Analyzing Structural Properties of Heterogeneous Cardiolipin-Bound

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Analyzing structural properties of heterogeneous cardiolipin-bound cytochrome c and their regulation by surface-enhanced infrared absorption spectroscopy Li Zeng, Lie Wu, Li Liu, and Xiue Jiang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03360 • Publication Date (Web): 01 Nov 2016 Downloaded from http://pubs.acs.org on November 2, 2016

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Analytical Chemistry

Analyzing structural properties of heterogeneous cardiolipinbound cytochrome c and their regulation by surfaceenhanced infrared absorption spectroscopy Li Zeng,a, b Lie Wu,a Li Liua and Xiue Jianga, * a

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, Jilin, China b University of Chinese Academy of Sciences, Beijing 100049, China ABSTRACT: Apoptotic mechanisms are not fully understood due to limitations in present analytical methods. Such understanding may be advanced by unravelling the structural properties of heterogeneous cardiolipin (CL)-bound cytochrome c (cyt c) and the factors or events that regulate them. In this study, surface-enhanced infrared absorption spectroscopy (SEIRAS) was employed to probe the adsorption of cyt c on CL membranes in biomimetic conditions. The results clearly show that pure electrostatic interactions result in the unfolding of partial α-helices, while the synergy between hydrogen bonding and electrostatic interactions governs orientation homogeneity of adsorbed protein, and conformational transition between α-helices and β-sheet. Hydrogen bonding plays a dual role; along with hydrophobic interactions, it may disturb the microenvironment of some secondary structures such as the βturn type III, while it also triggers structural changes in lipid molecules likely resulting from the extension of CL acyl chains to the hydrophobic channels of cyt c. These findings provide the details of protein transitions in early apoptosis at the molecular level.

Proteins execute their function with specific structure in organism. In different circumstance, proteins are conformationally dynamic, resulting in the transition in their functions.1 Therefore, unravelling detailed structural changes and the factors or events that regulate such transitions is of great importance in understanding biological activity.2-4 Cytochrome c (cyt c) is a small hemeprotein that transfers electrons between complex III and cyt c oxidase in the mitochondrion. The electron-transfer mechanism between cyt c and its partners has been extensively studied by performing direct electrochemistry of cyt c adsorbed on alkanethiolsmodified self-assembled monolayer (SAM).5-12 Recently, the focus on cyt c has intensified in understanding mechanism of early apoptosis due to its enhanced peroxidase activity resulting from the structural transition when associating with cardiolipin (CL).13-20 Unfortunately the mode of interaction between cyt c and CL and structural properties of CL-bound cyt c still remain unclear. It is suggested that cyt c adsorbs to CL-containing membranes through A site formed by Lys72, Lys73, Lys86 and Lys87 or C site close to Asn52, followed by the insertion of CL acyl chains into hydrophobic channels between the two non-polar polypeptide strands at positons 67– 71 and 82–85 or near Asn52.21-23 In contrast, some studies have reported that cyt c binds to CL-containing membranes by peripheral binding and/or deep insertion mechanism.17,24,25 Some advanced techniques, including magnetic circular dichroism (MCD) spectroscopy and time-resolved fluorescence resonance energy transfer (TR-FRET) enable the exploration of different heme configurations, and conformational diversity of the CL-bound cyt c ensemble.17,18 The structural properties of CL-bound cyt c, however, are only deduced by evaluating the structural configurations of cyt c in acidic19 and alkaline26 forms as well as conformer competent of cyt c15 regardless of the heterogeneity in the CL-bound cyt c ensemble. Besides, the motional restrictions of CL-bound cyt c

