Î±-Helix Facilitates Proteins To Form Langmuir Monolayer - American

Downloaded by CORNELL UNIV on September 23, 2016 | http://pubs.acs.org Publication Date (Web): December 8, 2015 | doi: 10.1021/bk-2015-1215.ch005

Chapter 5

α-Helix Facilitates Proteins To Form Langmuir Monolayer: Surface Properties and Orientation Studies of Aequorin and α-Synuclein at the Air−Water Interface Joseph Dale Combs and Chengshan Wang* Department of Chemistry, Middle Tennessee State University, 1301 E. Main Street P. O. Box 68, Murfreesboro, Tennessee 37132, U.S.A. *E-mail: [email protected].

Wide scientific attention has been focused on the interaction of proteins with amphiphilic membrane structures (e.g., cell membranes, vesicles, and micelles) because the complex of proteins with amphiphilic membranes participates in various metabolic processes in vivo. However, the factors which help proteins to complex with amphiphilic membranes are not clearly understood. Due to the amphiphilic nature, the air−water interface has been widely used as a model system to mimic amphiphilic membranes and Langmuir monolayer technique provides an accurate methodology to determine the biophysical properties (e.g., stability) at the interface. In addition, surface spectroscopic techniques such as Infrared Reflection-Absorption Spectroscopy (IRRAS) have been shown the power in the determination of the orientation of amphiphilic molecules at the air−water interface. This paper reviews the surface properties of cysteine-free recombinant aequorin as well as α-synuclein (α-syn). Aequorin was used as a model protein because it contains all of the typical conformations (such as α-helix, β-sheet, and unstructured conformation). α-Helix in aequorin was found to help aequorin form a stable Langmuir monolayer whereas unstructured conformation is destructive to the monolayer. α-Syn, which was shown to involve in the pathology of Parkinson’s disease, has been supposed to © 2015 American Chemical Society Wang and Leblanc; Recent Progress in Colloid and Surface Chemistry with Biological Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


be unstructured. However, α-syn was shown to form a very stable Langmuir monolayer. IRRAS results showed that α-syn transforms to α-helix when spread at the air−water interface, and the high stability of α-syn was due to the transformation. Although α-syn is abundant in vivo, the concentration of α-syn in plasma is very low. On the contrary, the accumulation of α-syn in presynaptic terminals has been reported and the reason for this accumulation is not clear. Thus, the high concentration of amphiphilic membranes (i.e., vesicles) in the presynaptic terminals may transform α-syn to α-helix. The stability of the Langmuir monolayer may explain the accumulation of α-syn in presynaptic terminals in vivo. IRRAS results also showed that the α-helix in the Langmuir monolayer of either aequorin or α-syn is parallel to the air−water interface.

Introduction Proteins/peptides consist of chains of amino acids covalently linked by amide bonds (i.e., CO-NH), formed between the amine group of one amino acid and the carboxyl group of the adjacent amino acid (1). The two termini of a protein/peptide chain are called the N-terminus (which has free amine group) and the C-terminus, which has free carboxylic acid group. The sequence of a protein/peptide is usually written from N-terminus to C-terminus and the primary structure of a protein refers to its sequence. The secondary structure of protein is the specific geometric shape caused by the intra- and intermolecular hydrogen bonding of amide groups (1, 2). Typical secondary structures (also called as conformations) are α-helix, β-sheets, and unstructured conformation (2, 3). Unstructured conformation is also called random coil, in which the chain of the protein/peptide is well dissolved in the aqueous solution and moves freely in the aqueous environment (2). As the most common secondary structure, α-helix looks like a spring as shown in Figure 1 (3). α-Helix is characterized by the intramolecular hydrogen bonds between the amino group of one amino acid and the carbonyl group of another amino acid located 3-4 residues away along the peptide/protein chain. These hydrogen bonds and carbonyl groups (C=O) in the backbone of the peptide/protein are usually parallel to the helical axis (3).

Figure 1. Illustration of α-helix. 90 Wang and Leblanc; Recent Progress in Colloid and Surface Chemistry with Biological Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


The structural units of β sheets are strands, which are fully extended structures characterized by multiple strands arranged side-by-side as shown in Figure 2 (4). The residue groups of each amino acid in the strand are above and below the plane of the chain. Interstrand hydrogen bonds are formed between the carbonyl oxygen of one chain and the amide hydrogen of the adjacent chain. β-Sheet includes two types, namely, parallel and anti-parallel sheets. In a parallel sheet, two strands run in the same direction with regards to their C- and N-terminus as shown in Figure 2a. In an anti-parallel β-sheet, the neighboring strands run in opposite directions (cf. Figure 2b). The directions of backbone carbonyl groups (C=O) are perpendicular to the direction of the strands (4).

