Secondary Structures of Ubiquitin Ions Soft-Landed onto Self

Article pubs.acs.org/JPCB

Secondary Structures of Ubiquitin Ions Soft-Landed onto SelfAssembled Monolayer Surfaces Qichi Hu and Julia Laskin* Physical Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352, United States S Supporting Information *

ABSTRACT: The secondary structures of multiply charged ubiquitin ions soft-landed onto self-assembled monolayer (SAM) surfaces were studied using in situ infrared reflection−absorption spectroscopy (IRRAS). Two charge states of ubiquitin, 5+ and 13+, were mass selected separately from a mixture of different charge states produced by electrospray ionization (ESI). The low 5+ charge state represents a nativelike folded state of ubiquitin, while the high 13+ charge state assumes an extended, almost linear conformation. Each of the two charge states was soft-landed onto a CH3- and COOH-terminated SAM of alkanethiols on gold (HSAM and COOH-SAM). HSAM is a hydrophobic surface known to stabilize helical conformations of soft-landed protonated peptides, whereas COOH-SAM is a hydrophilic surface that preferentially stabilizes β-sheet conformations. IRRAS spectra of the soft-landed ubiquitin ions were acquired as a function of time during and after ion soft-landing. Similar to smaller peptide ions, helical conformations of ubiquitin are found to be more abundant on HSAM, while the relative abundance of βsheet conformations increases on COOH-SAM. The initial charge state of ubiquitin also has a pronounced effect on its conformation on the surface. Specifically, on both surfaces, a higher relative abundance of helical conformations and a lower relative abundance of β-sheet conformations are observed for the 13+ charge state compared to the 5+ charge state. Timeresolved experiments indicate that the α-helical band in the spectrum of the 13+ charge state slowly increases with time on the HSAM surface and decreases in the spectrum of the 13+ charge state on COOH-SAM. These results further support the preference of the hydrophobic HSAM surface toward helical conformations and demonstrate that soft-landed protein ions may undergo slow conformational changes during and after deposition.

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studied for proteins adsorbed on metals,9 naturally occurring minerals,10 nanoparticles,11 amphiphilic lipid membranes,12,13 and self-assembled monolayer (SAM) surfaces.8,14 Because their physical and chemical properties may be tuned by varying the terminal functional group, SAMs are attractive model systems for such studies. As a result, SAMs often are used as model hydrophobic and hydrophilic surfaces for fundamental studies in protein adsorption.15 Conformational changes upon protein adsorption on surfaces traditionally are studied using Fourier transform infrared (FTIR) spectroscopy,16 circular dichroism spectroscopy,17 surface-enhanced Raman spectroscopy (SERS),18 and sum

INTRODUCTION

Protein adsorption on surfaces is an important process that determines their structure and function relevant to nanotechnology, biological sensing, materials science, and biology.1,2 Protein interactions with solid surfaces have been extensively investigated to achieve control over the binding energies, adsorption/desorption kinetics, and secondary structure of these complex molecules on surfaces.3−5 The adsorption of proteins on surfaces may be driven by both strong electrostatic and hydrogen-bonding interactions or weaker van der Waals forces.6 Regardless of the precise mechanism, such interactions may have a pronounced effect on the secondary and tertiary structures of adsorbed species.3,7 Chemical composition, surface morphology, and protein coverage are important factors affecting protein structural changes.1,3,7,8 Conformational changes induced by protein−surface interactions have been © XXXX American Chemical Society

Received: March 8, 2016 Revised: May 12, 2016

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DOI: 10.1021/acs.jpcb.6b02448 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B frequency generation (SFG) vibrational spectroscopy.19 Among these techniques, grazing angle infrared reflection−absorption spectroscopy (IRRAS) has been widely used to study the secondary structures of proteins adsorbed on surfaces.20,21 The amide I band region (1600−1700 cm−1), dominated by CO stretching modes of amide bonds in proteins, is used as a fingerprint of the protein conformation.22 The position of the carbonyl stretching vibration varies significantly as a result of hydrogen-bonding interactions in the vicinity of the CO bond. Different structural motifs in the protein contribute to the overall shape of the characteristic amide I band that typically is composed of several components. Detailed analysis of the amide I band, which often involves deconvolution and spectral fitting, provides valuable information about protein conformations. In addition, the kinetics of the conformational change induced by adsorption of proteins onto surfaces has been explored by acquiring IRRAS spectra at different times after adsorption.23 This approach has been used to interrogate the secondary structure of lysozyme adsorbed on solid substrates. A two-phase conformational transition was observed in that study, with the first phase involving fast conversion of the α-helix to the random coils/turns within the first minute after adsorption and the second phase corresponding to a relatively slow conversion from the α-helix to the β-sheet conformation on a time scale of 1−1200 min.23 Protein adsorption typically is studied by immersing a surface into a protein solution for a period of time. While efficient and convenient, this approach suffers from the presence of impurities as well as protein aggregation in solutions. Alternatively, protein adsorption may be studied using softlanding (SL)24,25 of mass-selected ions. SL enables preparation of intact, highly pure films of peptides and proteins on solid or liquid substrates, thereby overcoming the limitations of protein adsorption from the solution phase.26−38 This technique involves deposition of mass- and charged-selected gas-phase ions onto surfaces.25,39−41 The ability to select the specific mass and charge of the ion, and the precise control over the kinetic energy of the ion beam and deposition amount, makes SL an attractive approach for preparing protein layers on substrates. Using the SL technique, Cooks and co-workers demonstrated selective deposition of individual protein ions of a particular mass-to-charge ratio from a mixed protein solution at different positions on a gold substrate to form a microarray.26 The softlanded proteins were found to retain their biological activity. Later, the same group demonstrated retention of biological activity by protein ions soft-landed onto liquid surfaces.28 Similar results were reported by Turecek and co-workers, who demonstrated that SL of trypsin ions onto a plasma-treated metal surface resulted in a substantial retention of the enzymatic activity.30 To understand the retention of biological activity by softlanded proteins, it is important to determine their secondary structures following deposition. In these experiments, multiply charged protein ions are produced through electrospray ionization (ESI)42 and deposited either under ambient conditions43 or in vacuum.25 Although ESI is a gentle ionization technique that does not break covalent bonds,42 the protein’s secondary and tertiary structures may undergo significant changes upon transfer from solution into the gas phase.44 Furthermore, protein−surface interaction may have a significant effect on the conformation of soft-landed species. For example, our previous studies demonstrated that soft-landed polyalanine peptide, AcA15K, retains a dominant helical