in mitochondria are often ignored as suggested by Borsari group.27 Thus, it is essential to develop a label-free method to study in detail, the structural properties of heterogeneous CLbound cyt c and the underlying reasons by probing in situ the interactions of cyt c with CL membranes in physiological conditions. Surface-enhanced infrared absorption spectroscopy (SEIRAS) is a surface sensitive technique that exploits the electromagnetic properties of nanostructured metal ﬁlms to enhance the vibrational bands of adsorbed monolayer by a factor of 10-10000.28-30 The rough metal film has been served as a working electrode to elucidate the surface adsorption and reaction in interfacial electrochemistry.31-34 The SEIRA difference spectroscopy can identify minor structural changes in biomolecules under similar physiological stimulations to reveal the underlying molecular mechanism that regulate these changes.7,35-38 More importantly, an additional advantage of SEIRAS is that it does not require reporter groups. In this study, we demonstrate the successful use of SEIRA technique to monitor in situ the formation of CL membrane of the solid support, the subsequent adsorption of cyt c and the conformational changes of protein under different physiological conditions at the motional restriction form. The results reveal the event that triggers heterogeneity in CLbound cyt c and the details of the structural transformation. EXPERIMENTAL SECTION Au film preparation. The flat surface of a triangular Si prism was polished with 1.0 µm Al2O3 slurry and then washed thoroughly with pure water. The cleaned Si substrate was treated by immersion in a 40 wt % aqueous solution of NH4F for 1 min. A thin gold film was prepared on the treated triangular Si prism by chemical deposition. The flat surface of the treated Si substrate was exposed to a 1:1:1 volume mixture of

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(1) 0.03 M NaAuCl4, (2) 0.3 M Na2SO3 + 0.1 M Na2S2O3 + 0.1 M NH4Cl, and (3) 2.5 vol % HF solution for 90 s. After being rinsed with water, the gold film surface was electrochemically cleaned in 0.1 M H2SO4 solution by several oxidation-reduction cycles in the range between 0.1 and 1.4 V (vs. Ag/AgCl). Then the gold-coated prism was mounted on a poly(trifluorochloroethylene) cell with a Viton O-ring. A Cu plate served as the electric contact to the gold film. The IR beam from the interferometer of the FT-IR spectrometer (IFS 66 V/S, Bruker, Ettlingen, Germany) was coupled into the silicon prism at an incident angle of 60°C, and the reflected beam intensity was recorded with a liquid nitrogen-cooled mercury cadmiumtelluride (MCT) detector. Preparation of CL vesicle. Several microliters of CL (TLC, ≥97% from bovine heart, Sigma-Aldrich) in ethanol solution were injected into a glass vial and the solvent was removed via a N2 stream, followed by treatment in a vacuum chamber for at least 4 h. Then the lipid film was hydrated in 10 mM phosphate buffer solution (PBS, pH 7.0) at 59 °C and the suspension was sonicated at room temperature until a clear solution was obtained.39 The final concentration of CL was 1 mg/mL. The vesicle solution was stored at 4°C and always used within 24 h after preparation. Kinetic SEIRAS to monitor the adsorption of cyt c on CL hybrid lipid bilayer membranes and MUA monolayers. To monitor the formation of 1-dodecanethiol (DT, ≥98%, Sigma) monolayer on the freshly prepared gold film, a reference spectrum of blank ethanol was firstly recorded. Then a series of sample spectra were acquired at intervals of 10, 60, and 300 s concomitant with the addition of 20 µM DT into the ethanol. After thorough rinsing with ethanol and water, 500 µL of 10 mM PBS (pH 7.0) was added and scanned as the reference spectrum. The sample spectra were recorded after addition of 500 µL of CL vesicle solution. Adsorption of cyt c was monitored after adding 2 µM cyt c (ferric cyt c, 95% from horse heart, Sigma) without or with different additives and 10 mM PBS-incubated CL/DT/Au electrode without or with additives as references. Alternatively, a cleaned Au film was immersed in 0.1 mM MUA (Sigma) for 90 min to obtain MUAmodified Au electrode (MUA/Au). To immobilize the protein, 2 µM cyt c in PBS solution was directly added to the MUA/Au electrode for 90 min. Sequential adsorption was monitored with SEIRAS in the same way. For each spectrum, 512 scans were collected with a spectrum resolution of 4 cm-1. Potential-induced SEIRA difference spectra under steady-state conditions. A CHI 830C electrochemical workstation (CH Instruments, Austin, TX) was used for electrochemical measurements with the Cu plate connected to the gold film as working electrode, a Pt wire and an Ag/AgCl electrode with saturated KCl solution as counter and reference electrodes, respectively. To obtain the potential-induced difference spectra, a reference SEIRA spectrum was taken at a reduction potential and sample spectra were acquired at a series of sequential oxidation potentials, after which the adsorbed cyt c is gradually oxidized. For each spectrum, 512 scans were collected with a resolution of 4 cm−1. The whole procedure was repeated nine times, and the difference spectra were averaged to improve the signal-to-noise ratio. RESULTS