Figure 2. Parallel (a) and antiparallel (b) β-sheet.

Amphiphilic membranes (e.g., cell membranes, micelles, and vesicles) widely exist in biological systems (5–8). The interaction of proteins with amphiphilic membranes has attracted extensively scientific interest because the complex of proteins with amphiphilic membranes is involved in many fundamental metabolic processes such as oxidative phosphorylation (9–11), cell signaling (12, 13), endocytosis (5, 14, 15), autophagy (16, 17), and so on (18, 19). However, several basic questions about the interaction of proteins with amphiphilic membranes have not been answered lucidly. For example, which conformation (or secondary structure) helps proteins to interact with amphiphilic membranes? What is the orientation of proteins around amphiphilic membranes? To address the questions above, a simple model system is necessary. The air−water interface has been accepted as a model to mimic the interface of amphiphilic membranes, because similar properties have been reported between the air−water interface and the amphipihlic membranes (6, 7, 20). Furthermore, the Langmuir monolayer technique provides an accurate methodology to determine the chemical and physical properties (e.g., surface pressure and stability) of amphiphilic biomacromolecules at the interface (21–24). In addition, surface spectroscopic techniques such as Infrared Reflection-Absorption Spectroscopy (IRRAS) combined with Langmuir monolayer technique can determine the orientation of molecules at the interface (21–23). 91 Wang and Leblanc; Recent Progress in Colloid and Surface Chemistry with Biological Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


IRRAS spectroscopy is designed specifically to obtain the FTIR spectra of Langmuir monolayers by conducting the IR beam from an IR spectrometer to the air−water interface and then collecting the reflected IR beam. The IR beam can be polarized and the incident angle of IR beam to the air−water interface is variable. The s-polarized or p-polarized IRRAS spectra for a given monolayer may differ due to distinct selectivity rules. The electronic field of the s-polarized IR beam is perpendicular to the incident plane but parallel to the air−water interface. Thus, spolarized IRRAS can only detect the vibrations parallel to the air−water interface. The peaks in s-polarized IRRAS are always negative, with decreasing intensity when the incident angle increases. For the p-polarized IR beam, the electronic field is parallel to the incident plane and perpendicular to the forward direction of the incident beam. Therefore, the p-polarized IRRAS can provide information about the orientation of vibrations of various chemical bonds in the Langmuir monolayer. For vibrations parallel to the air−water interface, the peaks are initially negative and increase in intensity with an increase of the incident angle until the Brewster angle (e.g., 54.2º for IR light at 2850 cm-1) is reached. Above the Brewster angle, there is an inversion of the peaks to positive values and the intensity decreases with further increase of the incident angle. Regarding a vibration perpendicular to the interface, the opposite should be observed for both the direction and intensity of the peak when the angle of the incident light is varied (21–23). Several vibrations (e.g., stretching mode of carbonyls or C=O) in proteins/ peptides are IR active and generate various bands in the FTIR spectra of proteins/ peptides (25). For example, the amide I band between 1600~1700 cm-1 in the FTIR spectra of proteins/peptides mainly stems from the stretching mode of carbonyls (C=O) in the backbone amide bond in the chain of proteins/peptides. The amide II band between 1500~1600 cm-1 is mainly from the bending mode of N-H in the backbone amide bond. It is worth noting that the peak position of the amide I band is related to the conformation (i.e., secondary structure) of the protein/peptide. The characteristic position of the amide I band of α-helix is at 1650 cm-1 for globular proteins. The position of the amide I band of unstructured and β-sheet conformation is at 1640 and 1630 cm-1, respectively (25). To address which conformation helps the formation of Langmuir monolayer of proteins/peptides, the cysteine-free recombinant aequorin was used as a model protein at the air−water interface because it contains all of the typical secondary structures mentioned above (26). α-Helix was found to help aequorin to form a stable Langmuir monolayer at the air−water interface. Moreover, IRRAS was used to study the orientation of α-helix and the backbone carbonyls were parallel to the air−water interface. As shown in Figure 1, the stretching of backbone carbonyls (C=O) is parallel to the axis of α-helix. Thus, the α-helix in the aequorin Langmuir monolayer was also parallel to the interface. On the other hand, unstructured conformation was shown to be destructive to the Langmuir monolayer and make aequorin dissolve into the subphase (i.e., the bulk water under the air−water interface). This conclusion can help to understand why some proteins accumulate around or embed in the amphiphilic membranes in vivo (24). For example, α-synuclein (α-syn), which is the major protein component in the abnormal aggregation in the brain of the patients of Parkinson’s disease, was shown to be unstructured in aqueous solution (27). α-Syn is abundant in 92 Wang and Leblanc; Recent Progress in Colloid and Surface Chemistry with Biological Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


