Langmuir 2007, 23, 2037-2041
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Thermodynamic and Spectroscopic Studies on the Nickel Arachidate-RNA Polymerase Langmuir-Blodgett Monolayer Priya Rajdev and Dipankar Chatterji* Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560012, India ReceiVed August 23, 2006 The Langmuir-Blodgett (LB) monolayers offer a unique system to study molecular interaction at the air-water interface with reduced dimensionality. In order to develop this further to follow macromolecular interactions at equilibrium, we first characterized the Ni (II)-arachidate (NiA) monolayer at varying conditions. Subsequently, the interaction between NiA and histidine-tagged RNA polymerase (HisRNAP) were also studied. LB films of arachidic acid-NiA and NiA-RNAP with different mole fractions were fabricated systematically. Surface pressure versus area per molecule (P-A) isotherms were registered, and the excess Gibbs energy of mixing was calculated. The LB films were then deposited on solid supports for Fourier transform infrared (FTIR) spectroscopic measurements. The FTIR spectra revealed the change in the amount of incorporated Ni (II) ions into the arachidic acid monolayer with the change in pH and the increasing mole fraction of RNAP in the NiA monolayer with its increasing concentration in the subphase. The system developed here seems to be robust and can be utilized to follow macromolecular interactions.
Introduction Langmuir-Blodgett (LB)1,2 technique is very well recognized now to study insoluble films at the surface. The study of LB films is based on the alignment of amphiphilic molecules at the air-water interface in a monomolecular layer, where the hydrophilic groups anchor onto the aqueous subphase and the hydrophobic groups point toward the air.3 Long-chain fatty acids have been extensively used as LB monolayers at the air-water interface. Due to this property of LB monolayers to array the amphiphilic molecules in a particular manner, it can successfully serve as a template for two-dimensional reactions. The reactions can be both chemical and biological,4 and have the potential to probe the various functions of biological molecules. Surface pressure (P)-area per molecule (A) isotherms are usually studied to understand the behavior of LB monolayers. These P-A isotherms illustrate general conclusions regarding the phase behavior of the two-dimensional LB monolayer of amphiphilic molecules. A phase transition is indicated by the change in P-A isotherms, which is attributed to the alignment of molecules, forming the monolayer, from the gaseous phase to the liquid condensed phase.3 The phase transition is the most important element of the P-A isotherms with a characteristic signature of a plateau region in the isotherms. This phase transition point or plateau region changes with the change of certain external parameters such as temperature, pH, and ionic strength, and this gives general information regarding the phase transition behavior. Therefore, the isotherms can be utilized for the study of the thermodynamics of the system under study.5 One needs to ensure, however, that the system has reached equilibrium and that the * Corresponding author. Address: Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560012, INDIA. Tel: +91-80-2293-2836. Fax: +91-80-2360-0535. E-mail:
[email protected]. (1) Blodgett, K. B. J. Am. Chem. Soc. 1935, 57, 1007. (2) Blodgett, K. B; Langmuir, I. Phys. ReV. 1937, 51, 964. (3) Gaines, G. L. Insoluble Monolayers at Liquid-Gas Interfaces; WileyInterscience: New York, 1966. (4) Debreczeny, M. P; Svec, W. A; Wasielewski, M. R. Science 1996, 274, 584. (5) Kurnaz, M. L; Schwartz, D. K. J. Phys. Chem. 1996, 100, 11113. (6) Bhaumik, A.; Ramakanth, M.; Brar, L. K; Raychaudhuri, A. K; Rondelez, F.; Chatterji, D. Langmuir 2004, 20, 5891. (7) Cha, J.; Park, Y.; Lee, K. B.; Chang, T. Langmuir 1999, 15, 1383.