conformation on a methyl-terminated SAM (HSAM),33,45 while a significant fraction of AcA15K is converted into a βsheet conformation on a carboxyl-terminated SAM (COOHSAM) and a SAM of a fluorinated thiol (FSAM).45 In situ IRRAS characterization of soft-landed AcA15K confirmed the molecule is slowly converted from the initial α-helical conformation into the β-sheet conformation on COOHSAM.45 Herein, we examined the effect of the surface and initial conformation on the secondary structure of soft-landed proteins using ubiquitin as a model system. The single domain of ubiquitin, a protein with 76 amino acid residues and a molecular weight of 8433,46 was selected for this study because this relatively small protein has been extensively studied in both the gas and condensed phases.47−55 Ubiquitin’s native three-dimensional (3D) structure is extremely compact and includes one α-helix, five β-strands, a short piece of 310helix, and seven reverse turns connecting between these structural motifs.56 Charge states from 5+ to 13+ were produced by ESI of ubiquitin solutions, with the 13+ charge state being a fully protonated state of ubiquitin that has seven lysines, four arginines, one histidine residue, and the N-terminal amino group as protonation sites.57,58 Structures of gas-phase ubiquitin ions have been studied using ion mobility s p e c t r o m e t r y a n d e l e c t r o n c a p t u r e d is s o c i a t i o n (ECD).50,52,54,55,59 It has been demonstrated that the ubiquitin ion in the 5+ charge state has a compact nativelike tertiary structure with a cross-section of ∼900 Å2. In contrast, the 13+ charge state has an elongated “near-linear” tertiary structure with a much larger cross-section of ∼2000 Å2 and represents an unfolded state.54,55,60 The measured cross-section of the fully protonated 13+ charge state (ubiquitin has 13 possible basic sites) is in good agreement with the result obtained using molecular dynamics simulations.54,61 Secondary structures of ubiquitin ions also have been characterized by examining fragmentation patterns obtained following capture of low-energy electrons by the protein ion.44,50,52 ECD experiments indicate that the 13+ charge state exists in an elongated α-helical conformation, while the 5+ charge state has an “S”-shaped conformation with three helical regions and two extended bending parts connecting the helices.52 In contrast, ion spectroscopy experiments in helium droplets indicate that the high charge states of ubiquitin assume extended conformations without the helical component.62 In this study, we examined the effect of the surface on the secondary structures of proteins using the 5+ and 13+ charge states of ubiquitin as two extreme cases of gas-phase conformations for this system. Ubiquitin ions selected according to their mass-to-charge ratios were soft-landed onto two different SAM surfaces: a hydrophobic HSAM (SAM of 1dodecanethiol on gold) and a hydrophilic COOH-SAM (SAM of 16-mercaptohexadecanoic acid) previously shown to stabilize the α-helical and β-sheet conformations of deposited peptides, respectively.33 Secondary structures of soft-landed ions were characterized using in situ IRRAS.45 We demonstrate that both the initial charge state of ubiquitin ions and the surface properties have a noticeable effect on the secondary structure of soft-landed ubiquitin. Similar to the simpler peptide system examined in previous studies,33,45 we observe that HSAM preferentially stabilizes the α-helical conformation, while the βsheet conformations are more abundant on COOH-SAM. Moreover, the relative abundance of the α-helical conformation is shown to increase on both surfaces for the 13+ charge state of ubiquitin. This result is consistent with the extended B


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The Journal of Physical Chemistry B