human brain whereas the concentration of α-syn in plasma is low (28, 29). On the other hand, α-syn was reported to accumulate in the presynaptic terminals of neurons (30). The reason for the accumulation is not clear. α-Syn was spread at the air−water interface and it was found that α-syn formed a very stable Langmuir monolayer (23). As discussed above, proteins in unstructured conformation will not form a Langmuir monolayer. IRRAS was used to examine the Langmuir monolayer of α-syn and the conformation of α-syn was found to be α-helix at the air−water interface. Thus, α-syn transforms from unstructured conformation in aqueous solution to α-helix when spread at the air−water interface (23). The α-helical conformation at the amphiphilic interface keeps α-syn to stay stable at the interface. The high stability of the Langmuir monolayer of α-syn may help to explain the accumulation of α-syn in presynaptic terminals. In addition, p-polarized IRRAS also found that the α-helix of α-syn is also parallel to the air−water interface and the results are below (23).

Results Study of the Surface Properties and Orientation of Aequorin at the Air−Water Interface Study of the Surface Pressure-Area Isotherms and Stability of the Aequorin Langmuir Monolayer As mentioned above, some proteins can form Langmuir monolayer at air−water interface whereas other proteins cannot. The study of the surface properties of a protein/peptide will not only provide the biophysical behavior of the protein/peptide in vivo, but also assure that monolayer devices such as sensors can be fabricated from the protein/peptide. Thus, it will be interesting to investigate which conformation in a certain protein can affect its surface properties (i.e., stabilize the Langmuir monolayer of the protein/peptide). The bioluminescent photoprotein aequorin, which was originally isolated from the jellyfish Aequorea Victoria and consists of apoaequorin, coelenterazine and molecular oxygen, has been widely used as Ca2+ sensors. Because containing all of the three conformations mentioned above (26), aequorin was used here as a model protein to clarify which conformation can stabilize the Langmuir monolayer. The surface pressure-area (π-A) and surface potential-area (ΔV-A) isotherms of aequorin on Tris/HCl buffer at pH 7.6 are shown in Figure 3 (24). Tris/HCl buffer (pH 7.6) was chosen because this medium is usually used to observe the bioluminescence of aequorin in aqueous solution. The lift-off point of the π-A isotherm was detected at 5470 Å2·molecule-1, followed by a steady increase of the surface pressure up to a kink point at 2940 Å2·molecule-1. Further decreasing the surface area further caused a quick increase of the surface pressure and the Langmuir monolayer collapsed at 2100 Å2·molecule-1. The limiting molecular area was obtained at 3920 Å2·molecule-1 by extrapolating the higher surface pressures of the isotherm to nil surface pressure. 93 Wang and Leblanc; Recent Progress in Colloid and Surface Chemistry with Biological Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


The surface potential of the aequorin Langmuir monolayer increased from 12500 Å2·molecule-1 up to a kink point observed at 6100 Å2·molecule-1, a value bigger than the lift-off point of the π-A isotherm at 5470 Å2·molecule-1. This is normal because molecules “touch” each other at the lift-off point in the π-A isotherm, but “see” each other at the lift-off point in the ΔV-A isotherm. Surface potential increased sharply to 190 mV after the kink point in the ΔV-A isotherm at a molecular area of 2550 Å2·molecule-1. Further compression of the Langmuir monolayer did not increase the surface potential value significantly. Extrapolating the slopes before and after the kink point at 6100 Å2·molecule-1 shows an intercrossing point at 5510 Å2·molecule-1 which was very close to the lift-off point in the π-A isotherm (i.e. 5470 Å2·molecule-1). This indicated that the π-A and ΔV-A isotherms of aequorin in the Langmuir monolayer correlate well. This correlation is similar to that of the typical small amphiphilic molecules such as peptidolipids at the air−water interface (31).

