Formation and Properties; Kokubo, T., Nakamura, T., Miyaji, F., Eds.; Elsevier Science Ltd: Otsu, Japan, 1996; Vol. 9. (7) Nelson, D. G. A.; Featherstone, J. D. B.; Duncan, J. F.; Cutress, T. W. Caries Res. 1983, 17, 200-211.
salivary macromolecules onto tooth mineral surfaces.8 In addition to the regulation of enamel mineral homeostasis9 within the oral cavity, the functions of the AEP include lubrication10 and the regulation of oral microbial attachment.11 The predominant groups of salivary proteins that show such selective adsorption patterns are the acidic proline-rich proteins (PRPs),12,13 statherin,14 and histatins.15 Among these, acidic PRPs represent the major component of the AEP.4 On the basis of differences in electrophoretic mobility, PRPs are organized into three subgroups: basic, acidic, and glycosylated.16 Nine major isoforms of acidic PRPs have been identified (PRP1, PIFs, PRP2, PRP3, PIFf, Db-s, Db-f, PRPIV, and Pa),17 which together constitute up to 30% of the human parotid saliva protein content.18 These isoforms are encoded by two gene loci, namely, PRH1 and PRH2.19,20 Acidic PRP1 is a major pellicle-forming protein.21 It consists of 150 amino acids and has a unique composition and sequence (Figure 1), with proline (27.5%), glutamine (22.8%), and glycine (8) Hay, D. I. Arch. Oral Biol. 1967, 12, 937-946. (9) Zahradnik, R. T. J. Dent. Res. 1979, 58, 2066-2073. (10) Tabak, L. A.; Levine, M. J.; Mandel, I. D.; Ellison, S. A. J. Oral Pathol. Med. 1982, 11, 1-17. (11) Gibbons, R. J.; Hay, D. I.; Childs, W. C.; Davis, G. Arch. Oral Biol. 1990, 35, S107-S114. (12) Hay, D. I. J. Dent. Res. 1969, 48, 806-810. (13) Oppenheim, F. G.; Hay, D. I.; Franzblau, C. Biochemistry 1971, 10, 4233-4238. (14) Hay, D. I. Arch. Oral Biol. 1973, 18, 1517-1528. (15) Oppenheim, F. G.; Yang, Y. C.; Diamond, R. D.; Hyslop, D.; Offner, G. D.; Troxler, R. F. J. Biol. Chem. 1986, 261, 1177-1182. (16) Bennick, A. Mol. Cell. Biochem. 1982, 45, 83-99. (17) Oppenheim, F. G.; Salih, E.; Siqueira, W. L.; Zhang, W.; Helmerhorst, E. J. Ann. N.Y. Acad. Sci. 2007, 1098, 22-50. (18) Kauffman, D. L.; Keller, P. J. Arch. Oral Biol. 1979, 24, 249-256. (19) Azen, E. A.; Kim, H. S.; Goodman, P.; Flynn, S.; Maeda, N. Am. J. Hum. Genet. 1987, 41, 1035-1047. (20) Hay, D. I.; Ahern, J. M.; Schluckebier, S. K.; Schlesinger, D. H. J. Dent. Res. 1994, 73, 1717-1726. (21) Li, J.; Helmerhorst, E. J.; Troxler, R. F.; Oppenheim, F. G. J. Dent. Res. 2004, 83, 60-64.
10.1021/la7013978 CCC: $37.00 © 2007 American Chemical Society Published on Web 09/20/2007
Conformational Changes in Protein 1
Figure 1. Primary structure of PRP1 with amino acid repeats underlined. (Abbreviation: PCA, pyrrolidone carboxylic acid).
