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Direct Measurements of the Crowding Effect in Proteins by Means of Raman Optical Activity Evelien Van de Vondel, Carl Mensch, and Christian Johannessen J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 19 Jan 2016 Downloaded from http://pubs.acs.org on January 19, 2016

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The Journal of Physical Chemistry

Direct Measurements of the Crowding Effect in Proteins by Means of Raman Optical Activity Evelien Van de Vondel, Carl Mensch and Christian Johannessen* Department of Chemistry, University of Antwerp, Groenenborgerlaan 171 2020 Antwerpen, Belgium, telephone: 00322653505; e-mail: [email protected]

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ABSTRACT: The effect of crowding interactions on the structure of dephosphorylated α-casein was studied with Raman optical activity (ROA). It was found that ROA is sensitive to the structural changes in the protein, induced by the presence of crowding agents. This effect depends on the employed crowding agent and its concentration.


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INTRODUCTION Proteins have long been considered the most important group of biomacromolecules, due to their diversity in structure and function. Because of this diversity, but also because of the link between malfunctioning proteins and high-profile diseases, such as Alzheimer’s and Parkinson’s diseases, it is imperative to understand the behavior of proteins in realistic cellular environments. Traditional methods in molecular biology measure the activity of proteins in vitro under socalled physiological conditions, e.g. very low concentration. However, in these studies, the overall high concentration of biomacromolecules present (300-400 mg mL-1, depending on cell type) in the cell is often ignored.1 The influence of these biomacromolecules is described by macromolecular crowding theory.1 Crowding theory states that the space that macromolecules occupy is mutually impenetrable. This assumption induces a non-specific steric repulsion, rendering part of the intracellular volume unavailable for other macromolecules. The size of this excluded volume depends on the number, sizes and shapes of the macromolecules in the background, and this exclusion of volume should hence force molecules with a flexible structure, such as a large proportion of proteins, in a more compact structure.2 Previously, studies on the influences of crowded environments on the structure of proteins have been carried out. Traditionally, electronic circular dichroism (ECD) and NMR are used to this end.3-4 However, the detailed interpretation of ECD and NMR studies of crowding interactions is difficult and often induces an ambiguous interpretation of the data at hand. In this study, we employ the chiroptical spectroscopic technique Raman optical activity (ROA) to study the effects of crowding conditions on proteins. It is well documented that ROA exhibits unprecedented sensitivity towards distinguishing and identifying local structure in proteins, why


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the technique is suited to perform this type of experiment.5-7 However, due to the complicated composition of samples used in crowding studies, ROA studies of proteins in crowded conditions have not previously been reported. Hence, this study is the first to show the full potential of ROA as a tool in crowding studies.

EXPERIMENTAL METHODS All samples were supplied by Sigma-Aldrich ®. The Raman and ROA spectra were measured at ambient temperature in deionised water using the previously described ChiralRAMAN-2X instrument (BioTools, Inc.),8 which employs the scattered circular polarization (SCP) measurement strategy. The ROA spectra are presented as circular intensity differences (IR-IL) with IR and IL denoting the Raman intensities with right- and left-circular polarization, respectively, whereas the Raman spectra are presented as the sum of these polarized components. The spectrometer is wave-length calibrated by measuring a neon-arc lamp and band aligning to premeasured standard. The sample concentrations were ca. 60 mg mL-1 for dephosphorylated αcasein (dP-Acas) and ca. 300 mg mL-1 or 150 mg mL-1 of Ficoll 70, dextran 70 and sucrose. All reagents were dissolved in deionised water. Experimental conditions: laser wavelength 532 nm; laser power at the source 400 mW; spectral resolution 7 cm-1; acquisition times 72 h for each sample. Cosmic ray spikes were removed from the ROA spectra by means of a median filter, after which a spectrum of the mixture of crowding agent and protein was aligned with a spectrum of the crowding agent alone in solution by normalizing to a non-variable sugar band. Next, the spectrum of the crowding agent was subtracted from the spectrum of the crowding agent and protein together in solution. The resulting spectra were filtered by a third-order nine-point Savitzky-Golay filter. All these treatments were always critically evaluated in MATLAB ®. All


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data was reproduced. The CID values were calculated by dividing the absolute intensity of the ROA-signal for the wavenumber by the intensity of the corresponding Raman signal for the same wavenumber over a range. Subsequently, the average of these values was taken for the studied spectral region (Amide III: 1231 cm-1-1340 cm-1 and Amide I: 1630 cm-1- 1700 cm-1).

RESULTS AND DISCUSSION The effect of highly crowded environments on the protein dephosphorylated α-casein (dPAcas) is shown in Figure 1.


