- Original article
- Open Access
Probing the orientation of electrostatically immobilized cytochrome C by time of flight secondary ion mass spectrometry and sum frequency generation spectroscopy
© Baio et al.; licensee Springer. 2013
- Received: 19 June 2013
- Accepted: 10 July 2013
- Published: 26 July 2013
By taking advantage of the electron pathway through the heme group in cytochrome c (CytoC) electrochemists have built sensors based upon CytoC immobilized onto metal electrodes. Previous studies have shown that the electron transfer rate through the protein is a function of the position of this heme group with respect to the electrode surface. In this study a detailed examination of CytoC orientation when electrostatically immobilized onto both amine (NH3 +) and carboxyl (COO-) functionalized gold is presented. Protein coverage, on both surfaces, was monitored by the change in the atomic % N, as determined by x-ray photoelectron spectroscopy. Spectral features within the in situ sum frequency generation vibrational spectra, acquired for the protein interacting with positively and negatively charged surfaces, indicates that these electrostatic interactions do induce the protein into a well ordered film. Time of flight secondary ion mass spectrometry data demonstrated a clear separation between the two samples based on the intensity differences of secondary ions stemming from amino acids located asymmetrically within CytoC (cysteine: C2H6NS+; glutamic acid: C4H6NO+ and C4H8NO2 +; leucine: C5H12N+). For a more quantitative examination of orientation, we developed a ratio comparing the sum of the intensities of secondary-ions stemming from the amino acid residues at either end of the protein. The 50 % increase in this ratio, observed between the protein covered NH3 + and COO- substrates, indicates opposite orientations of the CytoC on the two different surfaces.
- Heme Group
- Protein Immobilization
- Electron Transfer Pathway
- Barium Borate
- Polarization Combination
The heme group within cytochrome C (CytoC) provides an essential pathway for electrons traveling across a cell membrane . Taking advantage of this electron transfer pathway electrochemists have developed a series of devices based on CytoC immobilized onto both modified metal oxides [2–4] and alkanethiol SAMs [5–7]. Combined this work has demonstrated that the electron transfer rate through the protein to the electrode is directly related to the distance between the heme group and the substrate . Therefore, the distance between the heme group and electron transfer pathway can be tuned by altering the protein’s orientation.
Some of the most detailed characterizations of surface immobilized CytoC have been published by the Saavedra group. In a series of papers Saavedra and coworkers tracked the orientation of the heme group, within the protein, with linear dichroism in combination with emission anisotropy measured in total internal reflection fluorescence (TIRF) . This TIRF work has led to a detailed library of mean heme tilt angles for CytoC immobilized onto SH-, OH-, CH3-, and SO3-SAMs [10–14]. However, this TIRF work only provides details about the tilt angle of the heme group within the protein and not any information about the overall oreintation of the protein. As multiple CytoC orientations could produce similar heme tilt angles, focusing only on the heme tilt angle may not provide the distance between the heme group and the electrode.
To test this hypothesis while also connecting previous electrochemical observations to CytoC’s structure, ordering and orientation, we electrostatically immobilized CytoC onto positively and negatively charged substrates then we characterized differences in protein orientation by time of flight secondary ion mass spectrometry (ToF-SIMS), as well as probed protein ordering by sum frequency generation vibrational spectroscopy (SFG). Defining the ordering and orientation of the protein at the electrode surface enabled us to characterize any change in distance between the heme group and the electrode.
ToF-SIMS and SFG are two surface analysis techniques that provide complementary information about surface bound proteins. Taking advantage of ToF-SIMS’ high mass resolution (>4000 m/Δm) and excellent surface sensitivity (~2 nm sampling depth) changes in conformation and orientation of relatively thick protein films (>10 nm) can be directly related to changes in the intensity of specific secondary ions [18–28]. Recently two model systems built upon a small covalently and electrostatically immobilized protein (B1 domain of Protein G, 6 kDa), illustrated that differences in orientation can be discerned through the comparison of ratios of intensities of secondary ions originating from amino acid residues at opposite ends of the protein [29, 30]. These model systems also proved that ToF-SIMS can determine protein orientation even when the thickness of the protein is similar to the SIMS sampling depth.
