Initial Conformation of Adsorbed Proteins at an AirâWater Interface

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Initial Conformation of Adsorbed Proteins at An Air-Water Interface Yohko F. Yano, Etsuo Arakawa, Wolfgang Voegeli, Chika Kamezawa, and Tadashi Matsushita J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b01039 • Publication Date (Web): 09 Apr 2018 Downloaded from http://pubs.acs.org on April 9, 2018

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The Journal of Physical Chemistry

Initial Conformation of Adsorbed Proteins at an AirWater Interface Yohko F. Yano,*† Etsuo Arakawa,‡ Wolfgang Voegeli,‡ Chika Kamezawa‡ and Tadashi Matsushita§ †

Department of Physics, Kindai University, 3-4-1 Kowakae, Higashiosaka City, Osaka 5778502, Japan

‡

Department of Physics, Tokyo Gakugei University, 4-1-1 Nukuikita-machi, Koganei, Tokyo

184-8501, Japan §

Photon Factory, Institute of Materials Structure Science, KEK, 1-1 Oho, Tsukuba, Ibaraki 305-

0801, Japan [email protected] Phone +81-6-6721-2332, ext. 4088; Fax +81-6-6727-4301

*

Corresponding author

†

Kindai University

‡

Tokyo Gakugei University

§

Photon Factory

ACS Paragon Plus Environment

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The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT: We present the results of time-resolved X-ray reflectivity measurements carried out to investigate the early stage of protein adsorption and deformation at an air-water interface. Three globular proteins (lysozyme, myoglobin, and BSA) were studied, and we observed that the proteins adsorbed at the air-water interface initially possessed a thinner conformation than their native structures. The degree of deformation increased in the order myoglobin < lysozyme < BSA, which was inconsistent with the order of molecular flexibility. The initial rate of protein adsorption increased in the order lysozyme < BSA < myoglobin as determined by dynamic surface tension. More flexible proteins generally adsorb at the interface more rapidly; however, proteins with hydrophobic patches on the protein surface, such as myoglobin, adsorb at the interface with little deformation. These results provide evidence that protein unfolding during adsorption only takes place if the kinetics of adsorption are similar to or slower than the kinetics of unfolding.


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INTRODUCTION When proteins adsorb at an air-water interface as amphiphilic molecules, many of them lose their biological activity due to subsequent denaturation.1 This phenomenon is important for several biological and chemical processes. The protein unfolding phenomena at interfaces are still not completely understood, compared with those occurring in bulk states.2-3 Wierenga et al. suggested that unfolding will only take place if the kinetics of adsorption are similar to or slower than the kinetics of unfolding.4 However, it was derived based on an indirect observation, a direct observation is necessary to verify their suggestion. The adsorption rate is generally controlled by the bulk concentration5-7 and pH of the solution8-10. The arrival of a protein at the interface is assumed to be driven by diffusion processes, which are dependent on the bulk concentration and diffusion coefficient.11 Adsorbed proteins subsequently undergo conformal changes. At low bulk concentrations, however, the area at the interface covered by adsorbed protein molecules is small and the protein molecules can occupy a maximum interfacial area that induces a higher degree of unfolding.6,12 The net charge of the proteins also affects the adsorption kinetics. The adsorption of proteins is fastest around the isoelectric point (pI), where the numbers of negative and positive charges are equal, resulting in higher surface packing densities. On the other hand, the increased electrostatic repulsion resulted in a larger apparent size of the adsorbed proteins.13 The

protein-dependent

factors

that

control

the

adsorption

rate

are:

(1)

hydrophobicity/hydrophilicity ratio of the protein surface; (2) flexibility of the polypeptide chain; and (3) ability to interact with neighboring molecules.14 The pattern of distribution of hydrophilic and hydrophobic groups on a protein surface affects the rapidity of its adsorption at the air-water interface.14 Furthermore, highly flexible proteins can undergo rapid conformational