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Figure 1. ROA spectra of an aqueous solution of dephosphorylated α-casein (A), and in the presence of 300 mg mL-1 co-solute Ficoll 70 (B), dextran 70 (C) and sucrose (D).

This protein was chosen as a suitable case study for crowding due to the fact that it is a commercially available intrinsically disordered protein, hence lacking traditional structural elements, and belonging to the same class of proteins as tau and α-synuclein, which are involved in neurodegenerative diseases. The Raman and ROA spectra of dP-Acas have been reported before.7,9-10 In both Jarvis et al. and Ashton et al. a comparison of the ROA spectrum of dP-Acas vs. the phosphorylated form of the protein were performed, and both articles conclude that while dephosphorylation may induce small structural changes in the backbone of α-casein, the overall structural content (poly proline II) remains the same upon dephosphorylation.7,9 In the present crowding studies, the concentration of crowding agents was chosen to be 300 mg mL-1. In this case, the overall concentration of macromolecules (being the protein plus the crowding agent) is near the theoretical maximum of a cellular environment.1 Corresponding Raman spectra were collected simultaneously for all measurements. As Raman spectroscopy is less sensitive to backbone conformation compared to ROA, these spectra differ only with respect to their base lines, and can be found in Figure S1 and S2 in the supplementary information. The bottom spectrum in Figure 1 is that of dP-Acas in solution without crowding agents, and shows spectral features traditionally associated with structural disorder. This spectrum exhibits the same features as the previously published spectra of dP-Acas, why it is reasonable to assume a similar overall structure of these samples.7,9-10 The amide I region exhibits a broad positive feature, with a maximum at 1677 cm-1, typically assigned to disordered proteins.11 The amide III region supports this disordered nature, as the signal at 1320 cm-1 is typically assigned to PPII helix.12 However, the protein seems to possess a small amount of α-helical structure, as


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suggested by the couplet from 1095 cm-1 to 1125 cm-1 in the skeletal stretch region. The second panel from the bottom in Figure 1 presents the spectrum of dP-Acas in a crowded environment, where Ficoll 70 (a sucrose-based polymer) was used as the crowding agent. The resulting spectrum differs significantly from the spectrum of the uncrowded protein. The amide I contains an extra, negative contribution at 1639 cm-1. The resulting couplet-like feature suggests an overall important contribution of α-helical structure. The broadness of this feature suggests however that part of the protein remains disordered. This partial shift towards α-helical structure of the protein appears to be supported by the amide III region, where the PPII characteristic at 1320 cm-1 remains but new features also appear: the new band at 1352 cm-1 can be assigned to α-helical structure. Furthermore, the band at 1381 cm-1 suggests the presence of tight turns in the protein11, hence also indicating a more compact structure, with a higher content of classical secondary structure elements. As these results imply that crowding interactions do occur in the test system, the experiment was repeated with dextran 70 (a glucose-based polymer) as crowding agent. This way, any specific (i.e. non-crowding) interactions between Ficoll 70 and dP-Acas can be identified. Spectral differences do emerge due to the presence of dextran 70 compared to the situation in uncrowded environment, but these induced changes differ from those in the presence of Ficoll 70. In the amide I region of the sample with dextran 70 as co-solute, an extra negative contribution is observed, but the distinction between the couplet, suggesting α-helical structure, and the positive feature, linked to disordered structure, is more prominent. Furthermore, the new bands in the amide III region appear to be tighter, smaller and redshifted compared to the Ficoll 70 sample. Also, the amide III region contains fewer features, as for example the band at 1282 cm-1 is not present in this spectrum. The remaining features suggest a tightening of the structure


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toward α-helical structure compared to the structure of the protein in solution without crowding agents. These arguments implicate that the overall structural transition induced by dextran 70 differs from the one induced by Ficoll 70. It appears that dextran 70 and Ficoll 70 both induce a tighter structure in the protein, going towards more α-helical elements, but Ficoll 70 induces further structural changes. It is therefore reasonable to assume that the effect of Ficoll 70 is of a more specific nature, rather than solely based on non-specific size-exclusion. This hypothesis is confirmed by the spectrum of dP-Acas in the presence of a similar concentration of sucrose, the monomer unit of Ficoll 70, presented in the top panel of Figure 1. As can be seen from Figure 1, the amide I region of dP-Acas in sucrose only shows the broad positive feature associated with disordered proteins. However, this band at 1675 cm-1 appears to be sharper than the corresponding band in the spectrum of the protein without co-solute. This could indicate that the structure of the protein is more conformationally confined, although it remains disordered. This seems to be confirmed by the amide III region, where broad features, not present in the spectrum of dP-Acas in solution, appear. These bands at 1323 cm-1, 1350 cm-1 and 1386 cm-1 are broader than the corresponding bands in the spectrum of the protein in solution with Ficoll 70 and are an important observation, as sucrose should have no crowding effect on dP-Acas. Additionally, in the amide III region, the shoulder at 1288 cm-1, which was missing in the spectrum of the protein in solution with dextran 70, appears. The skeletal stretch region remains similar to the one of the protein in solution without co-solutes. From these results, it is concluded that the overall influence of Ficoll 70 on the structure of proteins is a sum of two types of interactions, one similar to the influence of dextran 70 and the other similar to the influence of sucrose. The latter interaction was previously suggested by