The amide I band was analyzed by fitting the experimental data with a sum of Gaussian curves. To identify the number and positions of different bands underlying the amide I envelope, Fourier self-deconvolution was first applied to the 1600−1700 cm−1 region using the OPUS software. Subsequent fitting of the amide I band with a sum of Gaussian functions provided the positions and integrated intensities of the individual components, indicative of the secondary structure of the deposited protein. Simultaneous fitting of all time-resolved IRRAS data obtained in one experiment was performed in Excel using the generalized reduced gradient (GRG) nonlinear algorithm. For the fitting, all band intensities were normalized to unity to ensure equal contribution of each IRRAS spectrum to the sum of squares of deviations between the experimental and simulated data. Fitting involved iterative optimization of the band positions and intensities of individual components of the amide I band while keeping the bandwidths of all the peaks the same. Optimization was repeated for different values of bandwidths until the best fit was obtained. Fitting robustness was tested by allowing the individual peak positions to vary during the fit and comparing the result with the fit obtained by fixing band positions between the time points. This test indicated that band positions remain the same throughout the deposition experiment. Furthermore, simultaneous fitting of all the time points ensures a robust fit.

structure containing several helical segments predicted for the 13+ charge state both experimentally50,52 and theoretically.61

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EXPERIMENTAL SECTION The ion deposition apparatus used in our SL experiments combined with in situ IRRAS characterization of surfaces is described in detail elsewhere.63,64 Briefly, ions were produced by ESI of a 100 μM solution of bovine ubiquitin (SigmaAldrich, St. Louis, MO), transferred into the vacuum system through a heated capillary maintained at 80−120 °C, and focused using an electrodynamic ion funnel and a collisional quadrupole (CQ). The solution conditions were optimized for the formation of different charge states of ubiquitin. Specifically, a mixture of H2O/CH3OH/NH4HCO3 (49:49:2, v/v/v) was used to produce the lower charge states. The 2% NH4HCO3 buffer was added to maintain neutral pH ≈ 7. Higher charge states, ranging from 9+ to 13+, were produced using a CH3OH/CH3COOH/glycerol (98:1:1, v/v/v) mixture as a solvent with 1% CH3COOH added to keep the solution acidic (pH ≈ 3) and 1% glycerol added as a “supercharging” agent.65,66 The 13+ charge state ion intensity was very low without glycerol; it was improved by more than an order of magnitude by adding glycerol. The precursor ion (either the 5+ or 13+ charge state of ubiquitin) was mass selected using a quadrupole mass filter (Extrel, Pittsburgh, PA) and focused onto the SAM surface using two einzel lenses. The pressure in the ion deposition region was 6 × 10−5 Torr. The ion current on the SAM surface for both charge states was between 10 and 20 pA per charge. During each SL experiment, similar amounts of ubiquitin ions ((∼1−2) × 1012 ions) were deposited onto an 8 mm spot on the SAM surface to obtain comparable intensity of the IRRAS signal. Surface coverage of the 50−60% monolayer was estimated for deposition of 2 × 1012 ions using the cross-section of 1500 Å determined for ubiquitin ions in ion mobility experiments.59 Self-Assembled Monolayer Surfaces. Hydrophobic SAMs of 1-dodecanethiol (HSAM) and hydrophilic SAMs of 16-mercaptohexadecanoic acid (COOH-SAM) were prepared following literature procedures.67,68 1-Dodecanethiol and 16mercaptohexadecanoic acid were purchased from SigmaAldrich (St. Louis, MO) and used as received. SAMs were prepared on 10 × 10 mm gold-coated silicon wafers with a 100 nm gold layer deposited on top of a 10 nm chromium adhesion layer (SPI Supplies, West Chester, PA). Gold surfaces were thoroughly cleaned in an ultraviolet/ozone cleaner and immersed in 1 mM solutions of the corresponding thiols in ethanol for 12 h. Prior to use, the substrates were washed ultrasonically in ethanol (10%, v/v, acetic acid in ethanol was used for the COOH-SAM) to remove physisorbed molecules from the SAM surface and dried under a nitrogen stream. IRRAS spectra were recorded using a Bruker Vertex 70 FTIR spectrometer (Bruker Optics, Billerica, MA). The output beam from the spectrometer was focused onto the SAM surface with an incidence angle of 80° with respect to the surface normal, and the reflected light was refocused onto a liquid-nitrogencooled mercury−cadmium−telluride (MCT) detector using two parabolic gold mirrors. The IRRAS spectra were obtained at a resolution of 4 cm−1 using p-polarized light, and each spectrum was an average of 512 scans acquired in 2 min every 10 min. Reflection and transmission mode FTIR spectra of neutral ubiquitin were recorded by depositing a 2 μL droplet of 100 μM ubiquitin in H2O (pH of 6−7) onto a bare Au surface and ZnSe window, respectively.