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SEIRAS Monitoring of CL Membrane Formation and the Subsequent Adsorption of Cyt c. CL membranes were constructed by vesicle fusion onto a gold electrode premodified with hydrophobic molecules (Figure S1). The process was monitored in situ by SEIRAS (Figure 1A, a) using PBS-incubated DT-modified gold electrode as the reference (Figure S2A). Upon vesicle fusion, positive peaks around 3000 cm−1 were assigned to the ν (CHn) in CL acyl chains. The slightly high frequencies of νas (CH2) and νs (CH2) at 2930 cm1 and 2857 cm-1 respectively suggests a gauche conformation of acyl chains,40 likely due to the loose packing of acyl chains induced by the steric hindrance of four acyl chains per CL. The bands around 1800-1700 cm-1 and 1095 cm-1 in the fingerprint region were assigned to the head groups of ester carbonyl and phosphate in CL molecules, respectively. The negative band at 1650 cm-1 was associated with the OH bending vibration of water due to the removal of water molecules during the hydrophobic interactions between DT and CL acyl chains.37 All characteristics of the band suggest the successful construction of the CL membrane, which was further confirmed by cyclic voltammetry, electrochemical impedance spectroscopy and atomic force microscopy (Figure S2 B, C). After the formation of CL membranes, sample spectra were recorded following the addition of cyt c to PBS at the concentration of 2 µM (Figure 1A, b). The CL membranes spectrum immersed in 10 mM PBS was used as the reference (Figure 1B). In this manner, the sample spectra include contributions from protein adsorption and CL membrane fluctuations. The adsorbed cyt c exhibits two positive absorption bands at 1660 cm−1 and 1550 cm−1 (Figure 1A, b), which are derived from the C=O stretching vibration (namely, amide I) and the coupled mode of in plane N-H bend and C-N stretch (namely, amide II), respectively.41 In the C-H vibrational region however, some weak negative peaks can be resolved at 3010, 2960, 2917 and 2852 cm-1 overlapped with the respective positive bands. In SEIRA spectrum, the surface bound species is expected to produce positive absorptions after normalizing against the reference CL membranes in PBS. Therefore, we suggest that these negative peaks are derived from structural changes in the reference (i.e. the CL membrane) but not from the adsorbed protein. At the same time, we also observed a weak negative band at 1095 cm−1 in νs (PO2–) region accompanied by a very small positive peak at 1088 cm−1 that may result from the hydrogen-bonded νs (PO2–).42 This suggests the formation of a hydrogen-bond network around the PO2– group of CL after protein adsorption. To gather additional evidence for this finding, we exposed the membranes to protein in 10 mM PBS at pH 5.0 to enhance hydrogen bonding by increasing the degree of CL protonation since hydrogen bonding occurs between cyt c and protonated acidic phospholipids.43 As expected, more efficient absorptions were observed at 1660 cm-1 and 1550 cm-1 (Figure 1A, c), suggesting that cyt c adsorption was more effective due to enhanced hydrogen bonding. More importantly, the couplet at 1095 cm−1 and 1088 cm−1 was greatly enhanced in the νs (PO2–) region in acid condition. To exclude the possibility that local changes in phosphate concentration induce such spectral changes, we exposed the CL membranes in the presence of 25 mM PBS at pH 7.0 and 5.0 respectively. In this cases, 10 mM


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Figure 1. (A) The SEIRA spectra of CL vesicle fused on the DT-modified Au electrode (a), and subsequent adsorption of cyt c in the presence of (b) 10 mM PBS at pH 7.0, (c) 10 mM PBS at pH 5.0, (d) 150 mM NaCl solution at pH 5.0 and (e) 150 mM PBS at pH 7.0 at 10 s, 150 s, 13.5 min and 90.5 min (from bottom to top), respectively. Colored lines are the corresponding spectra of pure CL membrane in different additives for 90 min. (B) The adsorption spectra of cyt c were recorded by normalizing against the reference spectrum of CL membranes immersed in PBS. (C) Illustration to elucidate the possibility of insertion of a CL acyl chain into the hydrophobic pocket surrounding Asn52. Modified from Sinibaldi et al., J Biol Inorg Chem 2010.