To complement the protein orientation information provided by ToF-SIMS, conformational changes and the ordering of specific secondary structures (α-helices, β-sheets and β-turns) can be assessed by probing amide, N-H, C-H, and O-H vibrational modes with SFG [31–38] Additionally, tilt angles of helicies within the protein can be calculated from the polarization dependence of the SFG amide peaks [39, 40].
2.1 Functionalized substrates
1×1 cm2 silicon substrates (Microelectronics Inc., San Jose, CA) were cleaned by sequential sonication in deionized water, dichloromethylene, acetone, and methanol. In a high vacuum electron beam evaporator (pressure < 1×10-6 Torr), substrates were then coated with a thin layer of titanium (10 nm) followed by high purity gold (99.99%, 80 nm). Following established protocols , NH2-SAMs were assembled by submerging the Au coated substrates into a 0.5 mM 11-amino-1-undecanthiol, hydrochloride (Asemblon, Redmond, WA) ethanol solution. The substrates were allowed to soak in this ethanol–thiol solution for at least 16 h. Following assembly, the substrates were removed from solution, rinsed with pure ethanol, and then vortexed within a 10% v/v NH4OH ethanol solution. COOH-SAMs were assembled by soaking Au substrates in a 1-mM 11-mercaptoundecanoic acid (Aldrich, St. Louis, MO) ethanol solution for at least 16 h and then sonicated within pure ethanol. Following the rinse step, all substrates were dried and stored under nitrogen until analysis or protein adsorption.
2.2 Protein immobilization
Horse heart cytochrome c (12 kDa) was purchased commercially (Sigma, St. Louis, Missouri). For protein immobilization, the NH2- and COOH substrates were submerged in an ultra low salt phosphate buffered saline protein solution (1.37 mM NaCl, 2.7 mM KCl, 10 mM sodium phophate, and 2 mM potassium phosphate pH 6 and 8, respectively) at CytoC concentrations of 5, 25, 100, and 200 μM to produce surfaces with varying degrees of protein coverage. The two different pH’s were chosen to maximize the charge on the substrate  while maintaining both positive and negative charges within the protein (CytoC’s pI = 10) . Binding proceeded for 1 h at room temperature. Following protein immobilization, samples were washed by serial dilution in buffer, followed by submerging samples in a series of stirred water solutions. Samples were then air-dried and stored under nitrogen.
2.3 X-ray photoelectron spectroscopy (XPS)
XPS compositions were determined from an average of three spots on two replicate samples. The data were acquired on a Kratos AXIS Ultra DLD instrument (Kratos, Manchester, England) in the hybrid mode using a 0° take-off angle (angle between the surface normal and the axis of the analyzer lens) and a monochromatic Al Kα1,2 x-ray source (hv = 1486.6 eV). Atomic compositions were calculated from C 1 s and Au 4f peak areas obtained from survey scans (0–1100 eV) plus O 1 s (524–544 eV), N 1 s (390–410 eV), and S 2p (155–173 eV) peak areas from narrow scans (analyzer pass energy = 80 eV for both survey and narrow scan spectra). Energy scales were calibrated by setting the large CH x peak in the C 1 s region to 285.0 eV and a linear background was subtracted for all peak area quantifications. The peak areas were normalized by the manufacturer supplied sensitivity factors and surface concentrations were calculated using CASA XPS (Casa Software Ltd).
Where, Ns and Aus are the measured N and Au At %, respectively, from the NH2 SAM covered Au substrate prior to protein immobilization; Np and Aup are the measured N and Au At %, respectively, from the NH2 SAM covered Au substrate after protein immobilization; and Nnorm is the N At % corresponding to just the protein layer.
2.4 Time-of-flight secondary ion mass spectrometry (ToF-SIMS)
Positive secondary ion spectra were acquired on a TOF.SIMS 5–100 instrument (ION-TOF, Munster, Germany) using a pulsed 25 keV Bi3 + primary ion beam under static conditions (primary ion dose < 1012 ions/cm2). Spectra were collected from three 100×100 μm2 regions per sample. Secondary ions were collected over a range of 0–800 m/z at a mass resolution (m/Δm) between 4000 and 8000. Spectra were mass calibrated using CH2 +, C2H2 +, C3H5 + and AuSCH2 + peaks. Mass calibration errors were typically below 20 ppm.