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RESULTS AND DISCUSSION Secondary structures of gaseous ubiquitin ions have been extensively investigated. Many studies have demonstrated the dramatic effect of the charge state on the secondary structure of ubiquitin ions in the gas phase. For example, the 5+ and 13+ charge states of ubiquitin assume compact, nativelike and almost linear conformations, respectively.50,52,54,57,61 The 5+ and 13+ charge states of ubiquitin, [U + 5H]5+ and [U + 13H]13+, representing extreme cases of gas-phase secondary structures of this well-characterized protein, were deposited onto a hydrophobic HSAM surface and a hydrophilic COOHSAM surface. Different charge states of ubiquitin were generated by varying the solution composition. The native secondary structure of ubiquitin, producing predominately 5+ and 6+ charge states in ESI, is fairly stable under different solution conditions.55,69 It has been demonstrated that addition of a small amount of glycerol to an acidified electrospray solution significantly enhances the formation of higher charge states of proteins.65 Efficient charging of proteins in the presence of glycerol was attributed to its relatively high surface tension, which facilitates additional charging at the droplet surface prior to Rayleigh fission. Figure 1 shows mass spectra obtained by electrospraying ubiquitin from a neutral (pH ≈ 7) and an acidic (pH ≈ 3) solution containing a small amount of glycerol. Consistent with previous studies,69 ubiquitin ions produced from a neutral solution of H2O/CH3OH/NH4HCO3 (49:49:2, v/v/v) have a charge state distribution ranging from 5+ to 9+, while higher charge states that range from 10+ to 13+ are produced from a mixture of CH3OH/CH3COOH/glycerol (98:1:1, v/v/v). Either the 5+ or 13+ charge state was selected from the charge state distribution shown in Figure 1 by a quadrupole mass filter and subsequently was soft-landed onto two different SAM surfaces. Secondary structures of the soft-landed ubiquitin ions were characterized using in situ IRRAS.64 Figure 2 shows IRRAS spectra of the 5+ and 13+ ubiquitin ions soft-landed on HSAM (panel a) and COOH-SAM (panel C


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AcA15K peptide, indicating that helical conformations contribute substantially to the observed absorption of infrared light by the soft-landed ubiquitin ions. Differences in the shapes of the amide I bands observed after SL of [U + 5H]5+ and [U + 13H]13+ will be discussed later in this paper. Substantial broadening of the amide I and amide II ubiquitin bands compared with the helical bands of AcA15K indicates that other secondary structure motifs also are quite abundant. The major difference between the HSAM and COOH-SAM surfaces is observed in the amide III region. On the COOH-SAM surface, a narrow band at 1265 cm−1 is overlaid on top of a broad feature covering the 1210−1320 cm−1 range, whereas only weak absorption in this region without distinct features is observed on the HSAM surface. The narrow peak at 1265 cm−1 observed on COOH-SAM most likely corresponds to random coils or β-turns.70,71 Meanwhile, the broad featureless absorption between 1210 and 1340 cm−1 is contributed by other conformations. For example, a broad peak centered around 1263 cm−1 was observed in our previous study and was attributed to the β-sheet conformation of AcA15K on COOHSAM. A characteristic narrow amide III band of the α-helix centered at 1310 cm−1 (Figure 2c) is not observed in the IRRAS spectra of soft-landed ubiquitin ions. A low signal in the amide III region of the IRRAS spectra of ubiquitin on HSAM precludes any conclusive statements about the protein conformation based on this region of the spectrum. To assist band assignments, we collected and analyzed reflection and transmission mode FTIR spectra of neutral ubiquitin molecules deposited from aqueous solution onto gold and ZnSe substrates, respectively. Because ubiquitin retains its native state in water at pH 5.8 up to 355 K,72 it is reasonable to assume that FTIR spectra obtained by depositing ubiquitin molecules from aqueous solution represent the native conformation. In both reflection and transmission mode experiments, the droplet was allowed to dry prior to analysis to minimize water absorption during FTIR data acquisition. Figure 3 depicts FTIR spectra of neutral ubiquitin obtained in

Figure 1. Charge state distributions of ubiquitin ions produced by ESI from two different ubiquitin solutions. The lower charge states from 5+ to 9+ were produced from a neutral (pH ≈ 7) solution of 100 μM ubiquitin in a mixture of H2O/CH3OH/NH4HCO3 (49:49:2, v/v/v), and the higher charge states from 10+ to 13+ were produced from an acidic solution (pH ≈ 3) of 100 μM ubiquitin in a mixture of CH3OH/CH3COOH/glycerol (98:1:1, v/v/v). To amplify the intensity of the 13+ charge state, glycerol was added to the second solution.

Figure 2. IRRAS spectra obtained after soft-landing of ∼2 × 1012 ions of the 5+ (black) and 13+ (red) charge states of ubiquitin onto (a) HSAM and (b) COOH-SAM surfaces. (c) IRRAS spectrum of the αhelical AcA15K peptide on HSAM shown for comparison.

b) surfaces. Time-resolved IRRAS spectra are shown in Figure S1 of the Supporting Information. For comparison, the spectrum of the α-helical AcA15K peptide reported in our previous study33 appears in Figure 2c. Characteristic amide I, II, and III bands appear in this region of the spectrum. For both surfaces and charge states, the amide I and amide II bands are centered around 1666 and 1540 cm−1, respectively. Interestingly, the position of the amide I band of soft-landed ubiquitin coincides with the position of the amide I band of the helical

Figure 3. Reflection (red line) and transmission (black line) mode FTIR spectra of the neutral ubiquitin molecules deposited from solution onto a bare Au surface and ZnSe window, respectively. The inset shows the amide I band region. Bar graphs show the relative abundance of the individual amide I band components obtained by fitting the experimental data with a sum of Gaussian functions (see the text for details). D