(20.8%) comprising more then 70% of the total number of amino acids. These amino acids tend to be present in “homooligopeptide” repeat sequences containing from two to six monomers of either Pro, Gln, or Gly (Figure 1). The 30-AA-long N-terminal domain of PRP1 contains 14 acidic amino acids, including 2 phosphoserines. This N-terminal segment has been shown to have nearly the same high affinity for hydroxyapatite (HA) surfaces as does the intact PRP1 molecule.22 The C-terminal portion of PRP1 contains mainly neutral amino acid residues and is rich in Pro, Gln, and Gly. Despite the high content of Pro, Gln, and Gly amino acid residues known for their high propensity to form polyproline type II (PPII) helices,23-25 this type of secondary structure has not been reported for acidic PRPs. Using circular dichroism and NMR, PPII structures could not be ascertained in acidic PRPs or in synthetic proline hexapolymers, and these observations led to the notion that acidic PRPs assume an extended random coil conformation in solution.26-28 The only indirect evidence for acidic PRPs that suggests changes in secondary structure upon adsorption was noted in studies focusing on the binding of oral microorganisms to salivary protein-coated HA surfaces. Gibbons and co-workers29,30 made the surprising discovery that acidic PRPs, although not binding bacteria in solution, bind bacteria when acidic PRPs are first adsorbed to HA. These data suggested a structural change in acidic PRPs occurring during the adsorption process exposing protein domains that are not accessible in solution. It was proposed that the induced conformational change was responsible for revealing specific protein epitopes (called cryptitopes) with a high affinity for bacterial receptor sites.11 These findings emphasize the potential importance of protein structural changes in the formation and function of the AEP in the oral cavity. In the present work, we describe the conformation of PRP1 in solution and the conformational changes occurring upon binding to HA as well as CHA by employing Fourier transform infrared spectroscopy (FTIR). Materials and Methods Mineral Synthesis. HA was synthesized using the reported method of Gadaleta et al.31 with some modifications. Specifically, 3850 mL of a solution containing 0.022 M calcium chloride (Mallinckrodt) (22) Moreno, E. C.; Kresak, M.; Hay, D. I. J. Biol. Chem. 1982, 257, 29812989. (23) Stapley, B. J.; Creamer, T. P. Protein Sci. 1999, 8, 587-595. (24) Kelly, M. A.; Chellgren, B. W.; Rucker, A. L.; Troutman, J. M.; Fried, M. G.; Miller, A. F.; Creamer, T. P. Biochemistry 2001, 40, 14376-14383. (25) Williamson, M. P. Biochem. J. 1994, 297, 249-260. (26) Murray, N. J.; Williamson, M. P. Eur. J. Biochem. 1994, 219, 915-921. (27) Bennick, A. Biochem. J. 1977, 163, 229-239. (28) Wong, R. S. C.; Hofmann, T.; Bennick, A. J. Biol. Chem. 1979, 254, 4800-4808. (29) Gibbons, R. J.; Hay, D. I. Infect. Immun. 1988, 56, 439-445. (30) Gibbons, R. J.; Hay, D. I. J. Dent. Res. 1989, 68, 1303-1307.
Langmuir, Vol. 23, No. 22, 2007 11201 was dripped into 1650 mL of 0.031 M sodium hydrogen phosphate solution (Fischer Scientific) and placed on a stirrer for 6 h at 80 °C. The pH of both solutions was adjusted to 11 using an ammonium hydroxide solution (Fisher Scientific) before the reaction was initiated. The synthesized product (5.5 L) was then washed three times with distilled deionized water. After being washed, the samples were lyophilized. For the synthesis of CHA, 60 mL of 0.5 M ammonium carbonate was added to 0.031 M phosphate solution before the beginning of the reaction. The rest of the procedure was the same. Characterization of Mineral Products. HA and CHA structures were confirmed by FTIR and by chemical analyses. The calcium and phosphate contents of the mineral products were