2.5 Sum frequency generation spectroscopy (SFG)
SFG spectra were obtained by overlapping fixed visible (532 nm) and tunable IR (1000 – 4000 cm-1) pulses, in time and space, using an EKSPLA Nd:YAG laser operating at 50 Hz that also pumped an EKSPLA optical parametric generation/amplification and difference frequency unit based on barium borate and AgGaS2 crystals. The IR light had a bandwidth of 4 cm-1 and the visible light had a bandwidth of 1 cm-1. Both beams were focused to a diameter of ~1 mm at the sample surface at an energy of 160 μJ per pulse. All vibrational spectra were collected at 4 cm-1 increments and the generated SFG signal was first spectrally filtered and dispersed by a monochromator, then detected with a gated photomultiplier tube.
SFG spectra were collected at two different polarization combinations ssp (s-polarized SFG signal, s-polarized input visible, and p-polarized input IR beam) and ppp (p-polarized SFG signal, p-polarized input visible, and p-polarized input IR beam) through the backside of a CaF2 prism partially submerged into a protein buffer solution (protein concentration was 100 μM). A bare CaF2 prism was used as a model positively charged substrate while a negatively charged surface was created by oxygen plasma treating a CaF2 prism coated with polystyrene. The resulting signals were normalized by a reference signal generated in a ZnS crystal. The fitting routine used for SFG data analysis has been described previously .
In this study horse heart CytoC was immobilized onto positively and negatively charged substrates to induce opposite end-on orientations of CytoC, as done previously for Protein G B1 . The two different CytoC orientations are expected because of the distribution of positively charged lysine residues distributed about the protein surface and the collection of negatively charged glutamic acids at one end of the protein [15–17].
Overall, the SFG spectra provide a qualitative view of ordering and indicate that the electrostatic interactions between the protein and the substrates guide the adsorption of a well-ordered protein film. However, these results do not provide any detail about the structure and orientation of the protein. Electrostatically immobilizing CytoC onto positively (NH2-SAMs) and negatively charged (COOH-SAMs) substrates should induce the protein into opposite end-on orientations, thereby, changing the distance between the heme group and the substrate surface (Figure 1).
As in previous protein immobilization studies, differences in orientation were probed using ToF-SIMS by monitoring the intensity of specific secondary ions stemming from amino acid residues at either end of the protein [18, 22, 29, 30]. Here we apply this same strategy to probe any differences in CytoC orientation induced by the positively and negatively charged substrates (Figure 1).
Secondary ion fragments used in the ToF-SIMS analysis
Summary of XPS determined elemental composition (atom %) for NH 2 -SAMs and COOH-SAMs before and after CytoC adsorption
Normalized N 1s
CytoC on NH2-SAMs (100 μM)
CytoC on COOH-SAMs (100 μM)
As mentioned earlier, positively charged lysine residues are distributed around the surface of the protein . Yet, on the end of the protein, opposite the heme group, there is a cluster of glutamic acid residues creating a region with no net surface charge. Previous work has demonstrated that on the COOH-SAMs, the heme group should be pointed towards the substrate . Therefore, within the SIMS spectra collected from the CytoC-COOH-SAM samples, we should observe an enrichment of secondary ions from the leucine and glutamic acid residues. We hypothesize that on the NH2-SAMs, the heme group is pointed towards the vacuum interface. As a result, we should observe an enrichment of secondary ions from the cysteine residues.
The protein orientation inferred from the ToF-SIMS data agrees with previously published studies that showed when immobilized onto a COOH-SAMs electrode, the heme was pointed down towards the surface . Previous electrochemical studies have demonstrated that the redox potential between the heme group and the electrode shifts when comparing CytoC immobilized onto COOH-SAMs versus NH2-SAMs of the same height . Large differences in the reported ToF-SIMS ratios, across the two samples, also confirm that when CytoC interacts with a positively charged surface, this same dipole like distribution of charge induces the exact opposite orientation. As a result, the heme group is now pointing away from the surface, increasing the distance between itself and the substrate. The ToF-SIMS results illustrate that protein orientation can be directly related to electrochemical measurements.
