Conformation of Lysozyme Langmuir Monolayer Studied by Infrared

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Conformation of Lysozyme Langmuir Monolayer Studied by Infrared Reflection Absorption Spectroscopy Garima Thakur and Roger M. Leblanc* Department of Chemistry, 1301 Memorial DriVe, UniVersity of Miami, Coral Gables, Florida 33146 ReceiVed October 1, 2008. ReVised Manuscript ReceiVed December 2, 2008 The surface chemistry and spectroscopy of the reduced lysozyme Langmuir monolayer were investigated at different pH values to compare with the native one. It was found that the limiting molecular area of the reduced lysozyme was not subphase pH dependent as the native lysozyme. To explain this result in terms of the conformation and orientation of the lysozyme Langmuir monolayer at various subphase pH values, we have used infrared reflection absorption spectroscopy. The interpretation of the results make plausible change of the conformation and orientation of the native lysozyme Langmuir monolayer with the subphase pH 3, 6, and 11.

Introduction One of the most widely studied protein in biochemistry is hen egg white lysozyme (HEWL) because it shares 60% of its structure with the human lysozyme.1,2 Lysozyme is an essential protein found in various fluids like human milk, placenta, leukocytes, and most abundantly, in tears.3,4 Surface behavior is of extreme importance for the study of how lysozyme interacts with membranes. Since the Langmuir monolayer approach is a unique methodology to mimic biological membrane systems, it may serve as a model system for studying lysozyme at the air-water interface. In our previous manuscript we investigated lysozyme Langmuir monolayer utilizing surface chemistry and UV-vis absorption and fluorescence spectroscopy.5 To understand the function and properties of a protein, it is important to explore the structure adopted by the protein in a diverse range of configurations. X-ray crystal structure of HEWL is already known, and it contains 129 amino acid residues, including four disulfide bridges.6 R-Helix and β-sheet content in the protein is 40% and 10%, respectively.7 Prior studies used to determine structural conformation of lysozyme adsorbed at the air-water interface has been reported using neutron reflection spectroscopy, Fourier transform infrared spectroscopy, infrared reflection absorption spectroscopy (IRRAS), and polarization modulation infrared reflection absorption spectroscopy (PMIRRAS).8-11 * To whom correspondence should be addressed. Fax: +1- 305-2846367. Phone: +1-305-284-2194. E-mail: [email protected]. (1) Pepys, M. B.; Hawkins, P. N.; Booth, D. R.; Vigushin, D. M.; Tennent, G. A.; Soutar, A. K Nature 1993, 362, 553. (2) Booth, D. R.; Sunde, M.; Bellotti, V.; Robinson, C. V.; Hutchinson, W. L.; Fraser, P. E Nature 1997, 385, 787. (3) Sack, R. A.; Nunes, I.; Beaton, A.; Morris, C. Biosci. Rep. 2001, 21, 463. (4) Hankiewicz, J.; Swierczek, E. Clin. Chim. Acta 1974, 57, 205. (5) Thakur, G.; Wang, C. S.; Leblanc, R. M. Langmuir 2008, 24, 4888. (6) Blake, C. C. F.; Koeing, D. F.; Mair, G. A.; North, A. C. T.; Phillips, D. C.; Sarma, V. R. Nature 1965, 4968, 757. (7) Walsh, M. A.; Schneider, T. R.; Sieker, L. C.; Dauter, Z.; Lamniz, V. S.; Wilson, K. S. Acta Crystallogr., Sect. D 1998, 54, 522. (8) Lu, J. R.; Su, R. K.; Thomas, R. K.; Penfold, J.; Webster, J. J. Chem. Soc., Faraday Trans. 1998, 94, 3297. (9) Lad, M. D.; Birembaut, F.; Matthew, J. M.; Frazier, R. A.; Green, R. J. Phys. Chem. Chem. Phys. 2006, 8, 2179. (10) Alahverdjieva, V. S.; Grigoriev, D. O.; Ferri, J. K.; Fainerman, V. B.; Aksenenko, E. V.; Leser, M. E.; Michel, M.; Miller, M. Colloids Surf., A 2008, 323, 167. (11) Postel, C.; Abillon, O.; Desbat, B. J. Colloid Interface Sci. 2003, 266, 74.