PBS with the corresponding pH values were used as reference (Figure S3). Increasing phosphate concentration did not reduce but enhanced the band intensity at 1095 cm-1, and positive sign was not detected at 1088 cm-1. Therefore, we suggest that the couplet indicates the presence of hydrogen bonding between CL and cyt c. Besides, more obvious negative peaks in the ν (CHn) region were resolved, suggesting that enhanced hydrogen bonding facilitates structural changes of in underlying lipid membranes. Surprisingly, we observed the similar phenomena in the two regions by exposing the CL membranes to cyt c in PBS pH 5.0 at high ionic strength (Figure 1A, d). The screened electrostatic interaction largely weakened the adsorption of cyt c, but still resulted in negative peaks in the ν (CHn) region, suggesting definitive contribution of hydrogen bonding to the phenomenon. To further validate this, we screened the hydrogen bonding between the phosphate group of CL and protein by exposing the membranes to cyt c in 150 mM PBS (pH 7.0). As shown in Figure 1A, e, the negative peaks and couplet were not visible in the ν (CHn) and νs (PO2–) regions. This clearly suggests that the hydrogen bonding between the CL phosphate groups and protein is responsible for the structural changes in the underlying lipid membranes. Indeed, previous reports have also shown that the hydrogen bonding near Asn52 may lead to the extension of CL acyl chains to the hydrophobic channels in cyt c (Figure 1C).21,43 As a result, part of CL acyl chains will be far away from the metal surface (Figure 1C), resulting in negative peaks in the C-H vibrational region based on the optical near-field effect of SEIRAS.7 Therefore, the extension

of acyl chain into the hydrophobic channels of cyt c are more likely to occur in our system although the change in lipid orientation may also result in negative peaks and, once this happens, hydrogen bonding may play an important role in the process. The Kinetics of Cyt c on CL Membrane under Different Binding Forces. To further identify the binding mode, we investigated the adsorption kinetics of cyt c by observing timedependent integrated area of amide I band (Figure 2). We found that the kinetic data c fitted well to biexponential equation when cyt c was mainly adsorbed by electrostatic interaction (red, neutral pH with low ionic strength), hydrogen bonding (green, acidic pH with high ionic strength), or hydrophobic interaction (pink, neutral pH with high ionic strength), illustrating the heterogeneous nature of the CLbound cyt c ensembles.44 In contrast, the data from cyt c adsorbed on CL memranes in acidic pH and low ionic strength can be fitted well with the monoexponential expression (blue), suggesting the homogeneous nature of CL-bound cyt c. To gather supporting evidence, we also desorbed the bound cyt c with a high ionic strength solution after protein adsorption on CL membranes in 10 mM PBS at pH 5.0 and 7.0, respectively (Figure S4). We found that half of the adsorbed cyt c at pH 7.0 was desorbed, suggesting that at least two binding modes exist at neutral pH, one of which may be a pure electrostatic interaction. In contrast, at acidic pH the CL-bound cyt c was insensitive to ionic strength, thus excluding the existence of cyt c adsorbed by pure electrostatic interaction. It should be noted that electrostatic interaction cannot be completely



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screened at pH 5.0 and low ionic strength since CL is only partially protonated under these conditions (one pK value of CL was 2.845). Both electrostatic interaction and hydrogen bonding simultaneously result in the adsorption of cyt c on CL membranes. Thus, we deduce that synergistic interactions of hydrogen bonding with electrostatic interaction may promote the homogeneous orientation of CL-bound cyt c.

Figure 2. Adsorption kinetics presented by the time-dependent surface coverage of cyt c on CL membrane in the presence of 10 mM PBS at pH 7.0 (red), 10 mM PBS at pH 5.0 (blue), 150 mM NaCl solution at pH 5.0 (green) and 150 mM PBS at pH 7.0 (pink), respectively.