This investigation has taken advantage of the charge distribution around the protein to induce CytoC into two different orientations, thereby controlling the location of the heme group with respect to the substrate. SFG spectra collected from CytoC interacting with both negative and positively charged surfaces illustrate that electrostatic interactions drive the protein to form a well-ordered protein film. Observed differences in the intensity of secondary ions originating from amino acids asymmetrically distributed around the protein demonstrate that on the negatively charged substrate CytoC’s heme group is pointed down towards the substrate, consistent with previous studies of CytoC on COOH-SAMs. The secondary ion intensity differences also indicate that CytoC adopts an opposite orientation on the positively charged surface, which would point the heme group away from the substrate.
This work was supported by NIH Grant EB-002027 (NESAC-BIO). T.W. thanks the German Research Foundation (DFG grants We 4478/1-1 and We 4478/2-1). J.E.B. thanks the National Science Foundation for a research fellowship (NSF grant #1202620).
- Hardison R: The evolution of hemoglobin: studies of a very ancient protein suggest that changes in gene regulation are an important part of the evolutionary story. Am Sci 1999,87(2):126.Google Scholar
- Runge AF, Saavedra SS: Comparison of microcontact-printed and solution-adsorbed cytochrome C films on indium tin oxide electrodes. Langmuir 2003,19(22):9418–9424.View ArticleGoogle Scholar
- Willit JL, Bowden EF: Determination of unimolecular electron-transfer rate constants for strongly adsorbed cytochrome-c on tin oxide electrodes. J Electroanal Chem 1987,221(1–2):265–274.Google Scholar
- Willit JL, Bowden EF: Adsorption and redox thermodynamics of strongly adsorbed cytochrome-c on tin oxide electrodes. J Phys Chem 1990,94(21):8241–8246.View ArticleGoogle Scholar
- Tarlov MJ, Bowden EF: Electron-transfer reaction of cytochrome-c adsorbed on carboxylic-acid terminated alkanethiol monolayer electrodes. J Am Chem Soc 1991,113(5):1847–1849.View ArticleGoogle Scholar
- Xu JS, Bowden EF: Determination of the orientation of adsorbed cytochrome c on carboxyalkanethiol self-assembled monolayers by in situ differential modification. J Am Chem Soc 2006,128(21):6813–6822.View ArticleGoogle Scholar
- Chen XX, Ferrigno R, Yang J, Whitesides GA: Redox properties of cytochrome c adsorbed on self-assembled monolayers: A probe for protein conformation and orientation. Langmuir 2002,18(18):7009–7015.View ArticleGoogle Scholar
- Georg S, Kabuss J, Weidinger IM, Murgida DH, Hildebrandt P, Knorr A, Richter M: Distance-dependent electron transfer rate of immobilized redox proteins: A statistical physics approach. Phys. Rev. E 2010,81(4):046101.View ArticleGoogle Scholar
- Lee JE, Saavedra SS: Molecular orientation in heme protein films adsorbed to hydrophilic and hydrophobic glass surfaces. Langmuir 1996,12(16):4025–4032.View ArticleGoogle Scholar
- Edmiston PL, Saavedra SS: Molecular orientation distributions in protein films. 4. A multilayer composed of yeast cytochrome c bound through an intermediate streptavidin layer to a planar supported phospholipid bilayer. J Am Chem Soc 1998,120(8):1665–1671.View ArticleGoogle Scholar
- Edmiston PL, Saavedra SS: Molecular orientation distributions in protein films: III. Yeast cytochrome c immobilized on pyridyl disulfide-capped phospholipid bilayers. Biophys J 1998,74(2):999–1006.View ArticleGoogle Scholar
- Wood LL, Cheng SS, Edmiston PL, Saavedra SS: Molecular orientation distributions in protein films.2. Site-directed immobilization of yeast cytochrome c on thiol-capped, self-assembled monolayers. J Am Chem Soc 1997,119(3):571–576.View ArticleGoogle Scholar