Figure 3. Surface pressure and surface potential-area isotherms of the aequorin Langmuir monolayer on Tris/HCl buffer (pH 7.6). Adapted with permission from Reference (24).Copyright 2007 American Chemical Society.

From the π-A isotherm, the aequorin Langmuir monolayer was in a liquid condensed phase at surface pressures above 11 mN·m-1 and the surface pressure can reach more than 20 mN/m. Thus, aequorin can form a stable monolayer and it is interesting to check the stability of the monolayer. 15 mN·m-1 was chosen to run the compression-decompression cycles and the stability check of the Langmuir monolayer as shown in Figure 4. For the compression-decompression cycles shown in Figure 4a, the cycles overlapped within 50 Å2·molecule-1. This indicates that the aequorin Langmuir monolayer was stable after 4 cycles. When the surface pressure was held at 15 mN·m-1 for 100 min, the molecular area changed only by 94 Wang and Leblanc; Recent Progress in Colloid and Surface Chemistry with Biological Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


120 Å2·molecule-1 which was less than 5% of the limiting molecular area (Figure 4b). Therefore, we concluded that aequorin formed a stable Langmuir monolayer. The limiting molecular area of aequorin was found to reach a maximum value at pH 7.6. In other words, the optimal condition for aequorin to form a Langmuir monolayer is at pH 7.6 and the results are below.

Figure 4. Compression-decompression cycles (a) and stability studies (b) at 15 mN·m-1 of the aequorin Langmuir monolayer on Tris/HCl buffer (pH 7.6). Adapted with permission from Reference (24). Copyright 2007 American Chemical Society. 95 Wang and Leblanc; Recent Progress in Colloid and Surface Chemistry with Biological Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


pH Effect on the π-A Isotherm of the Aequorin Langmuir Monolayer The π-A isotherms of the aequorin Langmuir monolayer on Tris/HCl, phosphate, and potassium phthalate buffers are shown in Figure 5. The pH ranged from 9.0 to 7.0 for Tris/HCl buffer (Figure 5a). As for pH 9.0, the lift-off point of the π-A isotherm was at 3230 Å2·molecule-1 and the limiting molecular area was at 1880 Å2·molecule-1 which was much smaller than the value of pH 7.6. When the pH was 8.0 and 7.0, the π-A isotherm was similar to the one at pH 7.6 (24). When the subphase was phosphate buffer (Figure 5b), the lift-off point of the isotherm at pH 8.0 was observed at 5500 Å2·molecule-1 and the limiting molecular area at 3490 Å2·molecule-1. As for the pH at 7.6, the lift-off point and the limiting molecular area was at 5180 Å2·molecule-1 and at 3780 Å2·molecule-1, respectively. For pH 7.0, the π-A isotherm was similar to that at pH 7.6. As for pH 5.9, the lift-off point decreased significantly to 4200 Å2·molecule-1 and the surface pressure increased quickly after the lift-off point (24). Figure 5c shows the π-A isotherms on the potassium phthalate buffer. The shape of the isotherm at pH 5.9 was identical to the one on the phosphate buffer at pH 5.9 with a lift-off point at 4580 Å2·molecule-1 and a limiting molecular area situated at 3800 Å2·molecule-1. The isoelectric point of aequorin was at pH 4.8. Under this condition, the lift-off point and the limiting molecular area were at 3100 Å2·molecule-1 and 2580 Å2·molecule-1, respectively. As for pH 4.0, the lift-off point and the limiting molecular area further decreased. When the pH was lower than 4.0, no reproducible π-A isotherms were obtained (24). In general, the effect of pH and the nature of the buffers on the aequorin Langmuir monolayer can be concluded as following. 1) For Tris/HCl or phosphate buffers, the lift-off point at pH 7.0, 7.6 and 8.0 is at 5360 Å2·molecule-1 in average which is within ± 4% of each lift-off point value. Similarly, the limiting molecular area shows an average value of 3715 Å2·molecule-1, within ± 7% of each limiting molecular area value. Thus, the π-A isotherms were similar under these conditions. This means that in the range of pH 7.0 - 8.0, the π-A isotherm of aequorin depends on neither the pH nor the nature of the buffers. 2) Higher than pH 8.0 or lower than pH 7.0, the value of lift-off point and limiting molecular area decreased. How can we explain the fact that at high and low pH we observed a decrease in both the lift-off point and the limiting molecular area from the π-A isotherms? It may be because that there is a conformational change of aequorin at these pH values. To validate this, we have examined the conformation of aequorin in aqueous solutions of different pH values by circular dichroism (CD) spectroscopy (24). The CD spectra of aequorin aqueous solutions at concentration of 0.1 mg·ml-1 under different pH values are shown in Figure 6. When the pH was 7.6, the spectrum showed two negative peaks at 208 and 221 nm and a positive peak at 192 nm, all of which are characteristic peaks of α-helix (32). Both higher and lower pH values than 7.6 reduced the contribution of the two peaks at 208 and 221 nm. This indicated that the content of α-helix in aequorin reached maximum value at pH 7.6. To quantify the fraction of the secondary structures of aequorin in aqueous solutions, the CD spectra were analyzed by the program of CDPro, which includes the three popular methods for CD spectra analysis: SELCON3, CONTIN and CDSSTR (32). The assignment of CD spectra from CDPro gave the content of 96 Wang and Leblanc; Recent Progress in Colloid and Surface Chemistry with Biological Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