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Table 1. Analysis of the Amide I Band in FTIR Spectra of Neutral Ubiquitin (Reflection and Transmission Modes) and Ubiquitin Ions in the 5+ and 13+ Charge States Soft-Landed onto HSAM and COOH-SAM Surfaces (Reflection Mode)a band assignment turns turns β-hairpin β-sheet (ν⊥) α-helix β-sheet (ν∥) turns

reflection

transmission

1595 1620

8 8

1595 1620

13 15

1644 1660 1676 1689

22 27 18 17

1643 1663 1670 1686

33 18 5 15

HSAM [U + 5H]5+

HSAM [U + 13H]13+

COOH-SAM [U + 5H]5+

1603

9

1600

3

1608

12

1633 1643 1662 1679 1700

9 8 32 24 19

1634 1640 1662 1679 1699

12 2 49 28 6

1631 1647 1662 1680 1690

14 14 25 26 9

COOH-SAM [U + 13H]13+ 1600 1620 1636 1646 1664 1680 1690

4 3 11 6 41 23 13

Band positions (cm−1) and average relative intensities (%) were obtained by fitting the amide I band with a sum of Gaussian functions (described in the text). a

(1630−1640 cm−1) wavenumbers.75 Theoretical calculations have provided unique insights into the localization of the individual normal modes in proteins, assisting secondary structure assignments. For example, Chung and Tokmakoff76 examined eigenstates of the amide I band of several proteins, including ubiquitin. They found that the 1640 cm−1 mode is localized on the β-sheet, whereas the 1675 cm−1 mode is influenced by the α-helix and coils. The 1645 and 1650 cm−1 modes were found to be localized on the α-helix but mixed with other modes. The higher frequency modes of the amide I band were found to be far more mixed among the α-helix, β-sheet, and random coil than the lower frequency modes. Choi et al.77 used a combination of molecular dynamics simulations and quantum chemistry calculations to examine the vibrational modes of ubiquitin. On the basis of the localization and sign of the eigenvector elements, they assigned the six most abundant modes of ubiquitin. The most-abundant normal mode at 1650 cm−1 was found to be delocalized over the α-helical domain and was assigned to the A mode of the α-helix. The second feature at 1655 cm−1 delocalized over two β-strands and was assigned as a high-frequency β-hairpin mode, while the mode at 1628 cm−1 was assigned as a low-frequency β-hairpin mode. The higher frequency mode at 1670 cm−1 was found to be delocalized over two β-sheet strands and the α-helix. The low-frequency modes at 1600 and 1623 cm−1 were assigned as turn-region modes. Chung et al.53 examined temperature-dependent conformational changes of ubiquitin using FTIR and two-dimensional (2D) infrared spectroscopy. In that study, absorption bands of the antiparallel β-sheet conformation were observed at 1642 and 1676 cm−1. The low- and high-frequency β-sheet bands were shown to have a transition moment perpendicular and parallel to the β-strand, respectively. The parallel β-sheet band amplitude was insensitive to the geometry of the β-sheet. Meanwhile, both the amplitude and frequency of the perpendicular lower frequency band were strongly affected by the strength of interstrand couplings. It has been shown both theoretically53,77 and experimentally49,51,53,74,78 that the frequency of the ∼1640 cm−1 band shifts toward higher wavenumbers with a decrease in the interstrand coupling strength during protein unfolding. For example, thermal unfolding of ubiquitin following nanosecond laser irradiation resulted in decreased intensity and a blue shift of the lower frequency β-sheet band. These changes were attributed to the corresponding decrease in the number of folded strands in the β-sheet.53 Meanwhile, the higher frequency mode of the β-sheet did not change much following laser heating.

both reflection and transmission modes. The amide I region of the spectra is shown in the inset. Despite the general similarity between the FTIR spectra obtained in the reflection and transmission modes, there is an obvious shift in the band position by ∼15 cm−1 to higher wavenumbers in the reflection mode. This 5−15 cm−1 shift in the absorption bands’ position toward higher wavenumbers in the reflection mode often is attributed to longitudinal optical excitations with a transition moment perpendicular to the surface, which are inactive in the bulk but observed as surface modes in thin films.73 Detailed analysis of the amide I band has provided additional information about the secondary structure of ubiquitin. Previous infrared spectroscopy studies demonstrated that the amide I band of ubiquitin is composed of eight principal bands assigned to different secondary structure motifs of the protein.49,53,74 In this study, fitting of the amide I band was performed to access the relative contributions of different secondary structures to the observed absorption bands. Eight components were used to describe the band shape accurately. In the fitting process, the positions and amplitudes of individual components of the amide I band were allowed to vary within constraints to obtain the best fit to the experimental data. Bar graphs in Figure 3 show the relative abundances of the individual amide I band components of neutral ubiquitin, obtained from fitting the experimental data with a sum of Gaussian functions. Clearly, the relative abundance of the individual amide I band components changes depending on the acquisition mode. In the transmission mode, absorption is dominated by bands centered at 1620 (15%), 1643 (33%), 1663 (18%), and 1686 (15%) cm−1. In the reflection mode, the relative abundance of the major bands changes, while their positions are quite similar to those in the transmission mode. The major bands in the reflection mode are found at 1644 (22%), 1660 (27%), 1676 (18%), and 1689 (17%) cm−1. The band at 1620 cm−1 remains present, but its abundance is 2 times lower in the reflection mode than in the transmission mode. The individual amide I band components were assigned to secondary structural motifs on the basis of the literature data, accounting for observed shifts in the band positions in reflection mode compared to transmission mode FTIR. The amide I band shape has been widely used for characterizing the secondary structures of peptides and proteins.22 The α-helical band typically is observed in the range of 1650−1660 cm−1, and absorption at 1640−1650 cm−1 is attributed to the random coil conformation. The range of 1660−1695 cm−1 corresponds to β-turns, while the β-sheet structure contributes to two absorption bands at high (1670−1680 cm−1) and low E