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Benton et al, who found that sucrose and Ficoll have approximately the same stabilizing effect on chymotrypsin inhibitor 2.13 As a control study, the protein hen egg white lysozyme (HEWL) was also studied under crowding conditions, see Figure S3 in the supplementary information. As can been seen from Figure S3, there are some minor differences in the uncrowded vs. crowded ROA spectra of HEWL, indicating a restriction of the conformational fluidity of the protein, though without any actual changes in secondary structure propensity, which is observed in the case of dP-Acas. This further supports the notion that intrinsically disordered proteins can obtain a more ordered and compact structure in a cellular environment, detectable by ROA, while already ordered proteins are less affected by crowding.


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Figure 2. ROA spectra of dephosphorylated α-casein alone in solution (A), and in the presence of 150 mg mL-1 Ficoll 70 (B), dextran 70 (C) and sucrose (D).

The experiment was repeated with a concentration of 150 mg mL-1 of crowding agent study the specific nature of the interaction of Ficoll 70 with dP-Acas in more detail. With a similar concentration of dP-Acas as before, the overall concentration of macromolecules is still above the theoretical minimum to study crowding effects, i.e. ~ 100 mg mL-1.1 The effect of these crowded environments is shown in Figure 2. The bottom spectrum in Figure 2 is again that of dP-Acas in solution without crowding agents. At lower concentration of crowding agent, the spectral changes induced by Ficoll 70 (seen in the second spectrum from the bottom) are smaller, but still present. In the amide I region a negative contribution emerges, resulting in a couplet negative at 1637 cm-1 and positive at 1670 cm-1. This couplet, similar to that at a higher concentration of Ficoll 70, suggests the presence of some α-helical structure in the protein, however the signal seems to contain a contribution of disordered structure as well, rendering it broad. The most dominant feature in the amide III region is the PPII-signal at 1318 cm-1, suggesting that the protein is disordered overall. The shoulder at 1357 cm-1 indicates a contribution of α-helical structure, but is not as prominent as in the spectrum in the presence of 300 mg mL-1 Ficoll 70. The band at 1381 cm-1 does not seem to return in this spectrum, while a contribution of unknown nature appears at 1291 cm-1. This contribution lies between the two bands at 1282 and 1307 cm-1, in the spectrum of the protein in the presence of Ficoll 70 at 300 mg mL-1. However, no conclusive spectral assignments can be made at this point. The backbone region contains a feature with a negative contribution at 1084 and a positive contribution at 1136 cm-1, suggesting an α-helical contribution.


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In the amide I region of the sample with 150 mg mL-1 of dextran 70 as a co-solute (seen in the second spectrum from the top in Figure 2), a couplet is observed, suggesting α-helical structure, although the positive part of the coupled, linked to disordered structure, remains more prominent. In the amide III region the feature at 1386 cm-1, indicating tight turns, is present, while it was not observed in the spectrum in the presence of 150 mg mL-1 Ficoll 70. The other features in the amide III region are also more pronounced, indicating bigger structural differences in the case of dextran 70 crowding compared to the spectrum measured with 150 mg mL-1 Ficoll 70 as a cosolute. These results suggest that the crowding interaction of dextran 70 is stronger than the one of Ficoll 70 at this concentration, implying that the interaction of sucrose units is competitive with the crowding interaction. This assumption is confirmed by the spectrum of dP-Acas in the presence of 150 mg mL-1 sucrose, where the structural differences are also more pronounced than in the spectrum with 150 mg mL-1 Ficoll 70 as co-solute. The amide I region only contains the marker of a disordered protein, at 1677 cm-1. In the amide III region, the small positive band at 1303 cm-1 and at 1347 cm-1 indicate the presence of some α-helical structure. Finally, the couplet in the skeletal stretch region negative at 1096 cm-1 and positive at 1128 cm-1 confirms the α-helical contribution, similar to that one of the uncrowded protein. The overall changes in the spectrum are however smaller than the ones in the spectrum in the presence of dextran 70, indicating that the structural effect, induced by the presence of sucrose, is smaller in this set of experiments. At this concentration, crowding with an achiral crowding agent, polyethylene glycol (PEG 200), was also attempted in order to rule out residual ROA signals from the subtraction procedure (see experimental details in the supplementary information). Unfortunately, the highly anisotropic