six secondary structural classes: regular α-helix, αR; distorted α-helix, αD; regular β-sheet, βR; distorted β-sheet, βD; turns, T; and unordered, U. The analysis results of aequorin aqueous solutions are shown in Table 1.When the pH increased from 7.6 to 9.0, the fraction of α-helix (i.e., the sum of αR and αD) decreased from 37.8 to 32.2 %. On the contrary, the fraction of the unstructured conformation increased from 27.2 to 33.0 %. The fraction of other secondary structures changed minimally. This indicated that the α-helix conformation changed to unstructured conformation when the pH increased up to 9.0. When the pH decreased from 7.6 to 4.8, the content of α-helix decreased from 37.8 to 29.7 %. At pH 4.0, the fraction of α-helix further decreased to 23.9 %. On the contrary, the fraction of β-sheet (i.e., the sum of βR and βD) kept increasing when pH decreased down to 4.0. This indicated that the α-helix conformation changed to β-sheet under acidic conditions (24). Based on these results, the α-helix has the highest content at pH 7.6. The conformation change of aequorin in acidic or basic aqueous solution supports the hypothesis which explains the lower limiting molecular area observed in the π-A isotherms at these pH values. In general, α-helical conformation can help to increase the limiting molecular area of aequorin at the air−water interface. To determine the orientation of α-helix, IRRAS of aequorin Langmuir monolayer was studied.

IRRAS of the Aequorin Langmuir Monolayer at 15 mN·m-1 with Different Incident Angles The s-polarized and p-polarized IRRAS spectra of the aequorin Langmuir monolayer are shown in Figure 7a and 7b. In Figure 7a, two peaks of the amide I band were detected at 1673 and 1651 cm-1 and two peaks of the amide II band were at 1545 and 1515 cm-1. The peaks at 1651, 1545, and 1515 cm-1 were assigned to the α-helix conformation and the peak at 1673 was assigned to the turns conformation (25). The intensity of all peaks decreased with the increase of the incident angle. This result correlated to the theoretical calculation and interpretation as above-mentioned CD results. The peaks of the β-sheet conformation were not detected due to the low percentage of the β-sheet conformation in aequorin. For the IRRAS spectra of p-polarization shown in Figure 7b, the amide I band was detected predominantly at 1651 cm-1 and the amide II bands were detected at 1545 and 1515 cm-1. All the peaks were assigned to the α-helix conformation and the peaks were negative when the incident angle was below the Brewster angle (54.2 ° for the IR light at 2850 cm-1). When the incident angle was above the Brewster angle, all the peaks changed from negative to positive. As mentioned in the Introduction, the amide I band arises mainly from the stretching vibration of backbone carbonyls (i.e., C=O) in proteins, and is sensitive to the conformation of proteins as mentioned in Introduction. Thus, Figure 7b indicates that the vibration of C=O is parallel to the air−water interface. As shown in Figure 1, the orientation of C=O in α-helix is parallel to the axis of α-helix. Consequently, the axis of α-helix in aequroin is also parallel to the air−water interface (22). 97 Wang and Leblanc; Recent Progress in Colloid and Surface Chemistry with Biological Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Figure 5. The effect of pH on surface pressure-area isotherms of the aequorin Langmuir monolayer: (a) – pH 9.0, -▲- pH 8.0, -■- pH 7.6, and -●-pH 7.0 .in Tris/HCl buffer, (b) – pH 5.9, -▲- pH 8.0, -■- pH 7.6, and -●-pH 7.0 in phosphate buffer, (c) – pH 5.9, -■- pH 4.8, and -○- pH 4.0 in potassium phthalate buffer. Adapted with permission from Reference (24).Copyright 2007 American Chemical Society. 98 Wang and Leblanc; Recent Progress in Colloid and Surface Chemistry with Biological Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Figure 6. Circular dichroism spectra of aequorin at concentration of 0.1 mg·ml-1 under different pH values: -●- 9.0, -□- 7.6, -○- 4.8, -Δ- 4.0. pH 9.0 and 7.6 was controlled by 0.02 M Tris solution and 0.02 M HCl solution. pH 4.8 and 4.0 was controlled by 0.1 M potassium dihydrogen phosphate solution and 0.1 M phosphoric acid solution. Adapted with permission from Reference (24). Copyright 2007 American Chemical Society.

