Relevance of Internal Friction and Structural Constraints for the

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Biophysical Chemistry, Biomolecules, and Biomaterials; Surfactants and Membranes

Relevance of Internal Friction and Structural Constraints for the Dynamics of Denatured Bovine Serum Albumin Felix Ameseder, Aurel Radulescu, Olaf Holderer, Peter Falus, Dieter Richter, and Andreas M. Stadler J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.8b00825 • Publication Date (Web): 24 Apr 2018 Downloaded from http://pubs.acs.org on April 25, 2018

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The Journal of Physical Chemistry Letters

Relevance of Internal Friction and Structural Constraints for the Dynamics of Denatured Bovine Serum Albumin Felix Ameseder†, Aurel Radulescu‡, Olaf Holderer‡, Peter Falus$, Dieter Richter† and Andreas M. Stadler†,* †

Jülich Centre for Neutron Science JCNS and Institute for Complex Systems ICS, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany

‡

Jülich Centre for Neutron Science JCNS, Forschungszentrum Jülich GmbH, Outstation at MLZ, 85747 Garching, Germany $

Institut Laue-Langevin, 71 avenue des Martyrs, 38000 Grenoble, France

AUTHOR INFORMATION Corresponding Author *[email protected]

ACS Paragon Plus Environment

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The Journal of Physical Chemistry Letters 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

A general property of disordered proteins is their structural expansion that results in a high molecular flexibility. Structure and dynamics of bovine serum albumin (BSA) denatured by guanidinium hydrochloride (GndCl) were investigated using small-angle neutron scattering (SANS) and neutron spin-echo spectroscopy (NSE). SANS experiments demonstrated the relevance of intra-chain interactions for structural expansion. Using NSE experiments, we observed a high internal flexibility of denatured BSA in addition to centre-of-mass diffusion detected by dynamic light scattering. Internal motions measured by NSE were described using concepts based on polymer theory. The contribution of residue-solvent friction was accounted for using the Zimm model including internal friction (ZIF). Disulphide bonds forming loops of amino acids of the peptide backbone have a major impact on internal dynamics that can be interpreted with a reduced set of Zimm modes.

TOC GRAPHICS


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The term ‘protein folding’ derives from the field of structural biology describing the formation of a functional 3-dimensional structure of a polypeptide chain consisting of amino acids. Motions of the disordered peptide chain are considered essential for protein folding as they explore the conformational space during the folding process.1,2 The investigation of structural and dynamic properties of disordered and unfolded proteins, therefore, is an important aspect for a detailed molecular understanding of the basic physical characteristics of the unfolded protein.3 Dynamics of the highly denatured state at the first stage of folding is of special interest. Unfolded chain dynamics have been studied by single-molecule fluorescence correlation spectroscopy (FCS) depending on the presence of denaturant, and the experimental data have been interpreted by means of polymer theory.4 Dynamics of classical soft matter systems - such as polymers - have been investigated using neutron spin-echo spectroscopy (NSE) in the past.5 NSE has been used recently for the investigation of domain motions in folded multi-domain proteins or for motions of disordered proteins as well.6,7 NSE probes collective motions on the 100 ns time-scale up to the µs in the small-angle scattering range. Small-angle neutron scattering (SANS) experiments provide information on the overall size of the protein as well as on local peptide chain structure. NSE experiments directly probe the dynamic structure factor and are sensitive to the full mode spectrum, whereas FCS experiments, for example, are sensitive to motions of the comparatively bulky dye labels.

As a prerequisite for further studies on the dynamics of denatured BSA using NSE, we characterized the structural aspects of the protein under different denaturing conditions. Figure S1 in supporting information (SI) summarizes the results of circular dichroism (CD)


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measurements of the protein samples. To break disulphide bridges the reducing agent mercaptoethanol (β-met) is added to GndCl solutions. CD data confirm that BSA in 6 M GndCl (BSA6M), BSA in 4 M GndCl & 150 mM β-met (BSA4Mβ), and BSA in 6 M GndCl & 150mM β-met (BSA6Mβ) has lost its α-helical content and is in the fully denatured state under those solvent conditions. Structural properties of BSA samples in these solvent conditions have been further investigated using SANS. Structural parameters determined by SANS are listed in table 1. A typical SANS concentration series and structure factors of 3% w/v solutions used for NSE are given in figure S2.