- Walker DS, Hellinga HW, Saavedra SS, Reichert WM: Integrated optical wave-guide attenuated total-reflection spectrometry and resonance raman-spectroscopy of adsorbed cytochrome-c. J Phys Chem 1993,97(39):10217–10222.View ArticleGoogle Scholar
- Du YZ, Saavedra SS: Molecular orientation distributions in protein films. V. Cytochrome c adsorbed to a sulfonate-terminated, self-assembled monolayer. Langmuir 2003,19(16):6443–6448.View ArticleGoogle Scholar
- Edmiston PL, Lee JE, Cheng SS, Saavedra SS: Molecular orientation distributions in protein films.1. Cytochrome c adsorbed to substrates of variable surface chemistry. J Am Chem Soc 1997,119(3):560–570.View ArticleGoogle Scholar
- Banci L, Bertini I, Gray HB, Luchinat C, Reddig T, Rosato A, Turano P: Solution structure of oxidized horse heart cytochrome c. Biochemistry 1997,36(32):9867–9877.View ArticleGoogle Scholar
- Bushnell GW, Louie GV, Brayer GD: High-resolution 3-dimensional structure of horse heart cytochrome-c. J Mol Biol 1990,214(2):585–595.View ArticleGoogle Scholar
- Baio J, Cheng F, Ratner D, Stayton P, Castner D: Probing orientation of immobilized humanized anti-lysozyme variable fragment by time-of-flight secondary-ion mass spectrometry. J Biomed Mater Res A 2011,97A(1):1–7.View ArticleGoogle Scholar
- Wagner MS, Castner DG: Characterization of Adsorbed Protein Films by ToF SIMS with PCA. Langmuir 2001, 17:4649–4660.View ArticleGoogle Scholar
- Wagner MS, Castner DG: Analysis of adsorbed proteins by static time-of-flight secondary ion mass spectrometry. Appl Surf Sci 2004, 231:366–376.View ArticleGoogle Scholar
- Wagner MS, Horbett TA, Castner DG: Characterizing multicomponent adsorbed protein films using electron spectroscopy for chemical analysis, time-of-flight secondary ion mass spectrometry, and radiolabeling: capabilities and limitations. Biomaterials 2003,24(11):1897–1908.View ArticleGoogle Scholar
- Baio JE, Weidner T, Samuel NT, McCrea K, Baugh L, Stayton PS, Castner DG: Multi-technique Characterization of Adsorbed Peptide and Protein Orientation: LK310 and Protein G B1. J Vac Sci Technol B 2010,28(4):C5D1-C5D8.View ArticleGoogle Scholar
- Xia N, May CJ, McArthur SL, Castner DG: Time-of-flight secondary ion mass spectrometry analysis of conformational changes in adsorbed protein films. Langmuir 2002,18(10):4090–4097.View ArticleGoogle Scholar
- Xia N, Castner DG: Preserving the structure of adsorbed protein films for time-of-flight secondary ion mass spectrometry analysis. J Biomed Mater Res A 2003,67A(1):179–190.View ArticleGoogle Scholar
- Michel R, Pasche S, Textor M, Castner DG: Influence of PEG architecture on protein adsorption and conformation. Langmuir 2005,21(26):12327–12332.View ArticleGoogle Scholar
- Lhoest JB, Detrait E, Van Den Bosch De Aguilar P, Bertrand P: Fibronectin adsorption, conformation, and orientation on polystyrene substrates studied by radiolabeling, XPS, and ToF SIMS. J Biomed Mater Res 1998,41(1):95–103.View ArticleGoogle Scholar
- Lhoest JB, Wagner MS, Tidwell CD, Castner DG: Characterization of adsorbed protein films by time of flight secondary ion mass spectrometry. J Biomed Mater Res 2001,57(3):432–440.View ArticleGoogle Scholar
- Wang H, Castner DG, Ratner BD, Jiang SY: Probing the orientation of surface-immobilized immunoglobulin G by time-of-flight secondary ion mass spectrometry. Langmuir 2004,20(5):1877–1887.View ArticleGoogle Scholar
- Baio JE, Weidner T, Baugh L, Gamble LJ, Stayton PS, Castner DG: Probing the orientation of electrostatically immobilized protein G B1 by iime of flight secondary ion spectrometry, sum frequency generation and near-edge X-Ray absorption fine structure spectroscopy. Langmuir 2012,28(4):2107–2112.View ArticleGoogle Scholar
- Baugh L, Nguyen P-C, Baio JE, Weidner T, Gamble LJ, Castner DG, Stayton PS: Probing the Orientation of Surface-Immobilized Protein G B1 using ToF-SIMS, Sum Frequency Generation, and NEXAFS Spectroscopy. Langmuir 2010,26(21):16434–16441.View ArticleGoogle Scholar