As mentioned, this research is a continuation of the earlier studies. At that time we determined that 3 M salt concentration and subphase pH 11 were the ideal conditions for the formation of native lysozyme Langmuir monolayer.5 Lysozyme Langmuir monolayer was exceedingly stable over a period of 90 min and the solubility of the lysozyme into the subphase was not of importance. The linearity observed for absorbance as a function of surface pressure supported this result. Furthermore, the lysozyme Langmuir monolayer also showed the salt and pH effect. In situ UV-vis and fluorescence spectra indicated the presence of tryptophan and tyrosine amino acid residues in the lysozyme Langmuir monolayer. The interesting finding that came out of the lysozyme Langmuir monolayer investigation was the change in the behavior of the Langmuir monolayer at different pH subphase values, in particular the limiting molecular area.5 We hypothesized that this change could be interpreted by the conformation and the orientation of the protein at the air-water interface. The purpose of this work is to verify this hypothesis through the infrared reflection absorption spectroscopy12-14 measurements of the native and denatured lysozyme Langmuir monolayer. Disulfide bridges play a vital role in holding the conformation of the globular proteins.15 Reduction of the disulfide bonds of lysozyme will destroy its tertiary structure, thereby exposing the hydrophobic residues and consequently loosing its native conformation.16-18 Comparison of the native lysozyme with the denatured lysozyme Langmuir monolayer is most important to study the change in conformation of the native lysozyme Langmuir monolayer at various pH values.

Experimental Section Materials. All chemicals and methods used for the surface chemistry of lysozyme were described in the previous manuscript.5 Dithiothreitol (DTT), Trizma base, and iodoacetamide (IAM) were obtained from VWR Co. (Westchester, PA). R-Cyano-4-hydroxycinnamic acid, acetone, and acetonitrile were purchased from Sigma (12) Mendelsohn, R.; Brauner, J. W.; Gericke, A. Annu. ReV. Phys. Chem. 1995, 46, 305. (13) Blaudez, D.; Turlet, J. M.; Dufourcq, J.; Brad, D.; Buffeteau, T.; Desbat, B. Chem. Soc. Faraday Trans. 1996, 92, 525. (14) Dziri, L.; Desbat, B.; Leblanc, R. M. J. Am. Chem. Soc. 1999, 121, 9618. (15) Thoronton, J. M. J. Mol. Biol. 1981, 151, 261. (16) Tamburro, A. M.; Boccu, E.; Celotti, L Int. J. Protein Res 1970, 2, 157. (17) Gekko, K.; Kimoto, A.; Kamiyama, T. Biochem. 2003, 42, 13746. (18) Touch, V.; Hayakawa, S.; Saitoh, K. Food Chem. 2004, 84, 421.

10.1021/la803233p CCC: $40.75  2009 American Chemical Society Published on Web 01/27/2009

Conformation of Lysozyme Langmuir Monolayer Aldrich (St. Louis, MO). All chemicals were reagent grade and were used without any further purification. Reduction of Lysozyme. The reduction of lysozyme was carried out using DTT. Lysozyme solution in Tris-HCl buffer (10 mM, pH 8), at a final protein concentration of 1 mg · mL-1, was incubated with 2 mM DTT at 30 °C. An aliquot was withdrawn at 4 h, and immediately allowed to react with 3 molar equiv of IAM with respect to DTT at 30 °C for 1 h. The sample was then extensively dialysed against distilled water at 4 °C.18 The characterization of reduced lysozyme was completed using mass spectral analysis. Langmuir Monolayer Preparation. The solution of lysozyme and reduced lysozyme was prepared in pure water (pH 6) at a concentration of 1 × 10-5 M. KCl (3 M) solution was used as subphase at different pH’s. The preparation of the subphase pH was already described in ref 5. The volume of the spreading aqueous solution (1 × 10-5 M) was varied between 25 and 250 µL depending upon the experimental conditions. Equipment. All the isotherm measurements and in situ IRRAS were conducted in a clean room (class 1000) where temperature (20.0 ( 0.5 °C) and humidity (50% ( 1%) were kept constant. A Kibron µ-trough (Kibron Inc., Helsinki, Finland) with an area of (5.9 cm × 21.1 cm) was utilized for the surface pressure-area (π-A) isotherm. IRRAS measurements at the air-water interface were performed by the EQUINOX 55 Fourier transform infrared (FTIR) spectrometer (Bruker Optics, Billerica, MA) equipped with an XA511 external reflection accessory suitable for the air-water interface experiments. The IR beam was passed out of the spectrometer and focused at the air-water interface of the Kibron µ-trough. The reflected IR beam went to a HgCdTe (MCT) detector cooled by liquid nitrogen. The spectra were acquired with a resolution of 8 cm-1 by coaddition of 1200 scans. Mass spectra of native and reduced lysozyme was determined by matrix-assisted laser desorption ionization time-of-flight (MALDITOF) mass spectrometer (Biflex IV, Bruker Daltonics, Germany) equipped with a standard nitrogen laser (337 nm). A volume of 2 µL of native or reduced lysozyme sample (0.8 mg · mL-1) was mixed with the saturated matrix, 20 µL R-cyano-4 hydroxycinnamic acid (hcca, MW 189.17) solution prepared in water/acetone/acetonitrile (50:30:20). The resulting mixture (2-3 pmol) was spotted on stainless steel MALDI target (SCOUT 384) and dried. The calibration was checked with Bruker peptide calibration standard. Spectra were acquired with at least 200 laser shots (50 shots at a time) and were analyzed using Bruker XMASS software in linear mode. The resulting mass spectra for native lysozyme and reduced lysozyme showed the m/z signal at 14301 and 14716, respectively (figures not shown) that correspond to the previously reported results.18