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nature of the PEG 200 molecules in solution induces polarized Raman artefacts to occur in the ROA spectra, rendering these non-reproducible. To verify the accuracy of the presented results, the dimensionless circular intensity difference (CID) was calculated for each of the spectra in the amide I and the amide III region, as shown in table 1. All CID’s have a magnitude of 10-4, which is consistent with ROA theory.8 As seen in Figure S4 in the supplementary information, the protein bands and the subtracted sugar bands can be distinguished by intensity and region of appearance, hence validating the witnessed signals in the subtracted spectra. Table 1. CID Values for the Described Measurements Measurement CID Amide I

CID Amide III

dP-Acas

2.6 10

-4

1.9 10-4

dP-Acas in 300 mg mL-1 Ficoll 70

1.3 10-4

3.6 10-4

dP-Acas in 300 mg mL-1 dextran 70

1.1 10-4

3.4 10-4

dP-Acas in 300 mg mL-1 sucrose

1.3 10-4

6.2 10-4

dP-Acas in 150 mg mL-1 Ficoll 70

0.8 10-4

4.0 10-4

dP-Acas in 150 mg mL-1 dextran 70

0.7 10-4

5.1 10-4

dP-Acas in 150 mg mL-1 sucrose

6.6 10-4

3.4 10-4

CONCLUSIONS From the data presented above, we conclude that crowded environments have an effect on the structure of dP-Acas, and that this structural difference can be identified by means of ROA. This primary finding is an important step towards understanding the behavior of IDP’s under true physiological conditions, as our results indicate that the structure of this class of proteins could be influenced by crowding. Additionally, the results show that the induced structural difference depends on the concentration of the crowding agent. As this concentration has an influence on


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the amount of excluded volume, this conclusion is in line with the theory by Minton.1 Here, it is important to note that the observed changes in the ROA spectra of dP-Acas are unlikely to be due to the induction of long-chained secondary structure elements, but rather local changes in the back bone torsion angles, creating “pockets of structure” in the protein. Hence, these changes are unlikely to be observed by means of Raman or IR spectroscopy, but can be picked up by ROA. Furthermore, the effect also depends on which crowding agent is used in the experiment. There is a clear difference between the crowding agent based on sucrose monomer (Ficoll 70), and the glucose-based crowding agent employed in this study (dextran 70). Ficoll 70 seems to induce additional structural change, implying that the overall response is a combination of the crowding effect, where the interaction is similar to the one induced by dextran 70, and a specific interaction between the protein and sucrose. It is known that sugars, and especially sucrose, can stabilize a protein in solution.14 Hence, it is therefore reasonable to assume that this stabilizing effect and the observed structural changes are based on the same interaction. Lee and Timasheff proposed in 1981 that this stabilization by sucrose, is based on the preferential exclusion of sucrose from the interior of the protein, increasing the free energy of the system. Thermodynamically this leads to protein stabilization since the unfolded state of the protein becomes even less favourable in the presence of sucrose.15 It is important to consider these specific interactions in research on crowding interactions, as most commercially available crowding agents are sugar-based and therefore could cause unwanted structural perturbations in the studied proteins. Overall, we conclude that ROA is a promising tool in studying crowding effects in proteins, as the information content in the spectra gives unparalleled access to detailed interpretation of structural changes. Structural changes due to the crowding effect, which are dependent on the


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concentration of the crowding agent, can be observed directly with ROA. All the crowding agents employed in this study induce a similar structural change, though Ficoll 70 combines this effect with an interaction between the sucrose units and the protein.