Table 1. Fraction of Secondary Structures of Aequorin in Aqueous Solution under Different pH pH

αR

αD

βR

βD

T

U

9.0

17.2

15.0

8.0

6.8

20.1

33.0

7.6

21.2

16.6

6.7

6.8

21.7

27.2

4.8

17.8

13.1

11.9

8.2

21.7

27.6

4.0

13.7

10.2

18.8

9.0

20.9

26.6

αR: regular α-helix, αD: distorted α-helix, βR: regular β-sheet, βD: distorted β-sheet, T: turns, U: unordered structures. Adapted with permission from Reference (24). Copyright 2007 American Chemical Society.

99 Wang and Leblanc; Recent Progress in Colloid and Surface Chemistry with Biological Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Figure 7. IRRAS spectra of the aequorin Langmuir monolayer at a surface pressure of 15 mN·m-1 with different incident angles: (a) s-polarization, (b) p-polarization. Adapted with permission from Reference (22). Copyright 2008 American Chemical Society.

On the whole, we can conclude that α-helical conformation helps to increase the limiting molecular area of aequorin and, consequently, make the Langmuir monolayer more stable at the interface. Although the increase of 100 Wang and Leblanc; Recent Progress in Colloid and Surface Chemistry with Biological Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


the fraction of β-sheet decreases the limiting molecular area of aequorin as shown above, some protein major in β-sheet conformation has been shown to contribute to the Langmuir monolayer at the air−water interface (21). On the other hand, the increasing fraction of unstructured conformation will decrease the limiting molecular area and destabilize the Langmuir monolayer of the protein. Actually, we have studied the surface properties of other proteins in unstructured conformation. For example, gelatin derived from collagen is an unstructured protein. We have tried to spread gelatin at the air−water interface. However, gelatin was found to be unable to form a Langmuir monolayer and the surface pressure of gelatin cannot reach 5 mN/m, regardless of the pH values or the concentration of salt/buffer in the subphase. The surface pressure increased slowly and no reproducible π–A isotherm has been obtained. Interestingly, α-synuclein (α-syn) which has been reported to be unstructured in aqueous solution can form a stable Langmuir monolayer at the air−water interface and the results are below. Study of the Surface Properties and the Orientation of α-syn at the Air−Water Interface α-Syn is a presynaptic protein which is believed to play an important role in neuropathology in Parkinson’s disease (PD) (27, 28). PD is pathologically marked by the progressive loss of neurons in the substantia nigra (33, 34), a small brain region producing dopamine. A hallmark of PD is that surviving dopaminergic cells contain cytosolic filamentous inclusions known as Lewy bodies (33, 34). The major protein component in Lewy bodies is α-syn aggregates, of which the constituent monomeric peptide contains 140 amino-acid residues encompassing the positively charged N-terminus (residues 1-60), the aggregation-prone nonamyloid components (NAC, residues 60-95), and the negatively charged C-terminus (residues 96-140) as shown in Figure 8 below (35, 36).

Figure 8. The sequence of α-syn with the N-terminus in Italics and the NAC part underlined. α-Syn has been shown to be abundant in human brain and the average concentration of α-syn has been indicated to be ~100 μM (28). However, the concentration of α-syn in the plasma was shown to be very low around 20 nM (29) because most of α-syn enriches in presynaptic terminals (PST) (30). The reason for this enrichment was not clear. In addition, amphiphilic membranes (e.g., cell membranes, micelles, and vesicle interfaces) have been noted to be important among the various factors affecting the misfolding and aggregation of α-syn (37–43). Because PST contains abundant vesicles (8), it is of interest to investigate whether the presence of the amphiphilic interfaces enhances the accumulation of α-syn. 101 Wang and Leblanc; Recent Progress in Colloid and Surface Chemistry with Biological Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