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changes once they are adsorbed at the interface, enabling additional hydrophobic groups to bind to the interface.15 These behaviors have been mainly understood on a macroscopic basis5-7,11,16 and through the observation of secondary structures17-21. Recent theoretical studies suggested that the adsorbed proteins crawl along the interface and modify their conformations by pointing hydrophobic residues up toward the air. However, their movements become slow with increasing adsorption.22 Investigation of the correlation between the tertiary structures and adsorption rate is important for further understanding of protein unfolding at the interface. Previously, the adsorption process of hen egg lysozyme (LSZ) was examined using timeresolved X-ray reflectometry with a time resolution of several minutes to investigate the density profile of LSZ perpendicular to the air-water interface.12,23 The results showed that the LSZ molecules initially adsorbed at the air-water interface as monomolecular structures, with each molecule adopting a flat, distorted conformation. As adsorption continued, a second layer formed in which the molecules had a very loose structure. Recently, we developed a new X-ray reflectometer with an acquisition time of 1 s.24 Further experiments revealed that LSZ molecules unfolded into a flat shape within 1 s.25 In this report, we will apply this technique to other globular proteins to observe their initial conformations. Comparison with those native conformations, we can correlate the degree of unfolding with the adsorption rate. We will also calculate the hydrophobicity/hydrophilicity ratio of the Connolly surfaces26 of the proteins to correlate with their conformations at the interface. The three model globular proteins are summarized in Table 1. Lysozyme (LSZ) had the most rigid structure, with low foamability, and the strongest affinity for hydrophobic surfaces. Bovine serum albumin (BSA) had the most flexible structure with high foamability. Myoglobin (MYO) had intermediate flexibility and foamability.


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Table 1. Physical and Chemical Properties and Dynamic Surface Tension Parameters of Model Globular Proteins PDB No. LSZ MYO BSA

a

1VDQ 1BZR 3V03

Isoelectric point pI 11.1 7.0 4.7

12

10 βs

b

c

(cm ·dyn )

HIC (min)

Foam (mm)

4.67 8.98 10.5

18 8 6.7

0.1 1 15

2

-1

d

a

Structural data number obtained from Protein Data Bank.27 b Adiabatic compressibility.15 c Experimental retention time of proteins in a hydrophobic interaction chromatographic column.5 d Foam height of 5 mL of a protein solution shaken for 30 s.5 It is an indicator of protein hardness.

METHODS Materials. Three globular proteins, lysozyme from chicken egg white (Prod. No. L6876), myoglobin from skeletal muscle (Prod. No. M0630), and bovine serum albumin (Prod. No. A4059), were purchased from Sigma Aldrich and used as supplied. Protein solutions were made using phosphate buffer solutions (0.02 M NaH2PO4/Na2HPO4) at pH 7 using UHQ-grade water. In the present study, all the measurements were carried out under the conditions in which the proteins are in the native states. Protein solutions were prepared to a concentration of 43 mg/mL, from which 1 mL portions were added to 42 mL of a buffer solution in a Langmuir trough to give a final concentration of 1 mg/mL. Experiments. X-ray reflectivity measurements were performed using a simultaneous multiple angle-wavelength dispersive X-ray reflectometer24 at the AR-NE7A beamline of the Photon Factory at KEK, Japan. In this reflectometer, the incident beam is focused on the sample with incident angles α that change continuously in the range of 0 < α < 1.7°. The X-ray reflectivity


5


profiles in the range of the wave vector transfer 0 < q < 0.4 Å-1, where ‫= ݍ‬

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ସగா ௛௖

sin ߙ with the

energy region of E = 22.1 - 19.1 keV were simultaneously obtained. The protein sample solution was injected over 0.3 s using a liquid dispenser into a phosphate buffer solution. The X-ray reflectivity measurements were started before injection of the protein solution. The data collection time was 1.0 s. Within 10 s after protein injection, the X-ray beam was no longer ideally reflected because of the motion of the liquid surface.25 Surface tension was measured using a Wilhelmy plate mounted on the Langmuir trough. Computational Details. The X-ray reflectivity data were fitted using a 3-slab (or 2-slab for the BSA solution) model with the Parrat32 software28 using parameters of film thickness, electron density, and roughness for the three slabs.29 The hydrophobic and hydrophilic residues were identified based on data obtained from the Protein Data Bank27 using eF-site (electrostatic-surface of Functional site30), which is a database for molecular surfaces of proteins' functional sites that displays the electrostatic potentials and hydrophobic properties together on the Connolly surfaces.26 The data sets 1VDQ for LSZ, 1BZR for MYO, and 3V03 for BSA were used. Electron density profiles of the native proteins were calculated in the same manner by Tiemeyer et al31 based on the same data sets. Simulated one-dimensional density profiles of several orientations were calculated using Mathematica.32 Each area of the simulated density profile was normalized with respect to the area of the observed density profile.