Results and Discussion Effect of pH on Limiting Molecular Area. It was reported in ref 5 for the native lysozyme Langmuir monolayer that the limiting molecular area reached a minimum at pH 6 as shown in Figure 1. It has to be pointed out that the limiting molecular area value is the one extrapolated at zero surface pressure from the linear part of the surface pressure-area isotherm. As the pH of the subphase was increased from 6 to 11, the limiting molecular area increased from 2200 to 3600 Å2 · molecule-1, respectively. Whereas, when the pH was decreased from 6 to 3, the limiting molecular area of lysozyme Langmuir monolayer reached up to 3000 Å2 · molecule-1. The study of surface pressure-area isotherms of reduced lysozyme Langmuir monolayer was carried out in this work at various subphase pH values to compare with the pH effect of the native one. It is interesting to note that for the reduced lysozyme Langmuir monolayer the limiting molecular area remained substantially constant between subphase pH range 3 and 11. For example, the limiting molecular area at pH 3, 6, and 11 was 1163, 1500, and 1390 Å2 · molecule-1, respectively, for the reduced lysozyme Langmuir monolayer. In summary, it was

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Figure 1. Limiting molecular area of lysozyme Langmuir monolayer vs pH of the subphase at 3 M KCl concentration for native lysozyme (9, from ref 9, Figure 3) and denatured lysozyme (b), present work.

observed that the native lysozyme has a maximum limiting molecular area per molecule at subphase pH 11. It has to be noted that the isoelectric point of the lysozyme is at pH 11. From these results it is clear that the surface behavior of Langmuir monolayer of the native and reduced lysozyme differs significantly. Study of Native Lysozyme Langmuir Monolayer using IRRAS. To explain the pH effect on the limiting molecular area it is essential to study the change in conformation and orientation of the lysozyme at the air-water interface. IRRAS is an important technique to study the orientation and conformation change of monolayers at the air-water interface. Data for the IRRAS measurements were obtained as reflectance-absorbance (RA) vs wavenumber. RA is defined as -log10 (R/RF) where R is the reflectivity of the film covered surface and RF is the reflectivity of the water.12,19 When the vibrations are parallel to the air-water interface, for p-polarized radiation, which is parallel to the plane of incidence, the bands are initially negative and their intensities increase as the incident angle is increased until the Brewster angle (54.5° for 2920 cm-1 of IR radiation)20 is reached. Beyond the Brewster angle the bands become positive and their intensities decrease upon increasing the angle of incidence. If the vibrations are perpendicular to the air-water interface, bands are positive first and then become negative beyond the Brewster angle.20-23 If the vibrations are tilted to the plane of incidence at the air-water interface, the intensity of bands are weak or sometimes zero. However, for s-polarized radiation, which is perpendicular to the plane of incidence, only vibrations parallel to the air-water interface can be detected. The bands for s-polarized IRRAS are always negative, and the intensity of bands decreases upon increasing incident angle. The p-polarized IRRAS is more sensitive to the change in orientation of the monolayer at the air-water interface. Therefore, to prove our hypothesis, we have performed experimental studies using p-polarized IRRAS. By using IRRAS at different incident angles and surface pressures, we can investigate thus the change of orientation and conformation at different subphase pH 3, 6, and 11 (Figures 2 and 3). (19) Dulhy, R. A. Appl.Spectrosc. ReV 2000, 35, 315. (20) Du, X.; Miao, W.; Liang, Y. J. Phys. Chem. B 2005, 109, 7428. (21) Wang, C.; Zheng, J.; Zhao, L.; Rastogi, V. P.; Shah, S. S.; DeFrank, J. J.; Leblanc, R. M. J. Phys. Chem. B 2008, 112, 5250. (22) Wang, Y; Du, X.; Miao, W.; Liang, Y. J. Phys. Chem. B 2006, 110, 4914. (23) Gericke, A.; Michailov, A. V.; Huhnerfuss, H. Vibr. Spectrosc. 1993, 4, 335.

