When the Label Matters: Adsorption of Labeled and Unlabeled

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When the label matters: Adsorption of labelled and unlabelled proteins on charged surfaces Julia Romanowska, Daria B. Kokh, and Rebecca C Wade Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.5b03168 • Publication Date (Web): 22 Oct 2015 Downloaded from http://pubs.acs.org on October 23, 2015

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When the label matters: Adsorption of labelled and unlabelled proteins on charged surfaces Julia Romanowska,∗,†,§ Daria B. Kokh,† and Rebecca C. Wade∗,†,‡,¶ Molecular and Cellular Modeling Group, Heidelberg Institute for Theoretical Studies, Heidelberg, Germany, Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Heidelberg University, Heidelberg, Germany, and Interdisciplinary Center for Scientific Computing (IWR), Heidelberg University, Heidelberg, Germany E-mail: [email protected]; [email protected] Phone: +47 55 58 60 99; +49 6221-533 247. Fax: +49 6221-533 298

Abstract Fluorescent labels are often attached to proteins to monitor binding and adsorption processes. Docking simulations for native hen egg white lysozyme (HEWL) and HEWL labelled with fluorescein isothiocyanate show that these adsorb differently on charged surfaces. Attachment of even small labels can significantly change the interaction properties of a protein. Thus, the results of experiments with fluorescently labelled proteins should be interpreted by modelling the structures and computing the interaction properties of both labelled and unlabelled species. ∗

To whom correspondence should be addressed HITS ‡ ZMBH ¶ IWR § current affiliation: Department of Global Public Health and Primary Care, and Computational Biology Unit, University of Bergen, Norway †

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Keywords protein labelling, protein–surface interactions, protein adsorption, Brownian dynamics, molecular electrostatic potential, fluorescent label


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Protein–inorganic surface interactions play an important role in nature and in various applications in biotechnology and medicine. 1–3 These interactions have been studied using a wide range of experimental and theoretical techniques aimed at understanding the basis for specific protein adsorption and thereby aiding the design of materials having desired features. Experimental studies usually provide ensemble properties of the proteins during and/or after adsorption, such as the kinetics of layer formation, and the thickness and ordering of layer(s) (for comprehensive reviews, see e.g., Refs. 4,5 ). For protein and material design, however, it is important to determine the orientation(s) in which a given protein binds to a surface. For this purpose, a range of fluorescence detection techniques have been developed (see e.g., Refs. 4,6 ). Usually the studies focus on naturally abundant proteins, such as bovine serum albumin (BSA), β-lactoglobulin, human growth hormone (hGH) or hen-egg white lysozyme (HEWL). These proteins do not have high fluorescence capabilities, and therefore fluorescently labelled proteins or mixtures of labelled and unlabelled species are used. The labels vary in shape and size 6 from small molecules (e.g., maleimide, coumarin) to complete proteins (e.g., green fluorescent protein). For determining the orientations of the adsorbed proteins, the labels should be small to avoid conformational changes upon label attachment and steric hindrance during adsorption. Many studies have been done for semi-transparent surfaces such as glass, silica, mica or silicon with various modifications, either because the method requires a certain transparency of the adsorption substrate, or because of the ubiquity of these surfaces and their relevance for protein adsorption in various nanotechnological applications. 7,8 One of the most widely used labels is fluorescein isothiocyanate (FITC), a relatively small molecular dye that can be easily attached to reactive primary amine groups, i.e., on lysine residues and at the N-terminus of proteins. The fused FITC-protein construct can then be monitored using techniques such as total internal reflection fluorescence (TIRF), 9–13 fluorescence correlation spectroscopy, 14 single-molecule visualization, 14 fluorescence recovery after photobleaching, 15 and fluorescence emission and quenching. 16 TIRF measurements of HEWL labelled with FITC (HEWLFITC) have been used to study the orientation of lysozyme on hydrophilic silica 11,13 by utilizing the decrease in fluorescence intensity of the label when placed near the surface. The silica surface