RESULTS


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Hydrophobicity/hydrophilicity Ratios. Figure 1 shows typical molecular surfaces with three different orientations of LSZ, MYO and BSA. The hydrophobic residues are shown in green. The positively and negatively charged hydrophilic residues are shown in blue and red, respectively. The hydrophobicity/hydrophilicity ratio corresponds to the ratio of the colored areas are shown in Table 2. The orientations that have the widest surface areas for each protein are shown in (A) and (C) in Figure 1, whereas the side orientations with the maximum hydrophobicity/hydrophilicity ratios are shown in (B). In the case of LSZ, the hydrophobic area of any orientation is relatively high, with the negatively charged area primarily located in the (C) orientation. MYO has both hydrophobic (A) and hydrophilic (C) orientations. For BSA, the

hydrophobic area of any orientation is much lower than those of LSZ.


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Figure 1. Molecular surfaces of different orientations for three proteins in which hydrophobic, positively and negatively charged residues are shown in green, blue and red, respectively. Table 2. Hydrophobicity/Hydrophilicity Ratio Corresponds to the Ratio of the Colored Areas in Figure 1

Surface Tension. Figure 2 shows dynamic surface tension for each of the three proteins. The initial rates of the surface tension decay reproduced those reported previously.5 The rate of protein adsorption could be inferred from these tension decay values. The initial rate of protein adsorption increased in the order LSZ < BSA < MYO. This is not the only measurement performed at pI for MYO, but MYO was also the most surface-active agent. It is known that the hydrophobic amino acid residues contribute the surface activity positively, whereas those of charged and polar residues contribute negatively.15 The lower percentage of hydrophobic


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residues in BSA (see Table 2) predicted a greater equilibrium value of surface tension.

Figure 2. Time dependence of surface tension changes for the globular proteins lysozyme (LSZ), myoglobin (MYO), and bovine serum albumin (BSA) at pH 7.

X-ray Reflectivity. Figures 3-5 show the time dependence of the X-ray reflectivity profiles vs. the wave vector transfer q for the three proteins divided by Fresnel reflectivity RF, along with the electron density profiles obtained by fitting the data. These data were obtained 10 s after the protein injection. The data of LSZ has already published.25 Figure 3 shows that the X-ray reflectivity profile remains unchanged with that of the buffer solution until 12 s after LSZ

2.0

(a)

LSZ

12 sec 14 sec 1.5 min

1.0

Electron density e/Å 3

injection and then increases rapidly over 2 s. As shown in Figure 3(b), the electron density

R/RF

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(b)

0.45

0.40

0.35 pure water

0

0.1

0.2 0.3 -1 q /Å

0.4

0.30

0

10

20 z /Å

30

profile for the initially adsorbed LSZ at 14 s has a dense upper layer with a diffuse lower layer, which is highly distorted compared to the native configuration, and this configuration does not change with increasing amount of adsorbed LSZ.


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Figure 3. (a) X-ray reflectivity profiles taken 12 s to 1.5 min after LSZ injection. Data were divided by the Fresnel reflectivity of the air-water interface. The solid lines indicate the best fits to the data. (b) Profiles of electron density best fitting the data. z = 0 corresponds to the air-water interface. The dashed line represents pure water. On the other hand, the electron density profile for the initially adsorbed MYO also consists of a dense upper layer with a diffuse lower layer, but the shape of the electron density profile changes during adsorption, suggesting that the conformation of MYO gradually changes. The total density and thickness are larger than those of LSZ, indicating that the surface amount of adsorbed protein is higher. This is also supported by the dynamical surface tension as shown in Figure2.


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Figure 4. (a) X-ray reflectivity profiles taken 10 s to 1.5 min after MYO injection. Data were divided by the Fresnel reflectivity of the air-water interface. The solid lines indicate the best fits to the data. (b) Profiles of electron density best fitting the data. z = 0 corresponds to

BSA

(a)

2.0

8 sec 10 sec 30 sec

1.0


the air-water interface.

R/RF

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(b)

0.45

0.40

0.35 pure water

0

0.1

0.2 0.3 -1 q /Å

0.4

0.30

0

10

20 z /Å

30

For BSA, the X-ray reflectivity around q ≈ 0.4 Å-1 is lower than the other proteins (Figure 5). In this case, the electron density profile for the initially adsorbed BSA does not have a dense upper layer.

Figure 5. (a) X-ray reflectivity profiles taken 10–30 s after BSA injection. Data were divided by the Fresnel reflectivity of the air-water interface. The solid lines indicate the best fits to the data. (b) Profiles of electron density best fitting the data. z = 0 corresponds to the air-water interface.