Table 1: Structural parameters determined from SANS experiments. Guinier radii RG,Debye and RG of the generalized Debye function and of the Guinier approximation, power law scaling coefficient ν. *BSA in 6 M GndCl forms a dimer. Denaturant

State

RG (Å)

RG,Debye (Å)

ν

0 M GndCl

Folded

37.8 ± 1.2

-

-

4 M GndCl & Denatured, 150 mM β-met no disulphide bonds

105.8 ± 2.9

102.4 ± 1.8

0.54± 0.03

6 M GndCl & Denatured, 150 mM β-met no disulphide bonds

114.2 ± 1.1

112.7 ± 2.1

0.55 ± 0.04

113.3 ± 1.0*

113.2 ± 1.4*

0.51 ± 0.03

6 M GndCl

Denatured, intact disulphide bonds

In figure 1 SANS data measured on KWS-2 at the Heinz Maier-Leibnitz Zentrum (MLZ) of native and denatured BSA at infinite dilution are displayed. The scattering pattern of native BSA


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can be described as a mixture of monomer and dimer using the theoretical form factor of BSA calculated from the known crystal structure (pdb 4f5s), see SI for details. For the interpretation of SANS data of unfolded BSA we used the form factor of a linear chain with excluded volume effects as a generalized form of the Debye function.8,9 The values for the Guinier radii RG,Debye and RG of the generalized Debye function and of the Guinier approximation, respectively, are given in table 1. Later ones are used for further NSE analysis.

Figure 1. SANS data of native and denatured BSA at infinite dilution. (a,b,c) Log-log plots of native BSA (circles), BSA in 4 M GndCl & 150 mM ß-met (squares), BSA in 6 M GndCl &150 mM ß-met (diamonds), and BSA in 6 M GndCl (triangles). The measured intensities of native BSA are fitted with the calculated form factor of the crystal structure (pdb 4f5s); the measured intensities of denatured BSA are fitted with the Debye equation for a generalized Gaussian chain. (d) Reduced Kratky plot of native BSA, denatured BSA in 6 M GndCl, and 6 M GndCl 150mM ß-met, respectively.


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Increase of GndCl concentration from 4 M to 6 M in the presence of 150 mM ß-met results in a structural expansion of denatured BSA. At a first glance, the presence of disulphide bridges in BSA6M does not appear to reduce the Guinier radius significantly as compared to BSA6Mß. However, scrutinizing the measured forward scattering, we observe that I(q=0)=0.12 cm-1 of BSA6M is twice as high as that of BSA6Mß with I(q=0)=0.06 cm-1. Thus, BSA6M is predominately forming dimers. For further NSE analysis the Rg value of BSA6M is weighted with a prefactor of 1/ √2. Power law scaling behavior of the SANS data at high q-values with

I(q)˜q-1/ν reports on chain statistics of denatured BSA. The values of ν of denatured BSA, see table 1, are in between the theoretical values of ν = 0.50 of ideal Gaussian chains and ν = 0.59 of a random chain with excluded-volume interactions. Figure 1d) shows data of BSA6M, and BSA6Mß as a reduced Kratky plot. Directly visible in the Kratky plot as well is that scattered intensities of BSA6Mß without disulphide bridges decay less steep than BSA with active sulfur bonds in BSA6M reflecting the change of statistics from excluded-volume to ideal Gaussianlike. Our observations can be compared to previous SANS experiments on phosphate glycerate kinase (PGK) denatured by high concentration of GndCl.10,11 In contrast to BSA, no crosslinking disulphide bridges exist in PGK. The structure of denatured PGK was described as random coil with excluded-volume interactions, which is also the case for denatured BSA without disulphide bridges.

Figure 2 shows the experimental NSE data of 3% w/v BSA solutions measured either on JNSE at MLZ or on IN15 at the Institut Laue-Langevin (ILL) together with the calculated dynamic structure factors based on different polymer theory models. The spectra were first fitted with a stretched exponential function I(q,t)/I(q,t=0)=A*exp(-(Γt)β) where Γ represents the process


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rate, and ß as a stretching exponent. Plots of Γ and ß as function of the scattering vector are reported in figure S3 in SI. A mean ß averaged over high q values of 0.73 ± 0.03 is found for BSA6Mß. For denatured BSA4Mß is 0.86 ± 0.07. For BSA6M with disulphide bridges an average value of β = 0.87 ± 0.02 is found being significantly less stretched than in the presence of ß-met indicating a different dynamic behavior that is caused by structural restrictions of disulphide bonds as we will see further below. All values are close to β = 0.85 as predicted by Zimm theory for a Gaussian chain in solution.5


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Figure 2. Experimental NSE data of denatured BSA and fits based on polymer theory. Zimm model (dashed lines), ZIF model (dotted lines), and Zimm model with reduced mode amplitudes (solid lines). Data measured on (a) J-NSE, (b) and (c) on IN15. (a) BSA in 4 M GndCl & 150


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mM β-met, (b) BSA in 6 M GndCl & 150 mM β-met without sulfur bonds and (c) BSA in 6 M GndCl with active sulfur bonds.