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is negatively charged, exhibiting a strong electrostatic field within a screening distance characterized by the Debye length. This distance can be manipulated in an experiment by adjusting ˚ the ionic strength (IS) of the solution. For example, at 5 mM IS, the Debye length is ca. 43 A, ˚ 3 ), thus the fluorescence which is comparable with the dimensions of HEWL (ca. 40 × 40 × 30 A intensity of the labelled protein will vary depending on its orientation when adsorbed. In such experiments, the results of measurements of the labelled proteins or mixtures of labelled and unlabelled proteins are used to draw conclusions about the native proteins. Control experiments are done to show that the label does not introduce significant deformations in the protein structure and sometimes to check that the labelled and unlabelled species have similar binding affinity to the surface used (see e.g., Refs. 4,13,17 ). Yet it has been shown experimentally, that such labels affect the physicochemical properties of the labelled proteins, such as charge and size. 18 Moreover, in some cases, label attachment has been found to affect the amount of protein adsorbed, as has been shown for lysozyme on chromatographic resins

19

and BSA on hydrophobic hydrogels. 15 It is

difficult to directly compare measurements for the “visible”, labelled species with the “invisible” unlabelled one. Here, we employ a computational approach to investigate the effect of labelling an experimentally well-characterized model protein, HEWL, with FITC on protein adsorption onto oppositely charged surfaces. Since these protein-surface systems are highly charged, we analyse their electrostatic properties. Furthermore, we perform Brownian Dynamics (BD) simulations of the diffusional association of the proteins with the surfaces, which give preferred docking orientations of the adsorbed proteins on the surfaces. We show that (a) even a small label that does not introduce changes in the protein structure can significantly influence protein-surface interactions; and (b) relatively straightforward computational methods can be used to systematically account for and understand the effects of the label when interpreting experimental data on protein–surface interactions. We calculated the molecular electrostatic potential (MEP) for the unlabelled and labelled HEWL using APBS 1.3. 20 The coordinates of HEWL were taken from the crystal structure (PDB 21 id 1HEL 22 ). We modelled the FITC label using the structures 4FAB 23 and 3Q9K from the Protein


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Already a simple visualization of the MEPs of unlabelled and labelled HEWL (Figure 1) shows that the label changes the electrostatic properties of HEWL. The total charge at pH 7 decreased from +8e for HEWL to +5e for HEWL-FITC. Moreover, the label is attached to the N-terminus of HEWL in the most positively charged region of the protein. As noted in several studies, 24–26 the adsorption of lysozyme is mainly determined by its specific surface charge distribution, creating surface patches of negative and positive charge that enable strong interactions with the charged solid surface and simultaneously minimize repulsive interactions between HEWL molecules forming adsorbed layer(s). This specific charge distribution facilitates formation of layers of HEWL on various surface types, including same-charged surfaces. 12,24 Attachment of the FITC label clearly alters this charge distribution. Next, we employed Brownian dynamics (BD) with the SDA 7 software 27,28 (http://mcm.hits.org/sda7/) to simulate the diffusion of lysozyme in the presence of negatively charged mica- and silica-like surfaces (see Supporting Information for details). The diffusion resulted in protein adsorption. We recorded and clustered these protein:surface docked complexes. The BD simulations show that HEWL and HEWL-FITC acquire different orientations when adsorbed to the negatively charged surfaces (Figure 2 A and B, and Table 1). On the more negatively charged, mica-like surface, the electrostatic attraction is the dominant force and thus, both HEWL and HEWL-FITC acquire only one preferred orientation, where they position their most positive patch towards the surface. For unlabelled HEWL, this is on the opposite side to the active site (labelled in Fig. 1), whereas for HEWL-FITC, this positive patch is weakened by the presence of the label, which also introduces steric hindrance, prohibiting the orientation observed for HEWL. Instead, HEWL-FITC preferred an orientation on the mica-like surface that positions a different positive patch close to the surface. Although both these orientations can be called “side-on” (see the orientation angle in Table 1), the N-terminus is significantly farther from the surface in HEWL-FITC and the interaction energy is less favourable than for HEWL. Clustering of the most preferred orientations of HEWL and HEWL-FITC on the mica-like surface at higher IS conditions (20 mM and 50 mM) gives little change in their orientation (see Supporting Information), showing that the difference


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different IS conditions, a second minor orientation appears for HEWL-FITC at higher IS (Figure S3). Table 1: Results of BD docking of HEWL and HEWL-FITC to mica-like and silica-like surfaces at 5 mM IS. Average energies and occupancies for each cluster, and the orientation angles of the cluster representatives are given. PROTEIN

CLUSTER

#

AVG .