analyzed using the cresolphthalein complexone32 and the ammonium molybdate methods,33 respectively. HA and CHA were found to have calcium to phosphate molar ratios (Ca/P) of 1.64 and 1.68, respectively, similar to the stoichiometry for HA (Ca/P ) 1.67). The carbonate content was determined from transmission FTIR spectra of mineral powders in KBr pellets34 using the ratios of CO3-2 ν3 to PO4-3 ν3 absorption peak areas.35 Approximately 0.5 mg of the lyophilized samples was mixed with 200 mg of KBr (Sigma, FTIR grade) and ground with a mortar and pestle and then pressed into pellets using a hydraulic press (F.S. Carver, Inc.). The samples were analyzed in transmission mode using a Spectrum One FTIR spectrometer (PerkinElmer). Thirty two scans per spectrum at a resolution of 1 cm-1 were acquired, and the resulting spectra were baseline corrected in automatic mode and normalized with standard processing routines using Spectrum 5 spectroscopy software (Perkin-Elmer). The carbonate content was calculated by determining the area under the carbonate peaks and comparing them with known standards of 2, 4, 6, 8, and 12 wt % (Clarkson Chromatography Products). Pure stoichiometric hydroxyapatite (National Institute of Standards and Technology, Gaithersburg, MD) was used as a 0 wt % carbonate standard. On the basis of these analyses, the carbonate content of CHA used in this study was found to be 6 wt %. FTIR analyses also revealed that CHA was type AB on the basis of the appearance of the carbonate ν2 absorbance band, which is composed of two peaks at 873 and 879 cm-1 corresponding to B- and A-type substitution, respectively.35 (See Supporting Information for further details.) The specific surface areas of CHA and HA were determined by BET nitrogen adsorption (Quantachrome, Inc) and were found to be 81 and 46 m2/g, respectively. Protein Purification and Binding Experiments. PRP1 was purified from human saliva using the method of Oppenheim et al.13 PRP1 was dissolved in a binding buffer (0.04 M TRIS and 0.05 M NaCl, pH 6.8) overnight to the final concentration of 10 mg/mL and was used as a stock solution. Protein adsorption to HA and CHA powders was carried out at pH 6.8 and 37 °C using the method of Moreno and Kresak22 with some modifications. Binding experiments were carried out using 5 mg of apatites (either HA or CHA) suspended in 0.5 mL of 0.5 mg/mL PRP1 protein solution, prepared by diluting the stock solution with binding buffer. The suspension was rotated end over end at 37 °C for 4 h to attain equilibrium.22 In addition, control samples containing suspensions of HA or CHA in the buffer without protein as well as the protein in the buffer and the buffer alone were also incubated under similar conditions. After equilibration, the controls and the apatite-protein mixtures were centrifuged at 14 000 × g for 20 min at 4 °C (Eppendorf Scientific, Westbury, NY). After centrifugation, the supernatants were removed, and the pellets were washed two times (10 min each) with 0.5 mL of buffer. The washed apatite was then resuspended in 0.5 mL of buffer. These suspensions were used for FTIR measurements. (31) Gadaleta, S. J.; Paschalis, E. P.; Betts, F.; Mendelsohn, R.; Boskey, A. L. Calcif. Tissue Int. 1996, 58, 9-16. (32) Gitelman, H. J. Anal. Biochem. 1967, 18, 521-531. (33) Presley, B. J. Appendix: Techniques for Analyzing Interstitial Water Samples. Part I: Determination of Selected Minor and Major Inorganic Constituents; Winterer, E. A., Ed.; Government Printing Office: Washington, DC, 1971; Vol. 7, pp 1749-1755. (34) Duerst, R. W.; Duerst, M. D.; Stebbings, W. L. Transmission Infrared Spectroscopy; Mirabella, F. M., Ed.; John Wiley & Sons: New York, 1998; pp 11-82. (35) Ou-Yang, H.; Paschalis, E. P.; Mayo, W. E.; Boskey, A. L.; Mendelsohn, R. J. Bone Miner. Res. 2001, 16, 893-900.