Figure 3. (a) Effective diffusion coefficient measured with NSE of the 3% BSA solutions and (b) corrected for interparticle and hydrodynamic interactions. Straight dashed lines represent linear regressions of IN15 data. Values for translational diffusion Dt measured by DLS of (a) the 3% BSA solutions and (b) at infinite dilution are displayed at q=0 (empty symbols).

The effective q-dependent diffusion coefficients Deff(q) were determined using a cumulant expression I(q,t)/I(q,t=0)=A*exp(K1t+0.5K2t2) with Deff(q) = -K1/q2 from the NSE spectra. The values of Deff are given in figure 3. Corrected values for interparticle and hydrodynamic interactions D0(q) = Deff(q)*S(q)/Ht are shown in figure 3 in comparison with the translational


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diffusion coefficient Dt measured by DLS at infinite dilution. The hydrodynamic function Ht(q) was approximated to be q-independent and Ht was determined experimentally at q0 Å-1 according to Ht=D3%*S3%/D0,7 where D3% and D0 are the diffusion coefficients of the 3% protein solution and at infinite dilution measured by DLS, respectively, and S3% is the structure factor at the DLS value of q=0.0026Å-1 (see SI, figure S2 and table S1). For BSA6Mβ the D0(q) follow a linear dependence as expected from Zimm theory from polymer physics12 and intersect Dt measured by DLS (fig. 3). Intact disulphide bridges in BSA6M results in significantly lower D0(q) values and do not extrapolate to Dt from DLS for q 0 Å-1.

To interpret the measured NSE spectra the Zimm model from polymer theory was used in a first approach.12 The Zimm model describes the diffusive motions of a finite chain consisting of N beads with uniform bond length l including hydrodynamic interactions between the beads. Solving the Langevin equation yields relaxation modes with mode number p and characteristic relaxation time τp given by τ =

√

, with η as the solvent viscosity measured

independently, and R = (2ν + 1)(2ν + 2)R as the average end-to-end distance of the chain.8 The values from the small-angle power law scaling coefficient = 0.54 and = 0.55

are used and give RE = 26.8 nm and 29.1 nm for first Zimm relaxation times τ1 of 2212 ns and 3760 ns for BSA4Mß and BSA6Mß, respectively. For BSA6M we used = 0.51 with RE = 22.2 nm and the first Zimm relaxation time τ1 is then 1664 ns. The dynamic structure factor of the Zimm model is given by @ABC

4R)+ .() πpn πpm = B(n, m, t) = (n − m)) l) + ) - )01 cos 5 7 cos 5 7 91 − exp ? p N 8 τ π @D1


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I(q, t) =

GHIJ K LMN OP Q

∑Y Z,[ ST 5

J K U(V,W,O) X

7,

(1)

with A(p) = 1 for the Zimm case. The number of beads used in the calculation is set to 40. Increasing the bead number did not change the spectra. The effective bond length l in equation 1 was calculated as l = R) /N ) giving l = 3.66 and 3.83 nm for BSA4Mß and BSA6Mß, respectively. For BSA6M we obtained l = 3.38 nm. Center-of-mass diffusion was measured by DLS. The structure factor S(q) of the 3% BSA solutions was determined by SANS, see figure S2 in SI. The values of D3% and D0 are reported in table S1 in SI. Those values were inserted into equation 1 according to \]W (^) =

L_,`ab cd e(J)%

. Fits to the measured NSE spectra are shown in figure

2. It is obvious that the Zimm model does not fit the measured NSE data. The obtained χ2-values quantify that observation (BSA4Mß : χ2 = 24.6, BSA6Mß: χ2 = 37.0, BSA6M: χ2 = 326.9).