Potential-Induced SEIRA Difference Spectroscopy Reveals the Structural Properties of Cyt c Adsorbed on the CL membranes under Different Binding Forces. To identify the structural properties of cyt c, we recorded the potentialinduced SEIRA difference spectra. The spectral features of CL-bound cyt c in 10 mM PBS (pH 7.0) were easily distinguishable from those of CL membranes (Figure S5). However, it was significantly different from that of native cyt c adsorbed on MUA SAM (Figure 3, black) upon reduction (−) and oxidation (+).7 Compared to the native cyt c, the band for α-helices46 at 1661 (+) cm−1 is red-shifted to 1656 (+) cm−1, and the bands at 1635 (+)/1626 (−) cm-1 for β-sheet7 are simultaneously intensified (red). We presume that the broader of the two bands is likely from the heterogeneous CL-bound cyt c species. As the adsorption mode was converted to homogeneity at pH 5.0 in 10 mM PBS due to the synergy of hydrogen bonding and electrostatic interaction, the band at 1661 (+) cm−1 was further red-shifted to 1652 (+) cm−1 and the bands at 1635 (+)/1628 (−) cm-1 were greatly intensified (blue), indicating the structural conversions between α-helices and β-sheet. However, the screening of electrostatic interaction (green) with high ionic strength (150 mM NaCl) reduced the amplitude of red-shift and the intensity of these bands, even in the presence of hydrogen bonding at pH 5.0. Consequently, the synergy of electrostatic interaction and hydrogen bonding could make definite contributions to the above structural conversions. Besides, the bands for β-turn type III comprising residues 67-71 and 14-19 are blue-shifted to 1676 (+) cm-1 accompanied with a red shift of the band at 1666 (-) cm-1 to 1664 (-) cm-1 (blue and green), suggesting that the existence of strong hydrogen bonding from the CL phosphate groups to protein has perturbed the intraprotein hydrogen-bond network

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of the secondary structures, leading to enhanced flexibility of some loops.

Figure 3. The potential-induced difference spectra of cyt c on MUA in 10 mM PBS at pH 7.0 (black ), and on CL membrane in the presence of 10 mM PBS at pH 7.0 (red ), 10 mM PBS at pH 5.0 (blue), 150 mM NaCl solution at pH 5.0 (green) and 150 mM PBS at pH 7.0 (pink), respectively. After washing with 150 mM NaCl, the difference spectrum of cyt c adsorbed on CL membrane at pH 7.0 was recorded in 10 mM PBS as brown line. The purple line is the subtraction spectrum of the red line from the brown line, which represents the difference spectrum of CL-bound cyt c by pure electrostatic interaction.

In 150 mM PBS at pH 7.0 (pink), screening of the electrostatic interaction and hydrogen bonding eliminated the structural conversion between α-helices and β-sheet, since hydrophobic-bound cyt c exhibits vibrations similar to native cyt c at 1666 (−)/1659 (+) cm−1. However, the bands at 1693 (−)/1675 (+) cm−1 and 1635 (+)/1628 (−) cm-1 were red-shifted to 1691 (−)/1672 (+) cm−1 and 1633 (+)/1626 (−) cm-1, respectively, suggesting a larger disturbance to the structure at the two frequencies, including residues 67-71, 37-40 and 5759. Based on the primary structure of cyt c, we can thus deduce that the hydrophobic site may be the likely hydrophobic channel between the two non-polar polypeptide strands, 67–71 and 82–85, and/or near Asn52 (Figure S6).47 Although the deduced hydrophobic interaction sites are consistent with the hydrophobic channels at the insertion of one of the CL acyl chains,21 we did not observe structural change in the underlying lipid in the absence of hydrogen bonding (Figure 1A, e), further confirming the importance of hydrogen bonding in mediating the possible extension of CL acyl chains into cyt c. Interestingly, the spectrum after the desorption of CL-bound cyt c in 10 mM PBS by pure electrostatic interaction had bands characteristic of CL-bond cyt c via hydrogen bonding and hydrophobic interaction at 1676 (+) cm1 and 1672 (+)/1659 (+) cm-1, respectively (brown). In contrast, the spectrum for adsorbed cyt c via pure electrostatic interaction, obtained by subtracting brown from red as shown in purple displayed a red-shift for the band at 1661 (+) cm-1 and 1628 (−) cm-1 to 1652 (+) cm−1 and 1626 (−) cm-1, respectively, suggesting that the electric interaction can lead to a structural