OCCUPANCY TERMINUS

ENERGY

[%]

DIST.

a

˚ [A]

[kcal/mol]

ORIENTATION ANGLEb

[deg]

MICA - LIKE SURFACE

HEWL

1

−103.2 ± 0.2

100

7.5

118.4 (−0.48)

HEWL-FITC

1

−73.9 ± 0.4

100

18.0

112.3 (−0.37)

SILICA - LIKE SURFACE

HEWL

HEWLFITC

1 2 3

−6.9 ± 0.1 −6.9 ± 0.1 −7.0 ± 0.1

62 16 15

8.6 14.0 11.7

105 (−0.26) 98 (−0.14) 101 (−0.18)

1 2 3

−2.5 ± 0.3 −2.8 ± 0.4 −2.7 ± 0.4

50 35 10

15.6 25.2 20.8

114 (−0.41) 99 (−0.15) 105 (−0.26)

a

distance of the centre of mass of the N-terminal amino acid (LYS1) from the surface; angle between the normal to the surface and the vector pointing from the protein centre of mass to the centre of mass of ARG128, the C-terminal residue (labelled in Figure 1); the number in brackets is the cosine value. b

The silica-like surface has an approximately 16-fold lower charge density and therefore the electrostatic contribution of the interaction energies is not as strong as for the mica model. The BD simulations results show several orientations for HEWL and HEWL-FITC (Figure 2 C and D, and Table 1). The preferred orientations of native HEWL on silica are similar to those on mica-like surface, but somewhat tilted. The silica-adsorbed orientations in the three clusters have similar interaction energies (Table 1 and Figure S5) and it is the subtle interplay between the shortrange non-polar attraction, proportional to the contact area, and the electrostatic interaction terms that result in several possible orientations of HEWL on silica. For HEWL-FITC, the attractive electrostatic interactions with the surface are even smaller, resulting in the non-polar interactions having a stronger influence on the most preferred orientations. This is in contrast to the orientations 8 ACS Paragon Plus Environment

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of HEWL, where on both types of surface, the attractive electrostatic term is clearly dominant and the positive patches are closely interacting with the surface. Furthermore, at higher IS, the orientations of HEWL-FITC are more diverse than those of HEWL. This indicates that the surface interactions with the labelled protein are less specific than those with the unlabelled one. These single-molecule BD docking simulations can be seen as the first step of protein layer formation at low protein concentrations. Two approximations used in the simulations that can potentially affect the outcome of these simulations are: (i) the implicit treatment of water and ions, and (ii) the rigid-body treatment of the proteins. While these approximations enable efficient simulations of large systems, they introduce certain limitations. The lack of explicit water and ions makes it impossible to study any bridged contacts between the protein and the surface. However, our simulations were conducted in very low IS to match the experiments, and thus the number of ions in the entire system is negligible. Moreover, we focus on how the protein orients during its approach towards the surface, a process that is steered by the long-range electrostatic interactions, which are reliably described in our model. When the protein is close to the surface, the orientation is already optimized and any short-range interactions, including those mediated by water or ions, would likely only result in side-chain adaptation. Several theoretical studies have investigated the role of water molecules and ions at the binding interface between biomolecules and solid surfaces (e.g., Refs. 29,30 ). These works show a clearly ordered structure of ca. two water layers next to the surface. Schneider et al. 29 noticed that only some amino acids penetrate this ordered structure to form direct bonds with the surface. Penna et al. 30 performed more than 200 molecular dynamics simulations of peptides approaching a metal surface and concluded that the penetration of this ordered water layer requires several anchoring steps, which suggests an energy barrier for forming direct interactions. Since we focus here on the diffusional approach of the protein towards the surface, we can safely use the implicit water model (which accounts for the different dielectric screening of these ordered surface water layers) keeping in mind that our docked complexes represent only intermediates after the first stage of a complete adsorption process.