11202 Langmuir, Vol. 23, No. 22, 2007 FTIR Spectroscopic Analysis of Protein Secondary Structure in an Aqueous Environment. The FTIR spectra of 10 mg/mL solutions of PRP1 in the binding buffer and of suspensions of the minerals with bound PRP1 in the binding buffer were collected in attenuated total reflection (ATR) mode. A 15 µL droplet of the solution was placed over the ATR crystal inside a small rubber O-ring (i.d. 3 mm) and covered with a glass slide that was pressed against the O-ring, using the ATR accessory press to minimize evaporation. The spectra were taken at a resolution of 1 cm-1 or 4 cm-1, when the noisiness of the spectra at 1 cm-1 resolution was unacceptable for further analysis, and 128 scans were collected per spectrum. The binding buffer was used to acquire the background spectrum. Spectra of the suspensions of HA and CHA with bound PRP1 in the binding buffer were obtained as described above for the protein solutions. Spectra of HA and CHA suspensions without bound protein, acquired in a same way, were used as background spectra. The 1725-1500 cm-1 regions of the spectra containing two major protein absorbance bands, amide I (1725-1600 cm-1) and amide II (1600-1500 cm-1), were selected for further analysis. Before analysis, the spectra were normalized using a normalization routine of the Spectrum 5.0 spectrum analysis software (Perkin-Elmer). The peak-fitting analysis of the spectra was performed using the methods of Susi et al.36 and Roach et al.37 The analysis was performed using the Origin 7.5 software package with the peak-fitting module. Spectra taken at 1 cm-1 resolution were smoothed using the FFT smoothing algorithm with 19-point sensitivity, whereas spectra taken at 4 cm-1 resolution were smoothed with 5-point sensitivity. Second derivative analyses were performed on the spectra, and the minimum wavenumbers were used as initial maximum positions for individual peaks. Before the peak-fitting procedure, the baseline was subtracted using a straight line connecting the starting and ending wavenumbers of the spectral fragments. Peak fitting was performed using a Gaussian peak type. The peak positions were initially fixed, and several rounds of peak fitting were performed until the χ2 value between the experimental and calculated spectrum outlines dropped to a value below 2 × 10-5. Then, the maximum values were released and several more rounds of peak fitting were performed. The fits were considered to be good when the differences between the peak maximum values from the second derivatives and those obtained during peak fitting were not more than (2 cm-1 apart and the χ2 value was less than 8 × 10-6. The percentage of peak areas with maxima in the amide I region between 1725 and 1600 cm-1 was calculated for each spectrum.
Results and Discussion PRP1 in the buffer solution exhibits a broad amide I band with two identifiable peaks at 1655 and 1619 cm-1 and a shoulder at 1670 cm-1 (Figures 2 and 3). To distinguish between overlapping vibrational bands in amide I, second derivative analysis of the spectra36 followed by a peak-fitting procedure38 was performed. Structural data from an earlier NMR and CD study26 and sequence prediction analysis of PRPs39 also aided in the interpretation of the spectra. Our analysis revealed seven major peaks at 1620, 1636, 1646, 1655, 1671, 1684, and 1695 cm-1. A very strong peak at 1620 cm-1 comprising 41% of the total amide I absorbance (Figure 3, Table 1) has been attributed to the hydrated PPII helix conformation.40,41 PPII structures have been previously reported in basic PRPs;42 however, this is the first time the PPII helix has (36) Susi, H.; Byler, D. M. Biochem. Biophys. Res. Commun. 1983, 115, 391397. (37) Roach, P.; Farrar, D.; Perry, C. C. J. Am. Chem. Soc. 2005, 127, 81688173. (38) Griebenow, K.; Klibanov, A. M. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 10969-10976. (39) Cid, H.; Vargas, V.; Bunster, M.; Bustos, S. FEBS Lett. 1986, 198, 140144. (40) Johnston, N.; Krimm, S. Biopolymers 1971, 10, 2597-2605. (41) Wellner, N.; Belton, P. S.; Tatham, A. S. Biochem. J. 1996, 319, 741747.
ElangoVan et al.
Figure 2. Normalized FTIR spectra of PRP1 in binding buffer (-), adsorbed on HA (‚‚‚), and with CHA (---) in the region of the amide I and amide II bands (1500-1725 cm-1).