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Figure 2. p-Polarized IRRAS of native lysozyme Langmuir monolayer on 3 M KCl subphase: (I) surface pressure at 20 mN · m-1 and different incident angles; (II) at an incident angle of 60° and different surface pressures. A, B, and C represent subphase pH 6, 3, and 11, respectively

Moreover, IRRAS can be useful to analyze structural features of the proteins, such as R-helix and β-sheet interpreted through amide I and amide II bands in the region of 1700-1600 and 1600-1500 cm-1, respectively. About 80% of CdO stretching vibrations and 20% in plane C-N bending modes account for the amide I band (1700-1600 cm-1), whereas for amide II these values are 40% C-N stretching and 60% N-H bending (1600-1500 cm-1). The dihedral angle and hydrogen-bond strength affect the electron density in the carbonyl groups of the protein backbone. These carbonyl group vibrations are mainly

related to amide I mode of vibration. The stretching vibrations for the carbonyl group in a protein depend upon the hydrogenbond strength of the characteristic secondary structure of the protein. That is, the higher the hydrogen-bond strength, the lower the amide I absorption.24-26 While interpreting the infrared spectra, we cannot ignore the side chain amino acids in the protein. The reason is due to the (24) Jackson, M.; Mantsch, H. H. Crit. ReV. Biochem. Mol. Biol. 1995, 30, 95. (25) Krimm, S.; Bandekar, J. AdV. Protein Chem. 1986, 38, 181. (26) Bandekar, J. Biochim. Biophys. Acta 1992, 1120, 123.

Conformation of Lysozyme Langmuir Monolayer

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Figure 3. p-Polarized IRRAS of reduced lysozyme Langmuir monolayer on 3 M KCl subphase: (I) surface pressure at 20 mN · m-1 and different incident angles; (II) at an incident angle of 60° and different surface pressures. A, B, and C represent subphase pH 6, 3, and 11, respectively.

fact that the side chain amino acids absorb in the same amide I and II spectral region as the protein backbone used for the secondary structure determination.27 For the globular proteins, prior research estimates that the contribution of the side chain amino acids in overall absorption spectra is 10-30%.28 Among the various amino acid residues, asparagine and glutamine are special because these side chains absorb in the amide I region. While interpreting the protein spectra, it is important to take in consideration the content and position of these residues in the protein. It has to be pointed out that the position of the bands for pure amino acid residues and side chains in the protein is

1.

(27) Barth, A. Prog. Biophys. Mol. Biol. 2000, 74, 141. (28) Rahmelow, K.; Hubner, W.; Ackermann, T. Anal. Biochem. 1998, 257,

different depending upon the environment, the arrangement and the conformation of the side chain residues in the protein.29 Scheme 1 shows the structure of lysozyme in cartoon and surface representation. The lysozyme is a globular protein and HEWL sequence has 129 amino acids. The amino acid residues in the lysozyme that are most important in relation to explanation of spectra in our studies are 14 asparagine (Asn), 11 arginine (Arg), 10 serine (Ser), 7 aspartate (Asp), 2 glutamate (Glu), 6 tryptophan (Trp), 6 lysine (Lys), 3 glutamine (Gln), 3 tyrosine (Tyr), and 3 phenylalanine (Phe) residues. Most of these residues are hydrophilic or aromatic and found on the outer surface of the lysozyme molecule. Presence of side chains of these amino (29) Chirgadze, Y. N.; Fedorov, O. V.; Trushina, N. P. Biopolymers 1975, 14, 679.

2846 Langmuir, Vol. 25, No. 5, 2009 Scheme 1. Cartoon Representation (A) and Surface Representation (B) of Native Lysozyme (Gray Color Represents r-Helix, Black, β-Sheet and White Loop) and Surface Representation (C) of Native Lysozyme Shows the Presence of Asparagine and Arginine Residues on the Outer Surface (black color represents Asn, Gray represents Arg)

acids, particularly Asn and Arg, on the outer surface of the protein has been checked through pymol viewer using 2vb1 molecule taken from the protein data bank (pdb).30 Presence of 14 Asn and 10 Arg residues on the outer surface of lysozyme is shown in representation 1C. The secondary structure of lysozyme consists of 7 R-helices containing 52 residues, whereas there are 9 β-strands with only 14 residues. The rest of the protein is loop or unordered structure.30 It is clearly noticed from the two representations (Scheme 1A and B) that there is much less β-sheet present in the secondary structure of the lysozyme compared with the content of R-helix and loop. We have studied the p-polarized IRRAS of native lysozyme Langmuir monolayer at the air-water interface. Figure 2A I show the IRRAS spectra of the native lysozyme Langmuir monolayer at different incident angles and a constant surface pressure of 20 mN · m-1 at the subphase pH 6. Whereas, IRRAS spectra at various surface pressures at an incident angle of 60° is shown in Figure 2 A II. The reason for choosing 60° as an angle of incident for studying the variation of surface pressure by p-polarized IRRAS is good signal-to-noise ratio at this incident angle. The spectra obtained at different incident angles (Figure 2A I) at a surface pressure of 20 mN · m-1 shows amide I bands at 1653 and 1658 cm-1 corresponding to R-helix, while bands at 1623 and 1620 cm-1 correspond to β-sheet. The band observed at 1670 at an incident angle of 40° may be assigned to sterically constrained CdO moiety present in β-turn or split in the peak is noticed due to transition dipole coupling in β-sheet. Amide II bands are noticed at 1543 and 1521 cm-1 that correspond to R- helix and β-sheet, respectively. While varying the surface pressure at pH 6, the most intense bands appeared at 1627 and 1659 cm-1 (Figure 2A II) that correspond to β-sheet and R-helix, respectively, in amide I region. In amide II region the bands located at 1535 and 1526 cm-1 are assigned to parallel β-sheet. (30) Wang, J.; Dauter, M.; Alkire, R.; Joachimiak, A.; Dauter, Z. Acta Crystallogr., Sect. D 2007, 63, 1254.