In previous studies using FCS the Zimm model including internal friction (ZIF) has been used to interpret the dynamics of intrinsically disordered and unfolded proteins depending on GndCl concentration.4 Dynamics in folded proteins measured by NSE have been interpreted using Zimm-like dynamics before.13 Therefore, in a second data analysis approach, we tested whether or not the ZIF is appropriate for NSE data analysis. Fits using the ZIF model are shown in figure 2. The ZIF model is an extension of the Zimm model.14,15 It considers internal friction of the polymer chain via a dash-pot that is installed in parallel to the entropic springs. The solution of the corresponding Langevin equation yields a mode independent relaxation time τi that is added to each Zimm mode τp such that τpZIF = τp + τi, which leads to a dampening of higher Zimm modes. Fitting the ZIF model to the whole set of NSE spectra simultaneously with τi as only free parameter gives for BSA4Mß τi= 147.08 ± 11.27 ns and for BSA6Mß τi= 111.64 ± 15.60 ns with χ2-values of 2.9 and 12.0, respectively. Those values of τi account to 6.8 ± 0.5 % and 3.0 ± 0.4 %


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of the first Zimm relaxation time in BSA4Mß and BSA6Mß, respectively. For BSA6M with intact disulphide bridges a large value of τi= 1038.24 ± 94.23 ns is obtained (χ2=15.6), which accounts to 62.4 ± 5.7% of the first Zimm time and, therefore, leads to the breakdown of the Zimm mode spectrum. As evidenced by comparison of the obtained χ2-values, the ZIF model gives a significantly better description of the measured data of BSA4Mß than the Zimm model. The obtained values of internal friction can be compared to solvent friction of one bead: For reduced BSA4Mß we obtain τDgh =5.6 ns. For BSA6Mß and BSA6M we obtain τDgh = 8.5

and τDgh = 5.9 ns. It turns out that internal friction in denatured BSA is large compared to

solvent friction. For BSA6Mß and BSA6M, however, significant systematic deviations of the ZIF model from the measured IN15 NSE data are still observable at Fourier times larger than 50 ns demonstrating that the ZIF model alone is not fully sufficient to interpret dynamics of denatured BSA.

Therefore, we considered the case that amplitudes of long-wavelength Zimm modes are reduced in denatured BSA due to structural constraints of disulphide bridges. For that purpose, we used a sigmoidal mode suppression function .() =

1

1 0 +i@[ ((@@_ )/k)]

in equation 1 as

empirical choice.16 We obtained values of p0 = 7.08 ± 0.46 and σ = 6.21 ± 1.59 for reduced

BSA6Mß, and p0 = 31.05 ± 3.46 and σ = 16.57 ± 2.73 for BSA6M. Using that approach we obtained a good fit the data as seen in figure 2 with χ2= 2.9 for BSA6Mß and χ2 = 3.5 for BSA6M. The amplitudes of first Zimm modes are significantly suppressed as can be seen in figure S4 in SI. This is particularly the case for denatured BSA6M with intact disulphide bridges, where modes below p = 31 are strongly reduced. The wave-length of a Zimm mode with p = 31 in denatured BSA with intact disulphide bonds can be estimated as n = o ∙ 8⁄ = 4.4 rs, where ACS Paragon Plus Environment

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l = 3.4 nm is the bead distance and N=40 is the number of beads used. Native BSA has 17 sulfur bridges forming loops of amino acids on the peptide backbone, and the 12 smallest loops incorporate 8 to 16 amino acids (number of loops and amino acids within loops see table S2). The effective length of that loops ranges from 3.04 nm to 6.08 nm. We see directly in that molecular picture that topological restrictions due to disulphide bridges create confinement effects: Long-wavelength Zimm modes are strongly reduced in amplitude due to loops formed by disulphide bridges.

In this letter, we report on a NSE study of chemically denatured BSA in solution. The observed dynamic processes can consistently be interpreted by means of polymer theory models including internal friction of the protein chain and suppression of low frequency Zimm mode amplitudes. Such characteristics concerning internal friction effects were also reported from FCS spectroscopy studies on small denatured proteins.4 It is important to point out that FCS experiments are sensitive to relative motions of rather bulky dye labels, whereas NSE detects the full mode spectrum of the unlabeled protein. Comparing NSE and FRET results reveals insight into the emergence of internal friction dependent on denaturant concentration. Computer simulation studies reported that at atomistic scale internal friction in unfolded proteins results from concerted dihedral rotations.17 Structural expansion due to higher GndCl concentration reduces internal chain friction. High quality NSE data demonstrate that amplitudes of lowfrequency Zimm modes acting on long length scales are reduced in denatured BSA. Mode suppression in ideally expanded polymers would not be expected. However, the ratio of hydrodynamic and Guinier radii RH/RG=0.81 (see SI) show that BSA6Mß is still more compact than ideally expanded polymers, which have RH/RG ratios of 0.66 and 0.64 in good and θ solvent,