Figure 3. FTIR spectrum of PRP1 in binding buffer in the region of the amide I and amide II bands (1500-1700 cm-1). The solid line represents the original spectrum, with individual fitted component peaks in gray.
been detected in an acidic PRP. The analysis has also revealed a very strong peak at 1655 cm-1 (Figure 3, Table 1). The peak in this region can be assigned to either random coil or R helix.43,44 However, because ∼50% of the total number of PRP1 amino acids are Pro and Gly, which are strong R-helix breakers,45,46 it is highly unlikely that this protein contains many R helices. The results of earlier CD spectroscopy analyses26 and secondary structure modeling39 studies also suggest that PRP1 has a large fraction of random coil motifs and none or very few R-helix domains. Hence, the 1655 cm-1 absorbance band has been assigned to a random coil with some impact of Gln side chain absorbance.41 A broad absorbance peak at 1646 cm-1 has also been assigned to the random coil configuration.43 Combined, these two peaks comprise around 34% of the total amide I band absorbance (Table 1), suggesting that a significant portion of PRP1 in solution exists in an extended, unordered conformation, in agreement with an earlier study.26 Two peaks observed at 1671 and 1684 cm-1 can be attributed to β turns.47 A peak at 1636 cm-1, comprising 7% of the total amide I band area, was assigned to the β sheet.43,44 A small peak at 1695 cm-1 can be assigned to either a β turn or high-frequency split band of the β sheet. Together, the results of our study suggest that PRP1 in solution exists in an extended form with alternating regions of PPII helix and random coil with a relatively small fraction of the β sheet. (42) Isemura, T.; Asakura, J.; Shibata, S.; Isemura, S.; Saitoh, E.; Sanada, K. Int. J. Pept. Protein Res. 1983, 21, 281-287. (43) Barth, A.; Zscherp, C. Q. ReV. Biophys. 2002, 35, 369-430. (44) Krimm, S.; Bandekar, J. AdV. Protein Chem. 1986, 38, 181-364. (45) Chou, P. Y.; Fasman, G. D. Biochemistry 1974, 13, 222-245. (46) Lehninger, A. L. Principles of Biochemistry; Worth Publishers: New York, 1982. (47) Vass, E.; Hollosi, M.; Besson, F.; Buchet, R. Chem. ReV. 2003, 103, 1917-1954.
Conformational Changes in Protein 1
Langmuir, Vol. 23, No. 22, 2007 11203
Table 1. Assignments of Amide I Absorbance Peak Maxima Wavenumbers for PRP1 in Aqueous Solution and Bound States Based on the Peak-Fitting Analysis PRP1 in solution
assignment hydrated PPII β sheet anhydrous PPII random coil random coil/Gln β turn β turn β turn β turn β turn β sheet/ β turn
PRP1 in the HA suspension
max peak wavenumber (cm-1)
peak area (%)
1620 1636
41 7
1645 1655
3 31
1671
10
1684 1695
6 1
max peak wavenumber (cm-1)
peak area (%)
1630 1639 1646 1653
13 7 3 13
1666
31
1677 1685 1692
9 3 20
PRP1 in the CHA suspension max peak wavenumber (cm-1)
peak area (%)
1620 1636 1642
10 12 2
1654 1660 1666 1672 1679 1684 1695
27 3 8 10 6 17 6
To further confirm the presence of PPII structures in PRP1, we have performed FTIR spectroscopic studies of this protein in a 6 M solution of CaCl2 (details in Supporting Information). It has been previously reported that the presence of Ca2+ leads to the breaking of the hydrated PPII structure48,49 and is accompanied by a high-frequency shift of ∼20 cm-1 to 16391643 cm-1.40 Our data are consistent with the results of this earlier study. Namely, in the presence of 6 M CaCl2, the peak at 1620 cm-1 disappeared, and a strong peak around 1642 cm-1 was detected, providing further evidence of PPII structure in PRP1. When incubated with HA, significant changes in the shape of the amide I band of PRP1 were observed (Figures 2 and 4), with the maximum amide I absorption shifting to 1666 cm-1(Figure 4, Table 1). The second derivative and peak-fitting analyses revealed the disappearance of absorbance in the 1617-1621 cm-1 region, characteristic of hydrated PPII, and a significant reduction in absorbance in the 1645-1655 cm-1 region, assigned to random coil (Figures 3 and 4, Table 1). At the same time, the absorbance in 1660-1695 cm-1 region, corresponding to β turns47 and an antiparallel β sheet,44 increased significantly (Figures 3 and 