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transformation from α-helices to loop/random coil48,49 thus distrubing the microenvironment of β-sheet. These results reveal the structural properties and the heterogeneous nature of CL-bound cyt c at neutral pH. DISCUSSION During early apoptosis, CL migrates from the inner leaflet to the outer leaflet of the inner mitochondrial membrane to facilitate the tight binding between CL and cyt c. It is generally accepted that the association leads to the disruption of iron-Met80 bond, protein unfolding, and thus enhancing peroxidase activity.50 The interaction mechanism, however, is ambiguous. While insertion of acyl chains into the two hydrophobic channels has been reported to unfold the protein and expose the heme,19,21,26 peripheral binding of cyt c on the membrane results in a structural equilibrium between extended and compact ensemble.17,44 The discrepancy is likely derived from the different experinment conditions including the ionic strength, pH and lipid/protein ratios.50 However, there is no study trying to unravel the factors or events that regulate them. Moreover, another factor, the motional restriction of CL-

bound cyt c in mitochondria,27 is often ignored under the simulated environments, which could lead to different changes in the heme configuration from that in solution.18 Likewise, the motional restriction could trigger a difference in the secondary structure changes of CL-bound cyt c as well, but there is little work to elucidate the structure transitions in immobilization state due to the limitation of present techniques. SEIRAS has been widely used to investigate the structural informations of immobilized protein on biomimetic membranes.7,35,41,51,52 In our work, cyt c was adsorbed onto the supported CL membranes to reproduce the CL-bound cyt c in mitochondria. We clearly show that the competition and/or the synergy of electrostatic, hydrogen bonding and hydrophobic interactions result in the adsorption of cyt c to CL membranes in heterogeneous and homogeneous modes. At neutral pH, the positive end of the dipole moment vector in the exposed heme edge still acts as the main driving force for the adsorption of cyt c since half of the cyt c was adsorbed to CL membranes by pure electrostatic interaction. Previous reports have designated Site A as the electrostatic interaction center, and it was presumed to include residues Lys72, Lys73, Lys86 and Lys 87

Figure 4. Cartoons to elucidate the interaction of cyt c with CL membrane. (A) Structure of cyt c and the possible binding sites with CL. (B) The pure electrostatic interaction mode between the positively charged protein and negatively charged membranes by the general proposed residues of Lys72, -73, -86 and -87 in cyt c. The strong Coulomb interaction may break the Met80-iron bond and the hydrogen bonding between the unstable loop ( residues 71-85 ) and the heme propionate-6 (HP-6), which further disturb the hydrogen bonding between the heme propionate-7 (HP-7) and β-sheet. The broken segments represent the disturbed structure and the black arrows indicate the possible route of the structural changes induced by the interaction. (C) The hydrophobic interaction mode near the residues 67-71 by partial insertion of the nearby hydrophobic segment, probably residues 81-85 into the CL membranes. The β-sheet may be disturbed by the hydrophobic interaction near residues 67-71 through the hydrogen-bond network as the electrostatic interaction does. (D) The hydrogen bonding and electrostatic interaction near Asn 52 may lead to the structural transition from α-helix to β-sheet structural. (E) The insertion of acyl chain into the hydrophobic channel near Asn 52 (Modified from Pletneva et al., J. Am. Chem. Soc. 2015). The cyt c structure was created by Swiss PDB Viewer 3.7 with crystallographic data taken from the Protein Data Bank (PDB entry 1HRC).