As mentioned in the Introduction, the air−water interface has been accepted as a simple model to mimic the amphiphilic interface in vivo and the physical properties (e.g., stability or aggregation) of amphiphilic biomacromolecules at the air−water interface can be determined in detail by the Langmuir monolayer technique (21–23). Thus, it is reasonable to study the surface properties of α-syn which is unstructured in aqueous solution. Different from gelatin, α-syn can form a stable monolayer at the air−water interface. As shown in Figure 9, the surface pressure has a lift-off point at 2450 Å2/molecule either on the surface of pure water or 0.15 M NaCl (pH 7.4) aqueous solution. Further compression caused a kink point at 1560 Å2/molecule, indicating a liquid expanded-liquid condensed phase transition in the π-A isotherm. The limiting molecular area about 1490 Å2/molecule can be obtained by extrapolating the linear portion of the isotherms. Considering the similarity of the two isotherms, the ionic strength of the subphase within our experimental conditions may not play any role on the surface properties of α-syn in the Langmuir monolayer.

Figure 9. Surface pressure-area isotherms of 0.3 mg/mL α-syn spread on 0.15 M NaCl Tris buffer (pH 7.4) (solid curve) and pure water (-□-) subphase. Adapted with permission from Reference (23). Copyright 2010 the Royal Society of Chemistry.

Also to further verify this stability, the α-syn Langmuir monolayer was compressed to a surface pressure of 10 mN/m (Figure 10) and the surface pressure was kept constant over an extended period of time (more than 100 min). The surface pressure (solid line curve) was monitored together with the area (dashed line curve) over the entire experiment and the molecular area decreased by less 102 Wang and Leblanc; Recent Progress in Colloid and Surface Chemistry with Biological Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


than 2% after 100 minutes of compression. The small decrease in the molecular area indicates the high stability of α-syn Langmuir monolayer, which assures that α-syn did not denature during the compression. As mentioned above, α-syn was shown to be in unstructured conformation which cannot form a stable Langmuir monolayer at the air−water interface. This is contrary to our results shown in Figure 10, which needs more spectroscopic results to explain. Thus, the Langmuir monolayer of α-syn was transferred onto quartz slides using the Langmuir-Blodgett (LB) technique of deposition for CD measurements. The inset in Figure 10 compares the CD spectra of the α-syn protein in the LB film on quartz slides and in aqueous phase. An unexpected result was observed regarding the conformation of α-syn in these two phases: the characteristic peaks of the α-helical conformation (two negative peaks at 209 and 222 nm together with one positive peak at 192 nm) were observed for α-syn LB films (32), whereas only one negative peak at 199 nm (the characteristic peak of unstructured conformation) was observed for α-syn in solution (32). Particularly, the CD spectrum of the LB films of α-syn transferred after holding the surface pressure at 10 mN/m for two weeks was still identical to the solid line curve shown in the inset of Figure 10. Thus, the α-helical conformation of the α-syn Langmuir monolayer neither dissolved in the subphase nor denatured to other conformations.

Figure 10. Stability curves when the Langmuir monolayer of α-syn was compressed up to10 mN/m and kept constant for more than 100 min on the Tris buffer (pH 7.4) subphase containing 0.15 M NaCl. Inset: CD spectra of an α-syn Langmuir-Blodgett monolayer on a quartz slide (solid line curve) and 0.015 mg/mL α-syn dissolved in Tris buffer at pH 7.4 (dotted line curve), respectively. Adapted with permission from Reference (23). Copyright 2010 the Royal Society of Chemistry. 103 Wang and Leblanc; Recent Progress in Colloid and Surface Chemistry with Biological Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Figure 11. S-polarized (a) and p-polarized (b) IRRAS spectra of the α-syn Langmuir monolayer at various surface pressures at an incident angle of 30º and at various incident angles at a surface pressure of 10 mN/m, respectively. Adapted with permission from Reference (23). Copyright 2010 the Royal Society of Chemistry.