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However, the band at 1553 cm-1 corresponds to the R-helix. With increasing surface pressure, the intensity of bands increased suggesting that there are a larger number of molecules per unit area as the monolayer was compressed (Figure 2A II). No substantial change in the position of bands suggests that there is no denaturation of the protein at subphase pH 6, while compressing. Important bands for the native lysozyme Langmuir monolayer at subphase pH 6 are shown in Table 1A. Intensity of bands remained constant at various surface pressures which indicates that the amide chains are oriented parallel to the air-water interface. The bands corresponding to β-sheet (1627, 1623 cm-1) are higher in intensity, though the β-sheet content of the protein is lower (10%) as compared to R-helix (40%), which is already known through X-ray crystal structure of the native lysozyme. One of the reasons for this observation might be due to the carbonyl groups present in β-sheet that are lying parallel to the air-water interface in comparison with the carbonyl groups present in the R-helix. Furthermore, if we examine the X-ray crystal structure of the native lysozyme shown in Scheme 1A, it can be easily noticed that even if most of the β-sheet strands are parallel to the surface, there are even more R-helices which are parallel to the surface. Thus, another possible reason for the higher intensity of stretching vibrations observed at 1627 and 1623 cm-1 might be due to the presence of 14 Asn side chains on the outer surface of lysozyme. It is possible that the band observed in the amide I region is a superimposition of NH bending vibration from the Asn side chain (1622 cm-1),29 as well as the CdO stretching vibrations from the β-strands. Also, relatively broader bandwidth at 1627 cm-1 corresponds to the flexibility of the structure having conformational freedom.31 We are aware of the fact that the vibrations of the side chain moieties can be distinguished from the secondary structure of the protein by use of D2O in the subphase. However, the purpose of our work is to examine our hypothesis of the conformational and orientation change of the protein monolayer observed at different range of pH. Hence, we have concentrated more on that aspect of the interpretation of the spectra; nonetheless, we have discussed, in general, the fingerprint of the lysozyme Langmuir monolyer. Band assignments for the major bands in the native lysozyme Langmuir monolayer at subphase pH 3 are presented in Table 2A. At subphase pH 3 for various incident angles (Figure 2B I), the important bands are observed for β-sheet at 1625 and 1529 cm-1 for amide I and II modes, respectively. Bands for amide I and II observed at 1656 and 1553 cm-1, respectively, were assigned to R-helix content present in the protein. A weak band at 1675 cm-1 might be due to the presence of antiparallel β-sheet or the β-turns in the protein. At an incident angle of 60° when the monolayer of native lysozyme was compressed from 0 to 20 mN · m-1, there is a prominent band at 1626 cm-1 corresponding to β-sheet (Figure 2B II). At a surface pressure of 15 mN · m-1, we can observe a band representing R-helix at 1658 cm-1. There are two possibilities for the emergence of a band at 1643 cm-1, one is due to the antiparallel β-sheet in the protein, and another is due to the unordered structure in the protein.24 Amide II bands are observed at 1563 and 1540 cm-1. Moreover, a weak shoulder appears at 1608 cm-1 when the native lysozyme Langmuir monolayer was compressed to a surface pressure of 1 and 5 mN · m-1, this could be due to the presence of some aggregated strands.24 As mentioned before, contribution of side chain residues become important in analyzing the spectra, another possible reason (31) Barth, A. Biochim. Biophys. Acta 2007, 1767, 1073.


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Table 1. Important Bands for the Native (A) and Denatured (B) Lysozyme Langmuir Monolayer at Subphase pH 6.0 A

B

native lysozyme band position cm-1 (surface pressure, incident angle)

band assignment

denatured lysozyme band position cm-1 (surface pressure, incident angle

band assignment

1627 (1 mN · m-1, 60°)

1631, 1688 (0 mN · m-1, 60°)

antiparallel β-sheet

1659 (1 mN · m-1, 60°) 1670 (20 mN · m-1, 40°)

parallel β-sheet and Asn residues (NH bending) R-helix β-turn

1657 (0 mN · m-1, 60°) 1624 5 mN · m-1, 60°)

1553 (15 mN · m-1, 60°) 1526 (10 mN · m-1, 60°)