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Figure 4. Relative abundances of the amide I band components as a function of time during and after soft-landing of the 5+ (a, c) and 13+ (b, d) charge states of ubiquitin onto HSAM (a, b) and COOH-SAM (c, d) surfaces. The end of ion deposition is marked with a black arrow.

interactions. Figure 4 shows the variation in the relative abundances of the individual bands as a function of time during and after ion deposition. Although the relative abundances for most of the bands do not show any measurable time dependence, the relative abundance of the β-sheet band at 1680 cm−1 gradually decreases in the IRRAS spectra of the 5+ charge state on both surfaces and in the 13+ charge state on HSAM. Meanwhile, the relative abundance of the α-helical band at 1662 cm−1 increases in the spectrum of the 13+ charge state of ubiquitin on the HSAM surface and decreases in the spectrum of the 13+ charge state on COOH-SAM. These results indicate that soft-landed ubiquitin ions undergo slow conformational changes on SAM surfaces. Protein aggregation on SAM surfaces also could be responsible for the observed trends in the relative abundance of individual bands. However, relatively constant band intensities observed after ion deposition (Figure 4) indicate that protein aggregation that should occur both during and after ion soft-landing is not a dominant process. Figure 5 illustrates the average relative abundances of the individual amide I band components averaged over the time after the end of ion deposition. The β-sheet band at ∼1645 cm−1 and the α-helical band at 1662 cm−1 show a systematic variation with the initial charge state of the projectile ion. Specifically, higher relative abundance of the α-helical

Table 1 lists the positions, relative abundances, and tentative assignments of the individual modes contributing to the amide I band of ubiquitin obtained by fitting the amide I band using a procedure described in the Experimental Section. We assigned the most prominent bands at 1643 and 1644 cm−1 in the transmission and reflection modes, respectively, to the β-sheet conformation with a transition moment perpendicular to the βstrand (ν⊥), which is the most abundant secondary structure of native ubiquitin.56 The band at 1676 cm−1 in the transmission mode is assigned to the higher frequency component of the βsheet conformation with a transition moment parallel to the βstrand (ν∥), which most likely is mixed with other secondary structural motifs. Band assignment of the 1670 and 1686 cm−1 features in the reflection mode is more problematic. Most likely, the 1670 cm−1 feature corresponds to the ν∥ mode. The higher frequency band at 1689 cm−1 in the transmission mode is assigned to turns mixed with other secondary structures. The respective bands at 1660 and 1663 cm−1 in the transmission and reflection modes are assigned to the α-helix. The same fitting procedure was used to analyze the amide I band obtained for both 5+ and 13+ charge states of ubiquitin during and after deposition onto HSAM and COOH-SAM. The fitting results, including peak positions and relative abundances, are listed in Table 1. Minor shifts in band positions may be attributed to differences in protein−surface F


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surface on the secondary structure of soft-landed proteins. Furthermore, STM experiments unambiguously demonstrated that the conformation of soft-landed proteins may be controlled by varying the charge state of the projectile ion.35 Specifically, an extended, almost linear secondary structure was observed by SL of the 19+ charge state of cyt c Meanwhile, SL of lower charge states of the same protein resulted in more compact secondary structures.35 Molecular dynamics simulations of the cyt c SL indicate that the kinetic energy of the ion during the collision may promote conformational change. Surprisingly, at higher kinetic energies of the 19+ cyt c ions, more compact conformations of this protein were trapped on the surface. In contrast, no change in the size of the soft-landed species was observed for deposition of lower charge states of cyt c at different kinetic energies.35 The results shown in Figure 5 also reveal the effect of the surface on the conformation of soft-landed ubiquitin ions. Specifically, we observe that the relative abundance of the βsheet band at ∼1645 cm−1 increases and the relative abundance of the α-helical band at 1662 cm−1 decreases on COOH-SAM compared to HSAM. These results indicate that the β-sheet conformation is more abundant on the COOH-SAM surface, while the α-helical conformation is more abundant on the HSAM surface. Preferential immobilization of the α-helical conformation on HSAM previously was reported for softlanded polyalanine peptides that contained a basic lysine residue on the C-terminus.45 In that study, stable polyalanine helices,80 [Ac-AnK + H]+ (n = 7, 15), were deposited onto HSAM, FSAM, and COOH-SAM. It was found that the HSAM surface preferentially stabilized helical conformations. Meanwhile, slow conversion of α-helices into β-sheets was observed on COOH-SAM. In this study, we observed slow decay of the α-helical component for the 13+ charge state of ubiquitin on COOH-SAM (Figure 4d) and a noticeable increase in the relative abundance of the α-helix for the same charge state on HSAM (Figure 4b). In contrast, for the 5+ charge state, no measurable variation in the relative abundance of the α-helix was observed on HSAM, while this band increased during SL of [U + 5H]+ on COOH-SAM.