4, Table 1). Because the stronger β-sheet split band at 1630 cm-1 has a relatively low intensity, it is unlikely that the weak high-frequency β-sheet band44,50 has a significant impact on the increased absorbance in the 1660-1695 cm-1 region. Importantly, a new absorbance band at 1639 cm-1 that is characteristic of dehydrated PPII40,41 was observed to be in sharp contrast to the solution spectra (Figures 3 and 4, Table 1). When PRP1 was incubated with CHA, the amide I band of PRP1 exhibited characteristics intermediate between solution and HA-bound (Figures 2 and 5, Table 1). The amide I band in this experiment has a maximum at 1666 cm-1, similar to the spectra of the protein bound to HA. At the same time, although the absorbance in the β-turn region in this sample is significantly higher than that of PRP1 in solution, it is 1.3 times less than that in the PRP1-HA sample, whereas the random coil absorbance at 1654 cm-1 in this spectrum is 2 times higher than in PRP1HA sample (Figure 3, Table 1). The percentage of β sheet in this sample appears to be higher than in solution and is similar to that in the PRP1-HA sample (Table 1). At the same time, there is still a significant amount of hydrated PPII structure in the PRP1CHA sample, which is characterized by a peak at 1620 cm-1 alongside a small peak at 1642 cm-1 assigned to dehydrated
PPII, which is prominent in the PRP1-HA sample. These data imply that the environment and structural arrangement of the bound protein in PRP1-CHA are different from those of PRP1 bound to HA. Specifically, these data suggest that mineral binding has a smaller effect on the conformation and hydration state of PRP1 adsorbed to CHA in comparison to those of PRP1 adsorbed to HA. The notion that the conformation and hydration state of PRP1 are less affected when adsorbed on CHA is further supported by our unpublished binding studies on the effect of carbonate on the adsorption of salivary proteins that indicate that for the chosen experimental conditions almost twice as much PRP1 would be adsorbed to HA per unit surface area in comparison to CHA and that the ratio of bound to unbound PRP1 at equilibrium would also be almost twice as great for HA as for CHA. In both cases, however, >95% of the protein equilibrated with HA or CHA would be mineral-bound, indicating that FTIR bands of the (washed) protein-mineral suspensions analyzed predominately reflect those of protein in the bound state. These estimates are calculated from adsorption parameters obtained from the corresponding PRP1 adsorption isotherms (presented in Supporting Information). Thus, these results indicate that the PRP1 density on HA is much higher than on CHA, implying a greater potential for protein-protein and protein-mineral interactions and less exposure to water. Consistent with our protein-binding observations, earlier studies have also reported a smaller degree of protein (BSA) adsorption on carbonated apatites with an increase in carbonate content, citing changes in crystal morphology and texture as a possible cause.51 Another interesting change that occurs upon binding is an increase in the amide I/II maximum height ratio from 1.35 in solution to 2 in the PRP1-CHA sample and 3.3 in the PRP1HA sample (Figure 2). Note that the PRP1-CHA sample again shows intermediate characteristics between the protein in solution and HA-bound protein. The increase in the amide I/II height ratio upon binding to surfaces has been previously described.52,53 Interestingly, a link between the increase in the amide I/amide II ratio and the strength of protein binding to a surface, determined as resistance to elution, has been proposed.53 However, the basis of the association between protein binding and changes in the amide I/II ratio remains to be revealed. Our results indicate that a large portion of PRP1 adopts a hydrated PPII conformation in solution, in agreement with an
(48) Steinberg, I. Z.; Harrington, W. F.; Berger, A.; Sela, M.; Katchalski, E. J. Am. Chem. Soc. 1960, 82, 5263-5279. (49) Harrington, W. F.; Sela, M. Biochim. Biophys. Acta 1958, 27, 24-41. (50) Kubelka, J.; Keiderling, T. A. J. Am. Chem. Soc. 2001, 123, 1204812058.