monolayers are stable in nature, responding to hysteresis in the P-A curves6 before extracting thermodynamic parameters from them. LB films are generated from LB monolayers deposited on a solid substrate layer upon layer.3 The resulting LB films have a high degree of stability and structural order. These LB films have been characterized by various methods such as electron microscopy, X-ray diffraction, atomic force microscopy (AFM), and infrared (IR) spectroscopy.7-9 AFM and IR have been successfully employed in many cases,10,11 where the former is used to study the morphology of the LB films, and the latter gives information regarding the packing of the alkyl chains in the monolayer. IR spectroscopy is useful to study the defects in the LB films and hence helps in determining the conformational orders in the LB monolayers.12,13 Recently, it has also been employed successfully to estimate the secondary structure content of proteins.14 In addition, the intensity of the IR band of the monolayer can be a marker to follow the concentration-dependent changes in the characteristics of the LB films. We have shown recently6,15 that a divalent metal complex of a fatty acid such as arachidic acid can form a monolayer at the air-water interphase where the metal ion coordinates to carboxylate ion points in the aqueous subphase and has a considerable effect in aligning the monolayer. When Zn (II) was used, DNA from the subphase was picked up6 and oriented on LB films, whereas when Ni (II) was used, histidine-tagged RNA polymerase (HisRNAP) was aligned on the monolayer upon coordination with Ni (II).15 As expected, the reaction was pHdependent and reached equilibrium at a certain molar ratio of the reactants at the monolayer and in the subphase. (8) Bourdieu, L.; Ronsin, O.; Chatenay, D. Science 1993, 259, 798. (9) Kimura, F.; Umemura, J.; Takenaka, T. Langmuir 1986, 2, 96. (10) Vollhardt, D.; Fainerman, V. B.; Liu, F. J. Phys. Chem. B 2005, 109, 11706. (11) Haro, M.; Giner, B.; Lafuente, C.; Lo´pez, M, C.; Royo, F. M.; Cea, P. Langmuir 2005, 21, 2796. (12) Gurau, M. C.; Castellena, E. T.; Albertorio, F.; Kataoka, S.; Lim, S. M.; Yang, R. D.; Cremer, P. S. J. Am. Chem. Soc. 2003, 125, 11166. (13) Hasegawa, T.; Umemura, J.; Takenaka, T. Thin Solid Films 1992, 210211, 583. (14) Byler, D. M.; Susi, H. Biopolymers 1986, 25, 469. (15) Brar, L. K.; Rajdev, P.; Raychaudhuri, A. K.; Chatterji, D. Langmuir 2005, 21, 10671.
10.1021/la062486o CCC: $37.00 © 2007 American Chemical Society Published on Web 01/06/2007
2038 Langmuir, Vol. 23, No. 4, 2007
RajdeV and Chatterji
Although attempts have been made in the past to evaluate thermodynamic parameters from a Cd (II)-arachidate LB layer,5 the system has not been used extensively for biological samples, such as the incorporation of enzymes in the LB layer.15 In this work, we have tried to quantitate and derive a set of values for the Ni (II)-arachidate (NiA) interaction and their ionic strength and pH dependence. Further, we have extended the system to Ni (II)-RNA polymerase interaction with an aim to study the macromolecular interaction at the interphase. Materials and Methods Preparation of Silicon Slides. The silicon slides (10 × 10 mm) were carefully cleaned in a 3:7 (v/v) mixture of 30% H2O2 and 95% H2SO4 for about 2 h. Then they were rinsed with Milli-Q water thoroughly and stored under water. The silicon slides were sonicated in Milli-Q water immediately before the transfer of the monolayers. Purification of the Enzyme and its Transcription Assay. HisRNAP was purified16 from a strain obtained as a gift from Robert Landick, University of Wisconsin, following the same protocol as reported before by us.15 Formation and Deposition of LB Films. The LB films were prepared on a LB trough purchased from Nima Technology (U.K.) and placed under a Perspex box. Arachidic acid and NiSO4 (both of purity 99%) were purchased from Sigma-Aldrich. The monolayers were formed by spreading the arachidic acid solution (1 mg/mL) in chloroform (HPLC grade, Merck) at the surface of the water subphase (water purified with a Milli-Q system, resistivity 18.2 MΩ), containing 10-4 M of NiSO4. LB films were formed and transferred onto hydrophilic silicon slides, at a constant surface