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respectively.12 BSA6M with active sulfur bonds is significantly stiffer with slower internal dynamics as compared to BSA6Mß without disulphide bridges. We conclude, that active disulphide bridges block longer wavelength Zimm modes. NSE experiments described in this manuscript reveal polymer-like dynamics of denatured proteins as also recently suggested in a neutron spectroscopy study.18 Internal friction and low-frequency Zimm mode suppression depend both on structural expansion and on structural constraints in denatured proteins regulating conformational motions that occur during the first stage of protein folding. However, direct comparison of dynamic properties of chemically denatured proteins to the thermodynamic unfolded state occurring in vivo merits caution. GndCl ions increase the solubility of hydrophobic residues, thus modifying local energetic barriers, a fact that does not occur for the biologically relevant unfolded state.

ASSOCIATED CONTENT Supporting Information. Experimental methods; SANS: concentration series and structure factors; NSE: stretching exponents, rates, mode amplitudes, mode dependence. Disulphide bonds in BSA

ACKNOWLEDGMENT This work is based upon experiments performed on the instruments KWS-2 and J-NSE operated by Jülich Centre for Neutron Science at the Heinz Maier-Leibnitz Zentrum, Garching, Germany and on IN15 at the Institut Laue-Langevin, Grenoble, France. We would like to thank Dr. Ralf Biehl for provision of data analysis software and helpful discussion.


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Hammouda, B. Small-Angle Scattering From Branched Polymers. Macromol. Theory


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Petrescu, A. J.; Receveur, V.; Calmettes, P.; Durand, D.; Desmadril, M.; Roux, B.; Smith, J. C. Small-Angle Neutron Scattering by a Strongly Denatured Protein: Analysis Using Random Polymer Theory. Biophys. J. 1997, 72, 335–342.

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Petrescu, A. J.; Receveur, V.; Calmettes, P.; Durand, D.; Smith, J. C. Excluded Volume in the Configurational Distribution of a Strongly-Denatured Protein. Protein Sci. 1998, 7, 1396–1403.

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Doi, M.; Edwards, S. F. The Theory of Polymer Dynamics; Birman, J., Edwards, S. F., LLewellyn Smith, C. H., Rees, M., Eds.; Clarendon Press: Oxford, 1986.

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Cheng, R. R.; Hawk, A. T.; Makarov, D. E. Exploring the Role of Internal Friction in the Dynamics of Unfolded Proteins Using Simple Polymer Models. J. Chem. Phys. 2013, 138, 74112.

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Ameseder, F.; Radulescu, A.; Khaneft, M.; Lohstroh, W.; Stadler, A. M. Homogeneous and Heterogeneous Dynamics in Native and Denatured Bovine Serum Albumin. Phys. Chem. Chem. Phys. 2018, 20, 5128–5139.


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SANS data of native and denatured BSA at infinite dilution. (a,b,c) Log-log plots of native BSA (circles), BSA in 4 M GndCl & 150 mM ß-met (squares), BSA in 6 M GndCl &150 mM ß-met (diamonds), and BSA in 6 M GndCl (triangles). The measured intensities of native BSA are fitted with the calculated form factor of the crystal structure (pdb 4f5s); the measured intensities of denatured BSA are fitted with the Debye equation for a generalized Gaussian chain. (d) Reduced Kratky plot of native BSA, denatured BSA in 6 M GndCl, and 6 M GndCl 150mM ß-met, respectively. 84x86mm (600 x 600 DPI)


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Experimental NSE data of denatured BSA and fits based on polymer theory. Zimm model (dashed lines), ZIF model (dotted lines), and Zimm model with reduced mode amplitudes (solid lines). Data measured on (a) JNSE, (b) and (c) on IN15. (a) BSA in 4 M GndCl & 150 mM β met, (b) BSA in 6 M GndCl & 150 mM β-met without sulfur bonds and (c) BSA in 6 M GndCl with active sulfur bonds. 190x439mm (600 x 600 DPI)



Effective diffusion coefficient measured with NSE of the 3% BSA solutions and (b) corrected for interparticle and hydrodynamic interactions. Straight dashed lines represent linear regressions of IN15 data. Values for translational diffusion Dt measured by DLS of (a) the 3% BSA solutions and (b) at infinite dilution are displayed at q=0 (empty symbols). 114x158mm (600 x 600 DPI)


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Relevance of Internal Friction and Structural Constraints for the

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