11


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DISCUSSION The density profiles were compared with those of the native structures. Figure 6 illustrates experimental electron density profiles compared to the simulated density profiles. The thinnest protein structures with the hydrophobic groups pointing in the air [side (A) is exposed to air] were selected.

Figure 6. Comparison of the electron density profiles experimentally obtained 10-14 s after protein injection [the solid red curves correspond to the red curves in Figure 3-5(b)] with the simulated profiles (dashed black lines) from PDB data, taking into account the protein orientation in which side (A) is exposed to air.

Initially adsorbed LSZ, which has the most rigid structure, had a thinner shape compared to the native structure in both orientations of sides (A) and (B). With decreasing concentration, we observed that the LSZ conformations became thinner.12 Previous surface-sensitive IR spectra revealed that some α-helices changed to β-sheets upon adsorption.19 Because LSZ has a strong


12

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affinity for hydrophobic surfaces, as shown in Table 1 and Figure 1, this denaturation is irreversible.22 In contrast, MYO produces uniform electron density profiles that exhibit little deformation compared to the native structure. Considering the distribution pattern of hydrophobic and hydrophilic patches on the surface (see Figure 1), MYO has the ability to adsorb at the interface in its native conformation. In the bulk state, MYO is unfolded at pH < 5 whereas it is in a native state at pH > 5.33 Some previous studies reported that MYO is partially34-35 or fully unfolded36 at the air-water interface. Since the present condition of pH 7 is equal to pI for MYO, the rate of adsorption becomes fastest. As the result, the conformation of MYO might have deformed very little. On the other hand, in the case of LSZ with slower adsorption rate at pI, we observed the conformation of the adsorbed molecules highly deformed.37 Thus, both distribution patterns of hydrophobic and hydrophilic patches on the surface and a high adsorption rate near pI cause less deformation. The initially adsorbed BSA, which has the most flexible structure, adopted a highly deformed structure at 10 s (Figure 6). Previous reports indicated that the secondary structure of BSA changes very little during adsorption,19 and the compression per residue was approximately 181.8 cal, which is five times larger than that of MYO.38

Therefore, we think that BSA

molecules are spread widely at the interface while retaining their secondary structure.

CONCLUSIONS The early stage of protein adsorption behavior at an air-water interface was examined using three typical globular proteins. All of the proteins became thinner than they were in the bulk state. Although the initial rate of protein adsorption increased in the order LSZ < BSA < MYO, the


13


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degree of unfolding increased in the order MYO < LSZ < BSA. These results are inconsistent with the order of protein structural rigidity.15 MYO, containing hydrophobic patches on the protein surface, rapidly adsorbed at the interface with little deformation. The other proteins became highly deformed, resulting in a faster adsorption rate for the more flexible BSA. These results provide direct evidence that protein unfolding only takes place if the kinetics of adsorption are similar to or slower than the kinetics of unfolding4. Acknowledgments. Experiments were conducted on the beamline AR-NE7A of the Photon Factory under the Photon Factory experimental program 2011G068 and 2016G80. This research was financially supported by the program “Development of Systems and Technology for Advanced Measurement and Analysis” of the Japan Science and Technology Agency. Y.Y. acknowledges financial support from Grants-in-Aid for Scientific Research (C) (No. 24540444 and No. 17K05616) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


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(19) Lad, M. D.; Birembaut, F.; Matthew, J. M.; Frazier, R. A.; Green, R. J. The adsorbed conformation of globular proteins at the air/water interface. Phys. Chem. Chem. Phys. 2006, 8, 2179-2186. (20) Alahverdjieva, V. S.; Grigoriev, D. O.; Ferri, J. K.; Fainerman, V. B.; Aksenenko, E. V.; Leser, M. E.; Michel, M.; Miller, R. Adsorption behaviour of hen egg-white lysozyme at the air/water interface. Colloids Surf. Physicochem. Eng. Aspects 2008, 323, 167-174. (21) Sankaranarayanan, K.; Dhathathreyan, A.; Kragel, J.; Miller, R. Interfacial viscoelasticity of myoglobin at air/water and air/solution interfaces: Role of folding and clustering. J. Phys. Chem. B 2012, 116, 895-902. (22) Cieplak, M.; Allan, D. B.; Leheny, R. L.; Reich, D. H. Proteins at air-water interfaces: A coarse-grained model. Langmuir 2014, 30, 12888-12896. (23) Yano, Y. F. Kinetics of protein unfolding at interfaces. J. Phys.: Condens. Matter 2012, 24, 503101. (24) Matsushita, T.; Arakawa, E.; Voegeli, W.; Yano, Y. F. A simultaneous multiple anglewavelength dispersive x-ray reflectometer using a bent-twisted polychromator crystal. J. Synchrotron Radiat. 2013, 20, 80-88. (25) Yano, Y. F.; Arakawa, E.; Voegeli, W.; Matsushita, T. Real-time investigation of protein unfolding at an air–water interface at the 1 s time scale. J. Synchrotron Radiat. 2013, 20, 980983. (26) Connolly, M. L. The molecular-surface package. Journal of Molecular Graphics 1993, 11, 139-143.