pressure of 25 mN/m by vertical dipping. The rate of compression was 20 cm2/ min, and the dipper speed while transferring the layers was kept at 2 mm/min. The pH of the subphase solution was kept at 7.4, adjusted by using Tris-HCl (Sigma) buffer (2 mM). The temperature was maintained at 20 ((0.1) °C. The transfer ratio was monitored and was greater than 0.8 in all the cases. For the immobilization of HisRNAP, we introduced the HisRNAP into the aqueous subphase. HisRNAP purified in the laboratory was used for this work. The concentration of the enzyme purified in the laboratory was 1 mg/ mL, and the enzyme was found to be fully active in DNA-dependent RNA synthesis in vitro. Toward thermodynamic studies, different amounts of the enzyme solution were injected into the aqueous subphase so that the obtained mole fractions of the enzyme, which were calculated using the set of isotherms in the monolayer, were also different. AFM Imaging. The AFM images were taken using a commercial atomic force microscope, Thermomicroscope CP-R (Veeco Technologies, USA), with a 5 × 5 µm scanner in intermittent-contact mode. Rectangular tip integrated cantilevers made of silicon with a spring constant of 4 N/m (Olympus Opt., Japan) were used. The cantilever was vibrated at a frequency of 60 kHz, and the scan speed was varied from 1 to 5 µm/sec. The slides were dried for 2 h in vacuum before imaging. The representative image is shown here. Fourier Transform Infrared (FTIR) Spectroscopy. All the IR transmission spectra were obtained in a Perkin-Elmer FTIR spectrometer, Spectrum 1000, where the incident rays were normal to the sample, and the resolution was 4 cm-1. For NiA LB films, the range of spectra was from 1500 to 1700 cm-1, and, for NiARNAP LB films, it was from 1600 to 1700 cm-1. The silicon chip with the NiA monolayer or NiA-RNAP monolayers were used as solid substrates and placed on the IR source for the measurements. Minimally, 50 layers were necessary to transfer before any detectable signals were noticed.
Results Our first aim was to follow the effect of monovalent cations or ionic strength in the P-A isotherm of NiA. In our previous (16) Kashlev, M.; Martin, E.; Polyakov, A.; Severinov, K.; Nikiforov, V.; Goldfarb, A. Gene 1993, 130, 9.
Figure 1. P-A isotherms of (a) NiA in the pH range of 7.4-4.8 and (b) changes in a NiA monolayer with varying concentration of NaCl.
work, we already reported the characteristics of P-A isotherms.15 Figure 1a shows the variation of the NiA P-A isotherm as a function of pH. This figure clearly shows an increase in the incorporation of Ni (II) into the monolayer of arachidic acid when pH increased from 4.8 to 7.4 (from left to right). It can be seen from the figure that, with increase of pH at the subphase, the liquid condensed region progressively disappeared until about pH 7.4. It is therefore expected that the incorporation of Ni (II) at this pH is maximum. At pH 4.8, the NiA monolayer behaves as arachidic acid, with no incorporation of Ni (II), which can be attributed to the pKa of arachidic acid, 5.5. This is in accordance with the skeletonization experiment, which measured the amount of Ni (II) in the monolayer, reported by us recently.15 Figure 1b shows the effect of varying the concentration of NaCl on the LB monolayer of NiA at a constant pH of 7.4. As the concentration of Na (I) increases from 5 to 50 mM in the subphase, the onset of the liquid phase commences, suggesting that the Na (I) from the subphase replaces the Ni (II) ion from the monolayer and reduces the condensation effect of Ni (II) due to its bivalent nature. We will discuss this point in greater detail in the next section. Since our main motive behind this study was to follow macromolecular interaction on LB monolayers, it was essential to fix a salt concentration, suitable for such studies, without hindering the formation of a monolayer with sufficient Ni (II) ion in it to capture and align the His-tagged protein. Figure 2a shows the P-A isotherms of NiA at pH 7.4 and 25 mM NaCl with varying concentration of HisRNAP in the subphase. It can
Thermodynamics at the Air-Water Interface
Langmuir, Vol. 23, No. 4, 2007 2039
Figure 2. (a) P-A isotherms of NiA-HisRNAP. (b) 1 × 1 µm AFM image of NiA-HisRNAP.