AUTHOR INFORMATION Corresponding Author * Department of Chemistry, University of Antwerp, Groenenborgerlaan 171 2020 Antwerpen, Belgium, e-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. All authors contributed equally. ACKNOWLEDGMENT The authors acknowledge the Agency for Innovation by Science and Technology in Flanders (IWT) for the pre-doctoral scholarship of E.V.d.V, the University of Antwerp (BOF-NOI) for the pre-doctoral scholarship of C.M. and the University of Ghent (IOF Advanced TT) for the purchase of the ChiralRaman spectrometer. Supporting

Information

Available: Raman

spectra of an

aqueous

solution

of

dephosphorylated α-casein (A), and in the presence of 300 mg mL-1 co-solute Ficoll 70 (B), dextran 70 (C) and sucrose (D); Raman spectra of an aqueous solution of dephosphorylated αcasein (A), and in the presence of 150 mg mL-1 co-solute Ficoll 70 (B), dextran 70 (C) and sucrose (D); ROA spectra of an aqueous solution of hen egg white lysozyme (HEWL), and in the presence of 300 mg mL-1 co-solute Ficoll. The differences in these spectra indicate a stabilization


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of this structured protein, though without the big structural differences, as witnessed in the case of unstructured proteins; ROA spectra of an aqueous solution of 300 mg mL-1 Ficoll 70 (A), dPAcas in the presence of 300 mg mL-1 Ficoll 70 (B), and the resulting subtraction (C). The difference in band intensities can be accessed by comparing the intensity of the amide I region of the protein (1600-1700 cm-1, where no sugar bands are present) and the intense sugar bands below 1500 cm-1. This material is available free of charge via the Internet at http://pubs.acs.org.


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REFERENCES 1. Ellis, R. J. Macromolecular Crowding: Obvious but Underappreciated. Trends Biochem. Sci. 2001, 26, 597-604. 2. Minton, A. P. How can Biochemical Reactions within Cells Differ from those in Test Tubes? J. Cell Sci. 2006, 119, 2863-2869. 3. Flaugh, S. L.; Lumb, K. J. Effects of Macromolecular Crowding on the Intrinsically Disordered Proteins c-Fos and p27 Kip1. Biomacromolecules 2001, 2, 538-540. 4. Li, C.; Charlton, L. M.; Lakkavaram, A.; Seagle, C.; Wang, G.; Young, G. B.; Macdonald, J. M.; Pielak, G. J. Differential Dynamical Effects of Macromolecular Crowding on an Intrinsically Disordered Protein and a Globular Protein: Implications for In-Cell NMR Spectroscopy. J. Am. Chem. Soc., Chem. Comm. 2008, 130, 6310-6311. 5. Barron, L. D. Structure and Behavior of Biomolecules from Raman Optical Activity. Curr. Opin. Struct. Biol. 2006, 16, 638-643. 6. Zhu, F.; Isaacs, N. W.; Hecht, L.; Barron, L. D. Raman Optical Activity: A Tool for Protein Structure Analysis. Structure 2005, 13, 1409-1419. 7. Ashton, L.; Johannessen, C.; Goodacre, R. The Importance of Protonation in the Investigation of Protein Phosphorylation using Raman Spectroscopy and Raman Optical Activity. Anal. Chem. 2011, 83, 7978-7983. 8. Barron, L. D.; Buckingham, A. D. Vibrational Optical Activity. Chem. Phys. Lett. 2010, 492, 199-213. 9. Jarvis, R. M.; Blanch, E. W.; Golovanov, A. P.; Screen, J.; Goodacre, R. Quantification of Casein Phosphorylation with Conformational Interpretation using Raman Spectroscopy. Analyst 2007, 132, 1053-1060.


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10. Byler, D. M.; Farrell, H. M. Jr.; Susi, H. Raman Spectroscopic Study of Casein Structure. J. Dairy Sci. 1988, 71, 2622-2629. 11. Barron, L. D.; Hecht, L.; Blanch, E. W.; Bell, A. F. Solution Structure and Dynamics of Biomolecules from Raman Optical Activity. Prog. Biophys. Mol. Biol. 2000, 73, 1-49. 12. Smyth, E.; Syme, C. D.; Blanch, E. W.; Hecht, L.; Vaák, M.; Barron, L. D. Solution Structure of Native Proteins with Irregular Folds from Raman Optical Activity. Biopolymers 2000, 58, 138-151. 13. Benton, L. A.; Smith, A. E.; Young, G. B.; Pielak, G. J. Unexpected Effects of Macromolecular Crowding on Protein Stability Unexpected Effects of Macromolecular Crowding on Protein Stability. Biochemistry 2012, 51, 9773-9775. 14. Arakawa, T.; Timasheff, S. N. Stabilization of Protein Structure by Sugars. Biochemistry 1982, 21, 6536-6544. 15. Lee, J. C.; Timasheff, S. N. The Stabilization of Proteins by Sucrose. J. Biol. Chem. 1981, 256, 7193-7201.


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1 2 0 0

1 3 0 0


w a v e n u m b e r/c m

1 4 0 0

-1

1 5 0 0

1 6 0 0

1 7 0 0

1 8 0 0

Direct Measurements of the Crowding Effect in ... - ACS Publications

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