Figure 5. Average relative abundances and standard deviations of the amide I band components observed in IRRAS spectra after softlanding of the 5+ and 13+ charge states of ubiquitin onto HSAM and COOH-SAM surfaces. The values were obtained by averaging the results shown in Figure 4 after the end of ion deposition, when the relative abundance of different bands was fairly constant.

component is observed when the 13+ charge state is deposited onto both HSAM and COOH-SAM, while the relative abundance of this band is lower for the 5+ charge state on both surfaces. Furthermore, for both charge states, the α-helical band is more abundant on HSAM compared with COOHSAM. The β-sheet band at ∼1645 cm−1 shows the opposite trend. Specifically, this band is more abundant for the 5+ charge state and shows a reproducible preference for the COOH-SAM surface. In contrast, the same relative abundance of the 1635 cm−1 band assigned to the β-hairpin and the ν∥ band of the βsheet at ∼1680 cm−1 is observed for both charge states on both surfaces. It has been demonstrated that the ν∥ band is not sensitive to the β-sheet53 geometry and probably contains contributions from other secondary structures,76 which may explain the lack of sensitivity of this vibrational feature to the secondary structure of the soft-landed ubiquitin ions. Because random coil and β-turn conformations are known to contribute to absorption at ∼1680 cm−1, this conclusion is further supported by the presence of a narrow absorption band at 1265 cm−1 in the amide III region observed for ubiquitin ions softlanded on COOH-SAM but not on HSAM. As discussed earlier, this band has been assigned to random coils and β-turns on the basis of the literature data. The higher abundance of the α-helical conformation of the soft-landed 13+ charge state of ubiquitin on both HSAM and COOH-SAM is consistent with the largely α-helical structure of this charge state predicted on the basis of fragmentation patterns of the [U + 13H]13+ precursor ion observed in electron capture dissociation experiments50,79 and confirmed by molecular dynamics simulations of the 13+ charge state of ubiquitin.61 Our results indicate that the initial extended structure of [U + 13H]13+, containing several helical segments, is largely preserved on the surface. Rauschenbach and coworkers observed unfolded structures of soft-landed cytochrome c (cyt c) and bovine serum albumin on Cu(001) substrates using scanning tunneling microscopy (STM).34 In contrast, more compact secondary structures were found on Au(111) substrates, indicating the pronounced effect of the

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CONCLUSIONS We examined the effect of the initial secondary structure and surface properties on the conformation of a well-characterized protein, ubiquitin, soft-landed onto SAM surfaces. We used hydrophobic HSAM and hydrophilic COOH-SAM surfaces as they previously have been shown to stabilize the α-helical and β-sheet conformations, respectively. The charge state of ubiquitin was used to control the initial conformation of the protein in the gas phase. The 5+ charge state was selected to represent compact, nativelike conformations, and the 13+ charge state was chosen to represent elongated conformations of ubiquitin containing several helical segments.61 Timeresolved IRRAS spectra of the 5+ and 13+ charge states of ubiquitin soft-landed onto two different SAMs were acquired during and after ion deposition. Secondary structures of softlanded ubiquitin ions were characterized by analyzing the shape of the amide I band in the IRRAS spectra. Both the initial conformation and properties of the SAM surface had a pronounced effect on the secondary structures of soft-landed ubiquitin ions. For example, SL of the extended ubiquitin conformation represented by the 13+ charge state on both HSAM and COOH-SAM resulted in preferential stabilization of the helical conformations. Meanwhile, a G


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The Journal of Physical Chemistry B substantially higher abundance of the β-sheet conformations was observed for the 5+ charge state of ubiquitin on both surfaces. Time-resolved IRRAS data demonstrated an increase in the relative abundance of the α-helical band for the 13+ charge state on HSAM during and after ion deposition, while no measurable change was observed when the 5+ charge state was deposited onto this surface. In contrast, an opposite trend was observed on the COOH-SAM surface, which showed no change in the abundance of helical conformations for the 13+ charge state and an increase of the helical bands during ion deposition for the 5+ charge state. These results are in agreement with the predicted structures of the 5+ and 13+ charge states of ubiquitin.52,54,61 The surface properties showed a less pronounced but noticeable effect on the conformation of soft-landed ubiquitin ions. Specifically, we found that, for both charge states, helical conformations are more abundant on HSAM and β-sheet conformations are preferentially stabilized on COOH-SAM. These observations are in agreement with our previous studies where we examined SL of stable peptide helices onto SAM surfaces.33,45 However, no clear trends were observed by comparing time-resolved IRRAS data obtained for HSAM and COOH-SAM surfaces. Thus, it is likely that major conformational changes occur at the time of collision, during which protein ions undergo substantial deformation as indicated by molecular dynamics simulations.35 Subsequent slow conformational changes observed in our IRRAS experiments may be affected by both protein−surface and protein−protein interactions. Our results highlight the complex nature of protein ion−surface interactions that determine the final conformation of the soft-landed species.