R-helix β-sheet

1642 (10 mN · m-1, 60°) 1509 (15 mN · m-1, 60°)

R-helix parallel β-sheet and Asn residues antiparallel β-sheet tyrosine (C-N stretching coupled to CH and NH bending) C-H scissoring of CH2 and CH3 Aspartic and glutamic residues (COO- symmetric stretch)

1450 (10 mN · m-1, 60 °) 1405 (0 mN · m-1, 60°)

Table 2. Important Bands for the Native (A) and Denatured (B) Lysozyme Langmuir Monolayer at Subphase pH 3.0 A

B


band assignment


band assignment

1626 (15 mN · m-1, 60°) 1643 (15 mN · m-1, 60°)

β-sheet antiparallel β-sheet or unordered loop R-helix aggregated strands or Arginine (CN3H5+ antisymmetric stretch)

1622 (20 mN · m-1, 60°) 1644, 1692 (20 mN · m-1, 60°)

β-sheet and Asn residues antiparallel β-sheet

1660, 1665 (20 mN · m-1, 60°) 1587, 1405 (15 mN · m-1, 60°)

β-turn and R-helix aspartic acid (COOantisymmetric and symmetric stretch) tyrosine ring vibrations tryptophan residues (CdC stretching vibration coupled with C-H bending vibrations)

1658 (15 mN · m-1, 60°) 1608 (5 mN · m-1, 60°) 1675 (20 mN · m-1, 60°) 1514 (15 mN · m-1, 60°)

β-turn or antiparallel β-sheet tyrosine ring vibrations

for the appearance of this weak band at 1608 cm-1 could be the antisymmetric stretching vibrations in the guanidine (CN3H5+) groups27 present in 11 Arg side chains on the surface of the protein. A band observed at 1514 cm-1 corresponds to side chain amino acid tyrosine ring vibrations. Presence of a weak band at 1452 cm-1 represents the CH scissoring in the methylene (CH2) groups.25 Maximum intensity of amide I and II bands is observed at a surface pressure of 20 mN · m-1, this shows that more amide chains are present per unit area at this surface pressure and are lying parallel to the air-water interface. On the whole intensity of amide II bands is found low at 0, 1, 5, and 15 mN · m-1 which show that the amide chains are tilted more at these surface pressures. The IRRAS spectra at subphase pH 11 are significantly different from the other two at subphase pH 6 and 3 (see Table 3A). The bands for amide I mode are observed at an incident angle of 60° at 1665, 1648, 1631, and 1615 cm-1 (Figure 2C I). Splitting in the amide I band is attributed to transition dipole coupling. The band at 1631 cm-1 corresponds to the antiparallel β-sheet, which is complimented by the higher absorbance of the band observed at 1689 cm-1 at an incident angle of 35°, 40°, and 45° (spectrum not shown). The band observed at 1665 cm-1 is assigned to the β-turn present in the protein. Since predominantly R-helical proteins exhibit amide I maxima between 1648 and 1658 cm-1, the band at 1648 cm-1 is assigned to an R-helix. Moreover, at an incident angle of 40° there is a band that appears at 1655 cm-1 that corresponds to an R-helix. The band observed at 1615 cm-1 could be due to the presence of aggregated strands or side chain Arg residues. Amide II bands are observed at 1531, 1534, and

1515 (10 mN · m-1, 60°) 1496 (15 mN · m-1, 60°)

1558 cm-1. Furthermore, the band observed at 1450 cm-1 is due to CH2 or CH3 scissoring. At various surface pressures as shown in Figure 2C II, splitting of amide I band is observed. The band position at 1627 cm-1 corresponds to the parallel β-sheet structure and the band observed at 1641 cm-1 is due to the presence of CdO groups involved in H-bonding that stabilize the turn or antiparallel β-sheet. In comparison to subphase pH 6 and 3, antiparallel β-sheet content increases for the conformation of native lysozyme Langmuir monolayer at subphase pH 11. The prominent band that is observed at 1680 cm-1 at a surface pressure of 15 mN · m-1 is due to the vibration of CdO groups not involved in H-bonding and which are sterically constraint. Thus, absorption at 1680 cm-1 is assigned to high-frequency antiparallel β-sheet. Intense band observed at 1656 cm-1 represents R-helix. Amide II bands are observed at 1550 and 1529 cm-1. Moreover, there is appearance of bands at 1587, 1592, and 1512 cm-1. The band at 1587 cm-1 is assigned to the side chain acidic amino acid, COO- antisymmetric stretching vibrations. Guanidine groups in the side chain Arg residues which are oriented parallel to the air-water interface at an incident angle of 60° are most likely responsible for the band at 1592 cm-1. At a surface pressure of 5 and 15 mN · m-1, the intensity of amide I and II bands is increased with respect to other surface pressures; this indicates that at these surface pressures the amide groups are lying parallel to the surface and tilting of the chains is to lesser extent. Upon increasing the surface pressure, position and intensity of bands changed to a smaller extent, which show that the tilting of the amide chains was not significant at pH 11.