be seen from this figure that, upon increasing the concentration of the enzyme, area/molecule increases concomitantly, and the concentration of NaCl used here has no effect on the P-A isotherm. Figure 2b detects the presence of the enzyme on the LB monolayer through AFM. The dimensions of the enzyme molecule measured by AFM matches well with the reported dimension of RNAP, as mentioned in our earlier work.15 The degree of dissociation of fatty acids and their interaction with Ni (II) can, in principle, be followed by FTIR spectroscopy, where COO- ions and undissociated COOH molecules have characteristic stretching frequencies at 1540 and 1700 cm-1, respectively.17 Figure 3a shows that the undissociated arachidic acid has a signature band at 1700 cm-1 at pH 4.8, which progressively decreases with increasing pH, due to the dissociation of the fatty acid. On the other hand, a peak at 1540 cm-1, due to asymmetric stretching of the COO- ion,17 was noticed with increase in pH, as expected. All the samples of LB layers subjected here for FTIR measurements were generated on silicon chips, which are transparent to IR frequencies, and the monolayers of arachidic acid at higher pHs will have Ni (II) ions coordinated to the COO- end due to the presence of Ni (II) ions in the subphase. Likewise, Figure 3b shows the FTIR spectra of LB films of the NiA-HisRNAP complex. Here, HisRNAP was in the subphase prior to the formation of the NiA-HisRNAP monolayer. FTIR had been successfully utilized before to study the different conformations of protein, that is, the content of β sheets or the R helix.13 Moreover, FTIR spectroscopy helps in determining the structural changes of an enzyme accompanying various types (17) Diaz, M. E.; Johnson, B.; Chittur, K.; Cerro, R. L. Langmuir 2005, 21, 610.
Figure 3. (a) FTIR spectra of LB films of NiA transferred on silicon slides. It is evident from the figure that, as the pH increases from (bottom to top), the intensity of the 1540 cm-1 band increases, and that of the 1700 cm-1 band diminishes, as expected. (b) FTIR spectra of LB films of NiA-RNAP transferred on silicon slides. Here, the NiA monolayer was formed first, which then picked up HisRNAP from the subphase. It is evident from the figure that, as the mole fraction of RNAP increases (bottom to top), the intensity of the 1660 cm-1 band increases.
of reactions such as ligand binding18 and redox reactions,19 as well as changes due to the formation of a variety of reaction intermediates.20 The characteristic bands in the FTIR spectra of a protein sample13,21 are the peaks seen at around 1650 cm-1 and at 1550 cm-1 due to CdO stretching vibrations (the amide I band) and N-H bending vibrations (the amide II band), respectively. Out of these, the amide I band is of particular significance, as it is fundamental to all the proteins and is located on the backbone of the peptide. Thus this band mostly remains unaffected in the presence of different side chains. Likewise, RNAP, when oriented as NiA-HisRNAP on LB films, can be detected within 1600 to 1700 cm-1, the region of the amide I band. Figure 3b shows that, as the mole fraction of HisRNAP increases in the NiA LB monolayer, the intensity of the amide I band, around 1665 cm-1, also increases. It appears from the FTIR spectra of the NiA-HisRNAP complex that, with the (18) Iwaki, M.; Rich, P. R. J. Am. Chem. Soc. 2004, 126, 2386. (19) Iwaki, M.; Osyczka, A.; Moser, C. C.; Dutton, P. L.; Rich, P. R. Biochemistry 2004, 43, 9477. (20) Iwaki, M.; Puustinen, A.; Wikstro¨m, M.; Rich, P. R. Biochemistry 2003, 42, 8809. (21) Lee, A. S; Galea, C.; DiGiammarino E. L.; Jun, B.; Murti, G.; Ribeiro, R. C.; Zambetti, G.; Schultz, C. P.; Kriwacki, R.W. J. Mol. Biol. 2003, 327, 699.