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(Figure 4A and B).17 The strong electrostatic interactions between the Site A and CL membranes induce the structural transformation from α-helices to loop/random coil. Pletneva et al. identified the importance of electrostatic interactions in mediating strong binding and protein unfolding of the polypeptide fragment between residues 66 and 92, a lower stability Met80-containing loop.17 Such interaction may further impose changes in the microenvironment of β-sheet via intraprotein hydrogen bonding network (Figure 4B). In addition to electrostatic interactions, hydrophobic interactions mediate another binding mode at neutral pH. This is evident from the CL-bound cyt c spectrum after desorption, which shows bands with characteristics similar to the CL-bound cyt c via hydrophobic interactions that disturb the microenvironment of β-turn type III and β-sheet. Note that the hydrophobic and the electrostatic interactions cause the same disturbance to the β-sheet microenvironment since the peak at 1628 (−) cm-1 was red-shifted to 1626 (−) cm-1 at the both cases, possibly resulting from the similar way. Therefore, it is most likely that the hydrophobic interactions only involve the hydrophobic channel between the two non-polar polypeptide strands 67–71 and 82–85 (Figure 4C), which then may impose a change in the β-sheet microenvironment via intraprotein hydrogen bonding network. Although the disturbance to the microenvironment of β-turn type III and β-sheet, CL-bound cyt c is expected to be compact due to the almost fixed frequency at 1660 cm-1. The third interaction mode should result from the synergy of electrostatic interactions with hydrogen bonding as that in acidic condition. In acidic condition, the presence of electrostatic interactions may lead to significant structural changes between CL-bound cyt c with and without electrostatic interactions. This may result in distinct absorption at the C-H vibrational region. However, nearly consistent characteristic peaks further suggest that the negative peaks in the positive absorption protein spectra may arise from similar structural changes in the underlying CL, likely including the extension of CL acyl chains.21 The enhanced hydrogen bonding between protonated CL and cyt c, likely from Asn5243 could modify the hydrogen bonding network that connects two loops consisting of residues 40–57 and 71–85 as well as Tyr67.53 In addition, adjustments of heme pocket may allow for possible extension of CL acyl chains to the hydrophobic channel near Asn52 (Figure 4D). In the absence of electrostatic interactions, the enhanced hydrogen bonding only causes disturbance to the β-turn type III microenvironment (residues 67-71), but maintains the compact state of CL-bound cyt c. In the presence of electrostatic interactions, the acidic conditions may activate additional electrostatic binding sites on cyt c including His26.54 The strong interaction of His26 with CL membranes could disrupt its hydrogen-bond network, triggering the increase of β-sheet (Figure 4E). Spiro et al. found that disruption of the His26/Pro44 H-bond marginally destabilizes the 40s Ω loop, triggering the extension of β-sheet into three β-turns, the short 50s helix, and back into the 60s and 70s helices after heating cyt c at pH 3.19 Although our study revealed the same possibility for CL-bound cyt c, we clearly identified that the synergy between the electrostatic interactions and hydrogen bonding in protein and CL is the driving force for the structural changes. More importantly, such synergy results in the homogeneity of protein adsorption orientation. Fluorescence correlation experiments have

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suggested conformational exchange between compact and extended structures,17 which could also form by modulating the synergy between electrostatic interaction and hydrogen bonding. Electrostatic interactions induce unfolding of partial α-helices, while the synergy between hydrogen bonding and electrostatic interaction is responsible for the conformational transition between α-helices and β-sheet. Hydrogen bonding and hydrophobic interactions disturb the microenvironment of only some secondary structures, while maintaining the compact state of adsorbed cyt c. Our study further suggests that the nature of lipid anchorage or protein penetration may have originated from the difference in the hydrogen bonding effect. CONCLUSION In conclusion, SEIRA has been proven to be an ultrasensitive technique to determine the nature of interaction between cyt c and CL membranes. Adsorption of cyt c on CL membranes is a complex process that results from the balance of three forces: electrostatic attraction, hydrogen bonding, and hydrophobic interaction. These forces determine at least three adsorption modes including site A, site C with possible lipid extension, and hydrophobic interactions via the hydrophobic channel between the two non-polar polypeptide strands, 67–71 and 82–85. In this study, we have identified for the first time that the orientation and structural interconversion between αhelices and β-sheet for CL-bound cyt c are largely determined by the synergic interactions between hydrogen bonding and electrostatic attraction, while electrostatic interactions alone induce unfolding of partial α-helices. Although hydrogen bonding and hydrophobic interactions disturb the microenvironment of some secondary structures including βturn type III and β-sheet, CL-bound cyt c is still in an compact state. These findings provide insights to explore the use of SEIRAS technique to determine the complex interactions between proteins and lipids in physiological environment.

ASSOCIATED CONTENT Supporting Information Supporting Information Available: Figures of DT absorption spectra, AFM and EIS of the membrane, PBS concentration change on CL membrane, desorption spectra, series of difference spectra and hydrophobic channels in cyt c. (PDF). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was financially supported by the National Science Foundation for Excellent Young Scholar of China (21322510), the National Natural Science Foundation of China (21675149), Frontier Science Key Research Project of Chinese Academy of Sciences (QYZDY-SSW-SLH019), and the Science and Tech-nology Innovation Foundation of Jilin Province for Talents Cultivation (Grants 20150519014JH).


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Analyzing Structural Properties of Heterogeneous Cardiolipin-Bound

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