(51) Kandori, K.; Saito, M.; Takebe, T.; Yasukawa, A.; Ishikawa, T. J. Colloid Interface Sci. 1995, 174, 124-129. (52) Zeng, H. T.; Chittur, K. K.; Lacefield, W. R. Biomaterials 1999, 20, 377-384. (53) Lenk, T. J.; Horbett, T. A.; Ratner, B. D.; Chittur, K. K. Langmuir 1991, 7, 1755-1764.
11204 Langmuir, Vol. 23, No. 22, 2007
Figure 4. Infrared spectrum of PRP1 in binding buffer after incubation with HA in the region of amide I and amide II bands (1500-1700 cm-1). The solid line represents the original spectrum, with individual fitted component peaks in gray.
Figure 5. Infrared spectrum of PRP1 in binding buffer after incubation with CHA in the region of amide I and amide II bands (1500-1700 cm-1). The solid line represents the original spectrum, with individual fitted component peaks in gray.
earlier structural prediction study39 and the results of our own PRP1 sequence analysis using the ConSol tetrapeptide database, designed to identify PPII structures54 (data not shown). PPII helices play a very important role in intermolecular interactions in structural and signaling proteins.25,55 This is not surprising, considering that PPII is an extended helical structure with very few intramolecular hydrogen bonds and with the solvent-exposed side chains and backbone.25 Such intermolecular interactions are potentially important in pellicle formation and function.56,57 Our data agree with earlier NMR studies of PRP1 mimic peptides,26 suggesting that the protein adopts an extended conformation, although they were unable to clearly identify the PPII helix. Using FTIR in the ATR mode, we have also shown that PRP1 undergoes dramatic conformational changes upon binding to HA (Figure 3 and 4, Table 1). The major band in solution, 1620 cm-1, corresponding to hydrated PPII40 disappears, and bands at 1645-1655 cm-1 assigned to random coil43 dramatically decrease, whereas absorbance in 1660-1695 cm-1 region, which is primarily due to β turns,47 increases significantly (Table 1). Previously, Moreno et al.58 also observed changes in the FTIR spectra of PRP1 upon binding to HA, showing some similarities to those observed in our study. More specifically, our observations of a high-frequency shift of the amide I absorbance maximum are in agreement with the results of this previous study, which also concluded that a large fraction of PRP1 in the bound state exists in the form of β turns. However, the solution concentration of PRP1 used in this earlier report was 2 orders of magnitude lower than in our study, resulting in a very low protein signal (absorbance). It is therefore difficult to make other direct (54) Vlasov, P. K.; Vlasova, A. V.; Tumanyan, V. G.; Esipova, N. G. Proteins 2005, 61, 763-768. (55) Shi, Z. S.; Chen, K.; Liu, Z. G.; Kallenbach, N. R. Chem. ReV. 2006, 106, 1877-1897. (56) Yin, A.; Margolis, H. C.; Yao, Y.; Grogan, J.; Oppenheim, F. G. Arch. Oral Biol. 2006, 51, 102-110. (57) Lamkin, M. S.; Arancillo, A. A.; Oppenheim, F. G. J. Dent. Res. 1996, 75, 803-808. (58) Moreno, E. C.; Kresak, M.; Hay, D. I. Biofouling 1991, 4, 3-24.