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RajdeV and Chatterji Table 1. The Estimated Values of the Total Free Energy of Mixing for NiA and NiA-HisRNAP (a) NiA System ∆Gmix (J/mol) mole fraction of NiA
4 mN/m
6 mN/m
8 mN/m
10 mN/m
0.3 0.5 0.6 0.7
-1493 -1684 -1638 -1487.7
-1494 -1681 -1641 -1491
-1498 -1685 -1646 -1495
-1490 -1686 -1671 -1518
(b) NiA-RNAP System ∆Gmix (J/mol) mole fraction of RNAP
4 mN/m
6 mN/m
10 mN/m
12 mN/m
0.2 0.4 0.6 0.8
-1212 -1643 -1629 -1224
-1215 -1642 -1625 -1227
-1224 -1649 -1627 -1227.4
-1241 -1635 -1621 -1227.6
∆Gmix is given by the equation
∆Gmix ) ∆GΕ + ∆Gideal
Figure 4. Plot of the free energy of mixing, ∆GE, at different surface pressures, P versus the mole fraction of (a) arachidic acid-NiA and (b) NiA-RNAP. It can be seen from the plots that ∆GE shows a maximum at a molar ratio of 1:1.
increase in HisRNAP concentration in the subphase, more HisRNAP is incorporated into the LB monolayer of NiA until it reaches the maximum value, at pH 7.4. Next, we attempted to estimate the free energy of formation of the NiA monolayer as well as that of the NiA-HisRNAP complex. In order to do so, we first established that the system reached equilibrium by monitoring the area per molecule value in the P-A isotherm over an extended period of time. The excess Gibbs free energy of mixing, ∆GE also allows one to quantitatively analyze the intermolecular interaction between the molecules of the mixed monolayer formed,5,10 and it is given by the expression
∆GE )
∫0P
(A12 - x1A1 - x2A2)dP
max
(1)
where A1 and A2 are the areas per molecule of pure arachidic acid and NiA, respectively, at a surface pressure P, and A12 is the area per molecule of NiA at an intermediate pH at the same surface pressure P. x1 and x2 are the mole fractions of arachidic acid and NiA, respectively, at that pH, and Pmax is the upper limit of surface pressure for the calculation of the integral. Similarly, for the NiA-HisRNAP system, A1 and A2 are the areas per molecule of pure NiA and HisRNAP, respectively, at a surface pressure P, and A12 is the area per molecule of the NiA-HisRNAP complex moiety at some intermediate concentration of HisRNAP, at the same surface pressure P. x1 and x2 are the mole fractions of NiA and HisRNAP, respectively. The total free energy of mixing,
(2)
where ∆Gideal ) RT (x1 ln x1 + x2 ln x2). It is sometimes preferable to give ∆Gmix values rather than the ∆GΕ values, as the absolute values corresponding to the latter are small in magnitude. ∆GE and ∆Gmix are the free energy changes associated with the incorporation of Ni (II) ions in the arachidic acid monolayer and HisRNAP in the NiA monolayer, respectively. Figure 4a shows the plot of ∆GE versus the mole fraction of NiA at different surface pressures, P. It can be seen from Figure 4a that there is a maximum at all values of P, corresponding to a mole fraction of 0.5 of NiA, which was obtained at a pH value of pH 5.5 and hence represents the monolayer having a nearly equal proportion of arachidic acid and NiA. This is the pKa of arachidic acid, and the mixing is least, which gives rise to positive values of ∆GE. At other values of mole fractions less than and above 0.5, the process of mixing is favored, resulting in negative values of the free energy of mixing. The mixing process is spontaneous at pH values less than and above 5.5 because, at any other pH, the proportion of one of the two components is greater, and this does not augment the formation of domains of any one kind of component. It was also noticed that the mixing becomes more favorable as the surface pressure increases for all molar ratios other than 1:1. The values for ∆GE reported here are much less; however, they are close to those reported earlier for other divalent systems.5 Figure 4b shows the plot of ∆GE versus the mole fraction of HisRNAP at different surface pressures and, in this case, the plot also shows a maximum at a NiA/HisRNAP mole fraction of 1:1, like the arachidic acid-NiA system. Although, the plot shows the excess Gibbs free energy of mixing values quite low in magnitude, the values of total Gibbs free energy of mixing lie within the range of 1200-1600 J/mol, considering ∆Gideal values.22 The values reported in Table 1 are less than the ∆G values estimated in solution for an RNAP-divalent cation interaction.23
Discussion Here, we have devised a system comprising a hydrophobic monolayer upon a hydrophilic subphase where the metal ion (22) Chou, T. H.; Chang, C. H. Langmuir 2000, 16, 3385. (23) Chatterji, D.; Wu, F. Y.-H. Biochemistry 1982, 21, 4657.