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(27) Berman, H.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T.; Weissig, H.; Shindyalov, I.; Bourne, P. The protein data bank nucleic acids research, 28, 235-242. URL: www. rcsb. org Citation 2000. (28) Braun, C. Parratt32, HMI, Berlin, 1997-2002. (29) Pershan, P. S.; Schlossman, M. Liquid surfaces and interfaces: Synchrotron x-ray methods.; Cambridge University Press, 2012. (30) Kinoshita, K.; Nakamura, H. eF-site and PDBjViewer: Database and viewer for protein functional sites. Bioinformatics 2004, 20, 1329-1330. (31) Tiemeyer, S.; Paulus, M.; Tolan, M. Effect of surface charge distribution on the adsorption orientation of proteins to lipid monolayers. Langmuir 2010, 26, 14064-14067. (32) Mathematica, version 10.3; Wolfram Research: Champaign, IL 2015. (33) Dill, K. A.; Shortle, D. Denatured states of proteins. Annu. Rev. Biochem 1991, 60, 795825. (34) Poon, S.; Clarke, A. E.; Schultz, C. J. Structure–function analysis of the emulsifying and interfacial properties of apomyoglobin and derived peptides. J. Colloid Interface Sci. 1999, 213, 193-203. (35) Cheung, D. L. Conformations of myoglobin-derived peptides at the air-water interface. Langmuir 2016, 32, 4405-4414. (36) Xiao, Y.; Konermann, L. Protein structural dynamics at the gas/water interface examined by hydrogen exchange mass spectrometry. Protein Sci. 2015, 24, 1247-1256. (37) Yano, Y. F.; Uruga, T.; Tanida, H.; Terada, Y.; Yamada, H. Protein salting out observed at an air−water interface. J. Phys. Chem. Lett. 2011, 2, 995-999.


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(38) Birdi, K. The determination of work of compression of protein monolayers at the air-water interface. Kolloid Z. Z. Polym. 1972, 250, 222-226.


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TOC IMAGE


20

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Distorted

slow

Hard

Less distorted

Unfolded

fast

Surface active

Soft



(A) Y

25 Å

(B)

45 Å

X 23 Å

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Page 22 of 27

X 45 Å

Z

43 Å

80 Å

(C) X Y

LSZ

MYO ACS Paragon Plus Environment

BSA

Page 23 of 27

0 LSZ

∆γ / mNm

-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


BSA

-10

MYO

-20 100

101

102 t/s


103

12 sec 14 sec 1.5 min

1.0

0

0.1

0.2 0.3 -1 q /Å

0.4

Electron density e/Å

2.0

(a)

LSZ

R/RF

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

3


Page 24 of 27

(b)

0.45

0.40

0.35

0.30

0


pure water

10

20 z /Å

30

3.0 MYO

R/RF

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


(a) 10 sec 12 sec 1.5 min

2.0

1.0

0

0.1

0.2 0.3 -1 q /Å

0.4


Page 25 of 27

(b)

0.45

0.40

0.35

0.30


0

pure water

10

20 z /Å

30

BSA

(a)

2.0 R/RF

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

8 sec 10 sec 30 sec

1.0

0

0.1

0.2 0.3 -1 q /Å

0.4



Page 26 of 27

(b)

0.45

0.40

0.35

0.30


0

pure water

10

20 z /Å

30

Page 27 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


-1

Myoglobin

-1

Lysozyme

water

water 0

0.1

0.2

0.3

1

-1

0

0.1

0.2

0.3

BSA

water 0

1

1

2

2

3

3

3

4

4

4

z / nm

z / nm

z / nm

2

side (A) side (B)


0.1

0.2

0.3

air-water interface native

Initial Conformation of Adsorbed Proteins at an AirâWater Interface

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