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Table 3. Important Bands for Native (A) and Denatured (B) Lysozyme Langmuir Monolayer at Subphase pH 11.0 A

B


band assignment


Band assignment

1627 (15 mN · m-1, 60°) 1641 (5 mN · m-1, 60°) 1633 (18 mN · m-1, 60°) 1659 (0 mN · m-1, 60°) 1648 (20 mN · m-1, 60°)

β-sheet and Asn residues antiparallel β-sheet antiparallel β-sheet R-helix R-helix

β-sheet and Asn residues antiparallel β-sheet β-turn antiparallel β-sheet antiparallel β-sheet

1631 (20 mN · m-1, 60°)


1622 (0 mN · m-1, 60°) 1644 (20 mN · m-1, 60°) 1664 (20 mN · m-1, 60°) 1680 (20 mN · m-1, 60°) 1692 (20 mN · m-1, 45°, spectra not shown) 1587 (5 mN · m-1, 60°)

1680 (15 mN · m-1, 60°)


1496 (5 mN · m-1, 60°)

1656 (15 mN · m-1, 60°)1655 (20 mN · m-1, 40°) 1661 (20 mN · m-1,v60 °) 1615 (20 mN · m-1, 60°)

R-helix

1592 (0mN · m-1, 60°) 1587 (0 mN · m-1, 60°)

Asp residues (COOantisymmetric stretch) tryptophan residues (CdC stretching vibration coupled with C-H bending vibrations)

β-turn aggregated strand or Arg residues Arg residues (CN3H5+) Asp residues (COOantisymmetric stretch)

Study of Reduced Lysozyme Langmuir Monolayer using IRRAS. IRRAS of reduced lysozyme Langmuir monolayer at subphase pH 6 is investigated at different surface pressures and incident angles (Figure 3A). The spectra of reduced lysozyme Langmuir monolayer at different incident angles (Figure 3A I) show splitting of both amide I and amide II bands. This splitting demonstrates the presence of R-helix, parallel β-sheet and antiparallel β-sheet. The band located at 1666 cm-1 is assigned to β-turn and the band observed at 1677 cm-1 is noticed due to the presence of antiparallel β-sheet. The bands observed at 1648 and 1625 cm-1 correspond to amide I stretching in R-helix and β-sheet, respectively. Amide II bands are located at 1555, 1540, and 1521 cm-1. The bands observed at 1657 and 1624 cm-1 (Figure 3A II) correspond to CdO stretching vibration (amide I) for R-helix and β-sheet, respectively. The appearance of a band at 1688 and 1631 cm-1 that was absent in the native lysozyme is assigned to antiparallel β-sheet. The band at 1688 cm-1 is observed due to the presence of sterically constraint non-hydrogen-bonded CdO groups within turns in the antiparallel β-sheet. Moreover, a new band at 1642 cm-1 that appeared with increase in surface pressure further confirms the existence of antiparallel β-sheet in the reduced lysozyme. The amide II bands are observed at 1524 and 1547 cm-1. The band at 1450 cm-1 may be assigned to C-H scissoring of CH2 and CH3 moieties. A little hump at 1405 cm-1 is characteristic of the symmetric stretch from the COO- groups present in seven Asp side chain amino acid residues. The band position at 1563 cm-1 is also assigned to COO- stretching vibrations due to three side chain glutamic acid residues.21 The band position at 1509 cm-1 is attributed to C-N stretching vibration coupled to in-plane bending vibrational modes of C-H and N-H moieties present in the six side chain tryptophan residues.21 The intensity of amide I and amide II bands was significantly constant at various surface pressures, which indicate that the amide chains are oriented parallel to the air-water interface. Table 1A and B show the bands for native and denatured lysozyme Langmuir monolayer at subphase pH 6. The bands for the antiparallel β-sheet at 1631 and 1688 cm-1 appear for the reduced lysozyme Langmuir monolayer which are absent for the