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As mentioned, our BD model includes atomically-detailed but rigid molecules. We consider this a reliable approximation, since HEWL is a small globular protein with four disulphide bridges stabilizing its 3D structure. Additionally, it has a high pI of ca. 11, resulting in strong electrostatic interactions between the negatively charged surface and the surface residues of lysozyme. It was shown that with decreasing pH 31 and increasing IS, 32 the structure of lysozyme becomes more compact and rigid. While some molecular dynamics (MD) studies indicate deformation of the protein under certain conditions, 33 many experiments and simulations have shown that HEWL does not denature upon adsorption to silica or mica. 13,34–36 In the BD simulations, the orientation in which the proteins associate with and bind to the surface is obtained while neglecting any conformational changes that may occur after the diffusional association. The orientations of the adsorbed proteins from Brownian dynamics simulations can reasonably be compared with experimental data since the native HEWL was shown to be stable when adsorbed at pH 7. 37,38 MD simulations of a few (up to three) HEWL molecules in the presence of a model mica surface 34,39 showed that the major adsorption site included residues LYS1, ARG5, ARG125 and ARG128 (labelled in Figure 1). Apart from these residues, ARG14, ARG21, ARG114 and GLN121 were also found to interact specifically with the mica model. Comparison of the contacting residues from our docking results with these MD simulations is shown in Figure 3. It can be noted that while the orientations of HEWL give a similar pattern of contacting residues to those reported in the MD 34,39 studies, the pattern of contacts of all the HEWL-FITC orientations is shifted. This shows that significant changes in surface binding are introduced by attaching the FITC label to HEWL. Moreover, this emphasizes the reliability of our approach, namely that the rigid-body BD with implicit solvent treatment gives a good description of the initial steps of protein adsorption. Dismer and Hubbuch investigated the preferred orientation of HEWL on two highly negatively 40 charged chromatography ion-exchange resins containing SO− The experiment involved 3 groups.

labelling HEWL in the absence and the presence of the resins and comparing the labelling patterns. The results showed that LYS33 is the most protected residue when HEWL is adsorbed on the resin surfaces, whereas LYS97 has the same or higher probability of being labelled on the resins as


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low IS, the electrostatic attraction is the main driving force for HEWL adsorption and the surface coverage stays at a similar level at a range of pH values. A neutron reflection study of HEWL adsorbing to silica 32 showed that when using a low protein concentration (0.03 g/L), the adsorbed ˚ for the IS range 0.02–1.0 M at pH 7. This indicates that layer thickness stays constant at ca. 30 A even at higher IS than used in our simulations, HEWL does not deform in the presence of silica surface. Moreover, the constant layer thickness measured in this experiment means that the orientation of HEWL stays the same with increasing IS, while the attractive interactions between silica and lysozyme weaken. This suggests that the difference between the orientations of HEWL-FITC and HEWL we observe in the simulations is general. In a recent study, Mathé et al. analysed over a hundred yeast proteins with regard to their binding affinity to a silica surface. 42 Their conclusion was that the proteins that preferentially bound to silica, expelling the other proteins, had more polar residues (mainly positively charged lysine and arginine) and fewer aromatic residues. This further supports our observation that the addition of an aromatic negatively charged label like FITC diminishes the affinity of HEWL to a silica surface. To conclude, through computational approaches, we show that even a relatively small label attached to a protein can significantly change the way it interacts with surfaces while adsorbing, even though the structure of the protein may remain undistorted. While the results presented here focus on HEWL adsorbing to oppositely charged surfaces, we argue that the implications of our study go beyond this specific system, as shown in several experimental works that investigate the influence of label attachment on proteins. We stress that the interpretation of experimental data on protein adsorption from such labelled proteins should be done by modelling and simulating both the labelled and unlabelled species. We show that using Brownian Dynamics and electrostatic potential calculations, one can easily identify and quantify changes in interactions and binding due to the attachment of a label when there are significant electrostatic interactions between the protein and the surface.


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Acknowledgement The authors thank Dr. Michael Martinez for programming help, and Dr. Anna Feldman-Salit and ¨ Musa Ozboyaci for critical reading of the manuscript. They gratefully acknowledge the support of the European Molecular Biology Organization (EMBO), Heidelberg Institute for Theoretical Studies (HITS), and the Klaus Tschira Foundation (KTS).

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Supporting Information Available Supporting Information contains the details of the methods, and figures and tables describing the BD docking results for different ionic strength conditions. This material is available free of charge via the Internet at http://pubs.acs.org/.


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When the Label Matters: Adsorption of Labeled and Unlabeled

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