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(3) Roach, P.; Farrar, D.; Perry, C. C. Interpretation of Protein Adsorption: Surface-Induced Conformational Changes. J. Am. Chem. Soc. 2005, 127, 8168−8173. (4) Roach, P.; Farrar, D.; Perry, C. C. Surface Tailoring for Controlled Protein Adsorption: Effect of Topography at the Nanometer Scale and Chemistry. J. Am. Chem. Soc. 2006, 128, 3939−3945. (5) Norde, W.; Favier, J. P. Structure of Adsorbed and Desorbed Proteins. Colloids Surf. 1992, 64, 87−93. (6) Haynes, C. A.; Norde, W. Globular Proteins at Solid/Liquid Interfaces. Colloids Surf., B 1994, 2, 517−566. (7) Ball, A.; Jones, R. A. L. Conformational-Changes in Adsorbed Proteins. Langmuir 1995, 11, 3542−3548. (8) Sethuraman, A.; Belfort, G. Protein Structural Perturbation and Aggregation on Homogeneous Surfaces. Biophys. J. 2005, 88, 1322− 1333. (9) Mandal, H. S.; Kraatz, H. B. Effect of the Surface Curvature on the Secondary Structure of Peptides Adsorbed on Nanoparticles. J. Am. Chem. Soc. 2007, 129, 6356−6357. (10) Capriotti, L. A.; Beebe, T. P.; Schneider, J. P. Hydroxyapatite Surface-Induced Peptide Folding. J. Am. Chem. Soc. 2007, 129, 5281− 5287. (11) Wu, X.; Narsimhan, G. Characterization of Secondary and Tertiary Conformational Changes of Beta-Lactoglobulin Adsorbed on Silica Nanoparticle Surfaces. Langmuir 2008, 24, 4989−4998. (12) Dziri, L.; Desbat, B.; Leblanc, R. M. Polarization-Modulated FTIR Spectroscopy Studies of Acetylcholinesterase Secondary Structure at the Air-Water Interface. J. Am. Chem. Soc. 1999, 121, 9618−9625. (13) Wang, X. M.; Mock, M.; Ruysschaert, J. M.; Cabiaux, V. Secondary Structure of Anthrax Lethal Toxin Proteins and Their Interaction with Large Unilamellar Vesicles: A Fourier-Transform Infrared Spectroscopy Approach. Biochemistry 1996, 35, 14939− 14946. (14) Michael, K. E.; Vernekar, V. N.; Keselowsky, B. G.; Meredith, J. C.; Latour, R. A.; Garcia, A. J. Adsorption-Induced Conformational Changes in Fibronectin Due to Interactions with Well-Defined Surface Chemistries. Langmuir 2003, 19, 8033−8040. (15) Ferretti, S.; Paynter, S.; Russell, D. A.; Sapsford, K. E.; Richardson, D. J. Self-Assembled Monolayers: A Versatile Tool for the Formulation of Bio-Surfaces. TrAC, Trends Anal. Chem. 2000, 19, 530−540. (16) Kerth, A.; Erbe, A.; Dathe, M.; Blume, A. Infrared Reflection Absorption Spectroscopy of Amphipathic Model Peptides at the Air/ Water Interface. Biophys. J. 2004, 86, 3750−3758. (17) Kondo, A.; Murakami, F.; Higashitani, K. Circular-Dichroism Studies on Conformational-Changes in Protein Molecules Upon Adsorption on Ultrafine Polystyrene Particles. Biotechnol. Bioeng. 1992, 40, 889−894. (18) Das, R.; Jagannathan, R.; Sharan, C.; Kumar, U.; Poddar, P. Mechanistic Study of Surface Functionalization of Enzyme Lysozyme Synthesized Ag and Au Nanoparticles Using Surface Enhanced Raman Spectroscopy. J. Phys. Chem. C 2009, 113, 21493−21500. (19) Ye, S. J.; Nguyen, K. T.; Le Clair, S. V.; Chen, Z. In Situ Molecular Level Studies on Membrane Related Peptides and Proteins in Real Time Using Sum Frequency Generation Vibrational Spectroscopy. J. Struct. Biol. 2009, 168, 61−77. (20) Mendelsohn, R.; Mao, G.; Flach, C. R. Infrared Reflection− Absorption Spectroscopy: Principles and Applications to Lipid− Protein Interaction in Langmuir Films. Biochim. Biophys. Acta, Biomembr. 2010, 1798, 788−800. (21) Tengvall, P.; Lundström, I.; Liedberg, B. Protein Adsorption Studies on Model Organic Surfaces: An Ellipsometric and Infrared Spectroscopic Approach. Biomaterials 1998, 19, 407−422. (22) Surewicz, W. K.; Mantsch, H. H.; Chapman, D. Determination of Protein Secondary Structure by Fourier-Transform InfraredSpectroscopy - a Critical-Assessment. Biochemistry 1993, 32, 389−394. (23) Sethuraman, A.; Vedantham, G.; Imoto, T.; Przybycien, T.; Belfort, G. Protein Unfolding at Interfaces: Slow Dynamics of Alpha-

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b02448. Time-resolved IRRAS spectra of the 5+ and 13+ charge states of ubiquitin soft-landed onto HSAM and COOHSAM surfaces (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: 509-371-6136. Fax: 509-371-6139. E-mail: Julia. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the U.S. Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences & Biosciences. The work was performed in EMSL, a national scientific user facility sponsored by the DOE’s Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory (PNNL). PNNL is operated by Battelle for the DOE under Contract DE-AC05-76RL01830.

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Secondary Structures of Ubiquitin Ions Soft-Landed onto Self

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