Thermodynamics at the Air-Water Interface
content in the monolayer can be changed with the change of pH and ionic strength. Consequently, the system can then be successfully employed for the interaction of biomolecules injected into the subphase in an LB trough with metal ions. The degree of the interaction of the Ni (II) ions with the arachidic acid as a function of the pH or the concentration of NaCl or the interaction of HisRNAP with the NiA monolayer has been monitored with the help of P-A isotherms. FTIR spectroscopy has been utilized to study the changes in the concentration of metal ions and the protein molecules in the LB monolayer. LB technique is, perhaps, one of the simplest techniques today to align molecules at the interphase, such as air-water in this case, with an increase in surface pressure. If the amphiphathic molecule at the surface is a macromolecule, then LB films can orient it too; it can then be transferred to a solid substrate, and the chip then would have an array of oriented molecules amenable to various studies.3 However, in this work, our approach has been different; we started with an aim to follow macromolecular interactions at the LB surface. The objectives are manifold in this case. First, there is a reduction in dimensionality at LB films, which then can be used appropriately for macromolecular interaction.24 Second, one of the reactants at the surface is oriented, arrayed in a crowded fashion, resulting in a situation (we presume) closer to in vivo conditions. If the reactants are biological macromolecules, the thermodynamic parameters so derived will have fascinating applications. We observed here that the presence of a divalent cation is necessary to coordinate with two tandem COO- ions, which (24) Berg, O. G.; von Hippel, P. H. Annu. ReV. Biophys. Biophys. Chem. 1985, 14, 13140. (25) Record, M. T., Jr.; Lohman, T. M.; deHaseth, P. L. J. Mol. Biol. 1976, 107, 145.
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results in the ordering of a NiA monolayer. With increasing Na (I) ion in the medium, this arrangement is destroyed, resulting in an altered P-A isotherm. Thus, a divalent cation induces a near disappearance of the condensed liquid-liquid phase, quite close to the sublimation phenomenon, and monovalent cations do not favor sharp phase transition as reported before in some other cases.3 Alkaline pH, above the pKa of the amphiphathic acid used here, is required to generate COO- ions, which then complex with Ni (II). Ni (II), in its turn, picks up RNAPs through histidine coordination and arrays them. Since our ultimate aim is to follow the promoter-RNAP interaction on the LB layer, minimal salt concentration in the subphase is necessary to avoid nonspecific interaction.25 We found that 25 mM NaCl is suitable for this purpose, and RNAP was found to be active at this salt concentration (not shown). The different free energy values reported here for Ni (II)RNAP interaction are lower than the values estimated in homogeneous solution in vitro.23 We believe that this should necessarily be the case due to the nature of interactions with restricted dimensions. We have shown before15 that RNAP in the NiA monolayer can selectively interact and orient promoter DNA molecules and align them under increasing surface pressure. It would be worthwhile to find out the ∆G values for such interactions and other thermodynamic constants like ∆H and ∆S from a simple system such as an LB monolayer. Acknowledgment. D.C. thanks the Department of Biotechnology, Government of India, for sponsored projects. P.R. thanks IISc for fellowship. We would also like to acknowledge Mr. I.S. Jarali, Department of SSCU, IISc, for his help with FTIR spectroscopy and Anupam Chakravarty for his help. LA062486O