- Chen XY, Wang J, Sniadecki JJ, Even MA, Chen Z: Probing alpha-helical and beta-sheet structures of peptides at solid/liquid interfaces with SFG. Langmuir 2005,21(7):2662–2664.View ArticleGoogle Scholar
- Chen XY, Clarke ML, Wang J, Chen Z: Sum frequency generation vibrational spectroscopy studies on molecular conformation and orientation of biological molecules at interfaces. International Journal of Modern Physics B 2005, 19:691–713.View ArticleGoogle Scholar
- Wang J, Clarke ML, Chen XY, Even MA, Johnson WC, Chen Z: Molecular studies on protein conformations at polymer/liquid interfaces using sum frequency generation vibrational spectroscopy. Surf Sci 2005,587(1–2):1–11.View ArticleGoogle Scholar
- Wang J, Chen XY, Clarke ML, Chen Z: Detection of chiral sum frequency generation vibrational spectra of proteins and peptides at interfaces in situ. Proc Natl Acad Sci USA 2005,102(14):4978–4983.View ArticleGoogle Scholar
- Mermut O, Phillips DC, York RL, McCrea KR, Ward RS, Somorjai GA: In situ adsorption studies of a 14-amino acid leucine-lysine peptide onto hydrophobic polystyrene and hydrophilic silica surfaces using quartz crystal microbalance, atomic force microscopy, and sum frequency generation vibrational spectroscopy. J Am Chem Soc 2006,128(11):3598–3607.View ArticleGoogle Scholar
- Weidner T, Samuel NT, McCrea K, Gamble LJ, Ward RS, Castner DG: Assembly and structure of α-helical peptide films on hydrophobic fluorocarbon surfaces. Biointerphases 2010,5(9):9–16.View ArticleGoogle Scholar
- Weidner T, Breen NF, Li K, Drobny GP, Castner DG: Sum frequency generation and solid-state NMR study of the structure, orientation, and dynamics of polystyrene-adsorbed peptides. Proc Natl Acad Sci 2010,107(30):13288–13293.View ArticleGoogle Scholar
- Weidner T, Apte JS, Gamble LJ, Castner DG: Probing orientation and conformation of α-helix and β-strand model peptides on self-assembled monolayers using sum frequency generation and NEXAFS spectroscopies. Langmuir 2010,26(5):3433–3440.View ArticleGoogle Scholar
- Nguyen K, King J, Chen Z: Orientation determination of interfacial beta-sheet structures in situ. J Phys Chem B 2010,114(25):8291–8300.View ArticleGoogle Scholar
- Nguyen KT, Soong R, Im SC, Waskell L, Ramamoorthy A, Chen Z: Probing the spontaneous membrane insertion of a tail-anchored membrane protein by sum frequency generation spectroscopy. J Am Chem Soc 2010,132(43):15112–15115.View ArticleGoogle Scholar
- Baio JE, Weidner T, Brison J, Graham DJ, Gamble LJ, Castner DG: Amine terminated SAMs: Investigating why oxygen is present in these films. Journal of Electron Spectroscopy and Related Phenomena 2009,172(1–3):2–8.View ArticleGoogle Scholar
- Fears KP, Creager SE, Latour RA: Determination of the surface pK of carboxylic- and amine-terminated alkanethiols using surface plasmon resonance spectroscopy. Langmuir 2008,24(3):837–843.View ArticleGoogle Scholar
- Van Gelder BF, Slater EC: The extinction coefficient of cytochrome c. Biochim Biophys Acta 1962,58(3):593–595.View ArticleGoogle Scholar
- Moore FG, Becraft KA, Richmond GL: Challenges in interpreting vibrational sum frequency spectra: Deconvoluting spectral features as demonstrated in the calcium fluoride-water-sodium dodecylsulfate system. Appl Spectrosc 2002,56(12):1575–1578.View ArticleGoogle Scholar
- Mantus DS, Ratner BD, Carlson BA, Moulder JF: Static Secondary-Ion Mass-Spectrometry of Adsorbed Proteins. Anal Chem 1993,65(10):1431–1438.View ArticleGoogle Scholar
- Xia N: Time-of-flight secondary ion mass spectrometry (ToF-SIMS) characterization of conformation and orientation of adsorbed protein films. Seattle: University of Washington; 2003.Google Scholar
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