native lysozyme. Moreover, the bands for Tyr, Asp, and Glu residues are present in the denatured lysozyme which are absent in the native lysozyme Langmuir monolayer. At subphase pH 3 amide I and amide II bands at various incident angles and different surface pressures are presented in Figure 3B. At an incident angle of 60° (Figure 3B I), 1627 and 1660 cm-1 represent amide I bands corresponding to parallel β-sheet and R-helix, respectively. Bands located at 1642 and 1692 cm-1 indicate the presence of antiparallel β-sheet in reduced lysozyme at subphase pH 3. Whereas, amide II bands are located at band position 1557 and 1537 cm-1 representing R-helix and β-sheet, respectively. Band observed at 1518 cm-1 might be due to the ring vibrations of tyrosine residue. In Figure 3B II, it is noticed that the band for R-helix is shifted upfield from the range 1648-1658 to 1660-1665 cm-1, it might be due to decrease in the strength of H-bonds in the R-helix and therefore decrease in the content of R-helix. As the surface pressure is increased from 0 to 20 mN · m-1, the band at 1660 cm-1 moved to 1665 cm-1 at a surface pressure of 20 mN · m-1, the reason might be the overlapping of the bands of R-helix and β-turn or the content of β-turn is increasing in the structure. The band positions in Figure 3B II at 1622 and 1644 cm-1 represent parallel and antiparallel β-sheets, respectively. Antisymmetric COO- stretching vibrations in the side chain amino acids are observed at 1587 cm-1, and symmetric a COO- stretch is noticed at 1405 cm-1. The band observed at 1496 cm-1 is due to CdC stretching vibration coupled with C-H in plane bending vibrations of side chain tryptophan residues. The band position at 1515 cm-1 is due to tyrosine ring vibrations. Amide II bands are observed at 1555 and 1535 cm-1, respectively. The intensity of amide II bands is relatively lower than the amide I bands which indicates that the C-N stretching vibrations and N-H bending modes are relatively tilted than the carbonyl stretching vibrations at the air-water interface. Table 2A and B shows the important bands for native and denatured lysozyme Langmuir monolayer at subphase pH 3. The important band that is absent in the reduced lysozyme Langmuir monolayer is the R-helix at 1658 cm-1. This band is moved upfield to 1660 cm-1, and upon compressing the reduced lysozyme Langmuir monolayer, this band is observed at 1665 cm-1. This


band at 1665 cm-1 is assigned to β-turns. Possible reason for this change is the breaking of S-S bridges in the reduced lysozyme and decrease in the content of R-helix. At subphase pH 11 for the reduced lysozyme Langmuir monolayer (Figure 3C), spectra are similar to the other two spectra observed for the reduced lysozyme at subphase pH 6 and 3. The major bands observed for amide I mode at an incident angle of 60° are 1622, 1648, and 1680 cm-1, corresponding to parallel β-sheet, R-helix, and antiparallel β-sheet, respectively (Figure 3C I). Amide II bands are observed at 1534 and 1524 cm-1. At an incident angle 45° (spectrum not shown), bands observed at 1692 and 1629 cm-1 indicate the presence of antiparallel β-sheet. Upon compressing the reduced lysozyme Langmuir monolayer, the band for parallel β-sheet is observed at 1622 cm-1 at 0 mN · m-1 and at 1624 cm-1 at 20 mN · m-1 (Figure 3C II). The band position at 1646 and 1644 cm-1 can be assigned to antiparallel β-sheet. Bands observed at 1660 and 1664 cm-1 is most likely the overlapped bands of R-helix and the β-turn. As mentioned earlier the band position at 1587 cm-1 is related to COO- antisymmetric stretching vibrations of the side chain acidic amino acids. Moreover, amide II bands are situated at 1546 and 1528 cm-1. Intensity of amide I and amide II bands is maximum at a surface pressure of 20 mN · m-1 which shows that the carbonyl groups are lying parallel to the air-water interface. Table 3 A and B shows the bands for the native and reduced lysozyme Langmuir monolayer at pH 11. The major deviation from the subphase pH 3 and 6 for the native lysozyme Langmuir monolayer at pH 11 is the increase in the content of antiparallel β-sheet. Moreover, the bands for the side chain amino acids Arg

Langmuir, Vol. 25, No. 5, 2009 2849

and Asp residues are more pronounced. In contrast, the bands for the reduced lysozyme Langmuir monolayer at pH 11 are similar to the one found at pH 3 and 6.

Conclusion Change in the conformation of native lysozyme Langmuir monolayer at pH 11 is a possible reason to explain the increase in limiting molecular area at this subphase pH. For the native lysozyme Langmuir monolayer conformation significantly changes to antiparallel β-sheet at its isoelectric point. Whereas, for the reduced lysozyme Langmuir monolayer the conformation change is of least importance at various subphase pH values. We observed substantial increase in the number of bands for various side chain amino acids for the reduced lysozyme Langmuir monolayer. This result correlates to the fact that the structure is no longer conserved for lysozyme after breakage of S-S bridges. Most significant result for the reduced lysozyme is reduction in the bands for R-helix. These results confirm that there is no variation of the value of limiting molecular area within the experimental error for the reduced lysozyme Langmuir monolayer. Our hypothesis that the variation of the limiting molecular area at various subphase pH was due to the change in conformation and orientation of the native lysozyme at the air-water interface is confirmed. Acknowledgement This work was supported by the “Forest Graduate Student in Aging Fellowship” awarded by the Center of Aging, University of Miami (G.T.). LA803233P

Conformation of Lysozyme Langmuir Monolayer Studied by Infrared

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