To confirm that the α-helical conformation of the α-syn Langmuir monolayer was not due to the transfer from the air−water interface to the quartz slides, we investigated the in situ s- and p-polarized IRRAS of the α-syn Langmuir monolayer. As shown in Figure 11a, three peaks characteristic of the α-helix 104 Wang and Leblanc; Recent Progress in Colloid and Surface Chemistry with Biological Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


conformation were observed for the s-polarized spectra, namely 1655, 1545 and 1515 cm-1. Moreover, the peak positions remain unchanged when the surface pressure was varied. This correlates well with the CD data, which demonstrate the stability of α-helix of α-syn at the air−water interface. Figure 11b shows the p-polarized IRRAS spectra of the α-syn Langmuir monolayer. The fingerprint of the spectra indicates that the axis of the α-helix is parallel to the interface. In general, the high stability of the Langmuir monolayer of α-syn stems from the α-helical conformation at the air−water interface. The high stability indicates that α-syn prefers to stay at the amphiphilic interfaces than to be dissolved in the cytoplasma. Thus, the reason why α-syn enriching in PST may be due to the high density of vesicles existing in the PST (8).

Discussion In general, external environmental factors such as the ionic strength in the subphase do not significantly affect the surface properties of either aequorin or α-syn. Among various conformations, α-helix can help proteins to form a Langmuir monolayer whereas unstructured conformation is destructive to the Langmuir monolayer. As for aequorin, β-sheet conformation decreases the limiting molecular area. However, the aggregates of β-amyloid (Aβ) peptide have been shown to contribute to the formation of Langmuir monolayer. Interestingly, β-sheet strands of the aggregates of Aβ peptide were shown to be parallel to the air−water interface (21). Because the orientation of α-helix of either aequorin or α-syn is also parallel to the interface, the parallel orientation of the conformations may help the hydrophilic part of proteins to form more hydrogen bonds with water molecules in the subphase. It is worth noting that the properties of Aβ peptide are very similar to those of α-syn. For example, both Aβ peptide and α-syn can form various aggregates in β-sheet conformation (44–47). The well developed aggregates are called mature fibrils and the early stage aggregates are referred as oligomers (48–51). The abnormal aggregation of α-syn in vivo forms Lewy bodies which are the hallmark of PD. Similarly, the aggregation of Aβ peptide is the major component of senile plaques which are found in the brains of patients of Alzheimer’s disease (AD) (44–47). Thus, both Aβ peptide and α-syn can be regarded as amyloidogenic proteins. Although there are treatments to relieve the symptoms of PD, there is still no cure for either AD or PD (45, 47). Consequently, the determination of the structure of the aggregates of Aβ peptide and α-syn is important for the development of therapeutic agent for AD and PD, respectively. Another similarity between Aβ peptide and α-syn stems from the structure of their mature fibrils, both of which have been shown to be in parallel β-sheet conformation (cf., Figure 2) by NMR (52, 53). Furthermore, the mature fibrils of both Aβ peptide and α-syn are not toxic whereas the oligomers of Aβ peptide and α-syn are toxic (48, 51, 54). Because the formation and development of the oligomers of Aβ peptide and α-syn are dynamic, limited results have been reported about the structure of the oligomers by NMR which usually requires substantial measurement time. Thus, FTIR spectroscopy may help to determine 105 Wang and Leblanc; Recent Progress in Colloid and Surface Chemistry with Biological Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


the dynamic structure of various oligomers of either Aβ peptide or α-syn. For example, it has been shown that the oligomer of α-syn may contain anti-parallel β-sheet conformation (cf., Figure 2) which shows a characteristic peak around 1685 cm-1 (54). Interestingly, the oligomers of both Aβ peptide and α-syn have been reported to form pore structures in the cell membrane (42, 55–58). The pore structure has been shown to cause ion leakage and kill neuronal cells. Although extensive studies have detected the presence and the morphology of the pore in the cell membrane (50), neither the conformation nor the orientation of the β-sheet strands in the oligomers has been reported. Therefore, surface IR spectroscopy such as IRRAS may be helpful to address the challenges above. The determination of the structure of the oligomers will provide useful clues for the development of therapeutic agents for AD and PD.

Conclusion α-Helix in aequorin was found to help the formation of the Langmuir monolayer of aequorin at the air−water interface. On the contrary, the unstructured conformation was found to dissolve aequorin into subphase from the air−water interface, because the limiting molecular area of the Langmuir monolayer of aequorin decreases when the fraction of unstructured conformation increases in aequorin. Although unstructured in aqueous solution, α-syn stays stable at the air−water interface because of its α-helical conformation when spread at the air−water interface. In addition, the α-helix in the Langmuir monolayer of both aequorin and α-syn was generally parallel to the air−water interface according to the IRRAS results.

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Î±-Helix Facilitates Proteins To Form Langmuir Monolayer - American

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