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comparisons between these earlier data and our results. An increase in the number of β turns upon adsorption has been also reported for lysozyme bound to self-assembled monolayers.59 Our data are also consistent with predictions from thermodynamic analyses of PRP1 adsorption.22 Results from these analyses clearly indicate that PRP1 adsorption is driven by an increase in entropy that was found to be primarily associated with the protein and, presumably, with changes in the protein secondary structure resulting from disruptions to internal ion pairs, hydrogen bonding, and the state of hydration, along with the displacement of organized water molecules from the mineral surface. It has been previously reported that the adsorption of another AEP protein, statherin, is similarly driven by entropy and the loss of water of hydration.60 The significant conformational changes upon mineral binding shown in our study are in agreement with these earlier predictions and might indicate that the interactions with the mineral affect the whole protein. Hence, we assume that although the negatively charged N terminus is primarily responsible for initial interactions with minerals,16,22,28 the rest of the protein might also be involved in protein-mineral interactions.22 Conformational changes upon binding associated with other proteins and surface types have also been reported.37,52,53,59,61 Although in a majority of reported cases proteins tend to “unfold” upon absorption,62 it has been recently reported that statherin, another AEP protein that is unstructured in solution, undergoes folding upon binding to hydroxyapatite crystals.63 An absorbance band at 1639-1642 cm-1 was detected in the mineral-bound protein. A number of studies of peptides and proteins with high proline content40,41 have shown that absorbance bands in 1639-1643 cm-1 region are characteristic of the anhydrous PPII-type structure. Johnston and Krimm40 have performed an extensive study of poly-L-proline in aqueous solutions and have found a shift in the PPII maximum absorption from 1619 to 1641 cm-1 upon addition of calcium ions. They have demonstrated that the 1619 to 1641 cm-1 shift occurs because of the substitution of hydrogen bonds between water molecules and proline backbone carbonyls with carbonyl-calcium complexes.40 Our data, therefore, suggest the possibility that some of the PRP1 PPII motifs in the bound state retain their structure but loose their hydration either by forming complexes with calcium ions on the surface of the crystals or via hydrophobic interactions with the crystal surface.22,61 Our study also indicates that PRP1 undergoes conformational changes upon binding to CHA in a manner similar to those observed with HA but to a lesser extent (Figures 3-5 and Table 1). These latter data demonstrate that PRP1 adsorbed onto CHA has structural properties intermediate between PRP1 in solution and HA-bound. These differences can be due to the differences in the strength of the interactions between PRP1 and the two minerals used in this study and suggest greater exposure of PRP1 on CHA to the solvent. Conformational changes in adsorbed proteins may have important biological consequences. For example, Gibbons and Hay11 discovered significant differences in the affinity of oral bacteria for salivary proteins bound to HA versus proteins in solution. They hypothesized that bacteria have developed specific molecular recognition mechanisms that selectively identify epitopes (so-called cryptitopes) that are exposed only on the (59) Sethuraman, A.; Belfort, G. Biophys. J. 2005, 88, 1322-1333. (60) Goobes, R.; Goobes, G.; Campbell, C. T.; Stayton, P. S. Biochemistry 2006, 45, 5576-5586. (61) Norde, W. AdV. Colloid Interface Sci. 1986, 25, 267-340. (62) Haynes, C. A.; Norde, W. J. Colloid Interface Sci. 1995, 169, 313-328. (63) Goobes, G.; Goobes, R.; Schueler-Furman, O.; Baker, D.; Stayton, P. S.; Drobny, G. P. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 16083-16088.
Conformational Changes in Protein 1
protein surface in the bound state but not in solution. Our results clearly demonstrate that PRP1 undergoes significant conformational changes upon binding to HA and provide support to these earlier hypotheses.
Conclusions We demonstrate here for the first time that acidic PRP1 in solution contains PPII helical motifs. Significant changes in protein conformation were observed upon binding to hydroxyapatite and carbonated hydroxyapatite surfaces. Our analyses suggest that the whole protein and not only the negatively charged N-terminus is affected by protein-mineral interactions. These conformational changes likely reflect protein-protein and protein-mineral interactions as well as changes in the level of hydration. The carbonate content of the mineral was found to have a notable effect on the extent of conformational changes
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upon binding but not on the nature of these changes. The significant structural changes in PRP1 upon binding to apatites, observed in this study, could be critical in elucidating the molecular events underlying the formation of the acquired enamel pellicle and the mechanism of oral microbial attachment to salivary proteins bound to tooth mineral surfaces. Acknowledgment. This study was supported by NIH/NIDCR grants DE 05672 (FO), DE 07652 (FO), DE 14950 (FO), and DE 15163 (HM). Supporting Information Available: FTIR spectra of mineral phases used in the study. Adsorption isotherms of PRP1 at HA and CHA. FTIR spectrum of PRP1 in 6 M CaCl2 solution. This material is available free of charge via the Internet at http://pubs.acs.org. LA7013978
