Small-angle neutron scattering experiments and molecular dynamics simulations
combined with an application of concepts from soft matter physics to complex
protein mixtures provide new insight into the stability of eye lens protein
mixtures. Exploring this colloid-protein analogy we demonstrate that weak
attractions between unlike proteins help to maintain lens transparency in an
extremely sensitive and non-monotonic manner. These results not only represent
an important step towards a better understanding of protein condensation
diseases such as cataract formation, but provide general guidelines for tuning
the stability of colloid mixtures, a topic relevant for soft matter physics and
industrial applications.Comment: 4 pages, 4 figures. Accepted for publication on Phys. Rev. Let

A. Stradner

G. Foffi

G. Thurston

G. B. Benedek

H. Mach

J. Meijering

J. Peterson

J. L. Meijering

N. Dorsaz

P. Schurtenberger

R. Piazza

English

arXiv

Small-angle neutron scattering experiments and molecular dynamics simulations combined with an application of concepts from soft matter physics to complex protein mixtures provide new insight into the stability of eye lens protein mixtures. Exploring this colloid-protein analogy we demonstrate that weak attractions between unlike proteins help to maintain lens transparency in an extremely sensitive and nonmonotonic manner. These results not only represent an important step towards a better understanding of protein condensation diseases such as cataract formation, but provide general guidelines for tuning the stability of colloid mixtures, a topic relevant for soft matter physics and industrial applications. © 2007 The American Physical Society.GR-F

Stradner, A.

Foffi, G.

Dorsaz, N.

Thurston, G.

Schurtenberger, P.

Infoscience - École polytechnique fédérale de Lausanne

New insight into cataract formation: Enhanced stability through mutual attraction

Small-angle neutron scattering experiments and molecular dynamics simulations combined with an application of concepts from soft matter physics to complex protein mixtures provide new insight into the stability of eye lens protein mixtures. Exploring this colloid-protein analogy we demonstrate that weak attractions between unlike proteins help to maintain lens transparency in an extremely sensitive and nonmonotonic manner. These results not only represent an important step towards a better understanding of protein condensation diseases such as cataract formation, but provide general guidelines for tuning the stability of colloid mixtures, a topic relevant for soft matter physics and industrial applications

Stradner, Anna

Schurtenberger, Peter

RERO DOC Digital Library

http://doc.rero.chNew Insight into Cataract Formation: Enhanced Stability through Mutual AttractionA. Stradner,1 G. Fofﬁ,2,* N. Dorsaz,2 G. Thurston,3,† and P. Schurtenberger1,‡1Physics Department and Fribourg Center for Nanomaterials, University of Fribourg, CH-1700 Fribourg, Switzerland2Institut Romand de Recherche Nume´rique en Physique des Mate´riaux (IRRMA) and Institute of Theoretical Physics (ITP),Ecole Polytechnique Fe´de´rale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland3Department of Physics, Rochester Institute of Technology, Rochester, New York 14623-5603, USASmall-angle neutron scattering experiments and molecular dynamics simulations combined with anapplication of concepts from soft matter physics to complex protein mixtures provide new insight into thestability of eye lens protein mixtures. Exploring this colloid-protein analogy we demonstrate that weakattractions between unlike proteins help to maintain lens transparency in an extremely sensitive andnonmonotonic manner. These results not only represent an important step towards a better understandingof protein condensation diseases such as cataract formation, but provide general guidelines for tuning thestability of colloid mixtures, a topic relevant for soft matter physics and industrial applications.PACS numbers: 87.15.Nn, 61.12.Ex, 64.70.JaCataract, sickle-cell, and Alzheimer’s represent a broadclass of ‘‘protein condensation diseases’’ in which inter-molecular attractions lead to dense phases that can com-promise cell and organ function [1]. Statistical and colloidphysics have led to considerable progress in understandingthe molecular origins of these diseases, in which a subtleinterplay between protein attractions, repulsions, and en-tropy governs condensation, and health or disease canhinge on intermolecular interaction changes of just thermalenergy, kBT. In complementary fashion, proteins haveproven useful as model colloids, for studying how short-range attractions and hard and/or soft repulsions combineto affect phase transitions, aggregation, and cluster forma-tion [2–4].Cataract, eye lens clouding due to light scattering, is aleading cause of blindness and can result from proteincondensation [1,5–8]. Statistical and colloid physics havehelped rationalize eye lens transparency, liquid structure,and thermodynamics [7–11]. Mammalian eye lens cellscontain concentrated solutions of proteins called crystal-lins, of three major classes: , , and  [9,10]. The lens isnormally highly transparent and refractive, but loss oftransparency due to protein aggregation or phase separa-tion can lead to cataract. Quantifying and understandingcrystallin interactions and their impact on lens transpar-ency is therefore a ﬁrst step towards cataract prevention[6].The -crystallins are globular, polydisperse, multisubu-nit, 800 kDa proteins with a diameter of about 18 nm,whose interactions are well described with a simple hard-sphere colloid model [12,13]. The -crystallins are mono-meric, with a molecular weight of 21 kDa and a diameterbetween 3 and 4 nm for B-crystallin. The discovery of ametastable liquid-liquid phase separation provided evi-dence for short-range attractions between -crystallins,and use of the corresponding colloid model has led to aquantitative description of the phase behavior [11,14–18].Although the chaperone activity of -crystallin cansuppress aggregation of - and -crystallin [19,20], littleis known about interactions between unlike crystallins athigh concentrations. Recently, -crystallin has been foundto enhance phase separation of B-crystallin and lead topartial segregation of proteins by type in the separatedphases [21]. Therefore, we have started a systematic studyof - and B-crystallin mixtures up to concentrationsfound within the eye lens that combines small-angle neu-tron scattering (SANS) and molecular dynamics (MD)simulations of a coarse-grained colloidal model, appropri-ate given the hard cores of these folded, globular proteins.This approach allows for complementary experimental andcomputational investigation at the required large lengthscales and high number of proteins (30 000–60 000).We prepared different mixing ratios from stock solutionsof 230 mg=mL -crystallin (denoted as C  100) and260 mg=mL B-crystallin (denoted as C  0) in 0.1 Msodium phosphate buffer in D2O at a pH of 7.1, with20 mM dithiothreitol to inhibit protein oxidation and oli-gomerization [21]. With these precautions, B-crystallinremained monomeric under all conditions used. We per-formed SANS using 1 and 2 mm Hellma quartz cells andvaried wavelengths, sample-to-detector distances, and col-limation lengths to cover a q range of 0:02–3 nm1. Allexperiments were performed at a temperature of 25 C, i.e.,10 C above the critical temperature Tc of B-crystallin inthe present buffer. The choice of an experimental tempera-ture close to Tc of B-crystallin ampliﬁes thermodynamicstability variations due to the presence of alpha crystallin,and thereby enhances sensitivity for discerning effectiveprotein interaction potentials.We established a model for the protein interaction po-tentials by comparing MD computer simulations with theexperimental scattering intensity Iq as a function of thescattering vector q. We performed MD simulations in peri-odic cubic boxes with N  32 000 particles for the pure= = =1Published in "Physical Review Letters 99(19): 198103, 2007"which should be cited to refer to this work. http://doc.rero.ch-crystallin solution and the mixtures, and N  64 000particles for the pure B-crystallin solution. The scatteringintensities were calculated directly from many independentMD conﬁgurations using the experimental form factorsand a properly derived general resolution function to ac-count for the experimental smearing [22]. From the threedifferent mixing ratios (C  50, C  25, and C 12:5, where C is deﬁned as 100 	volume of the C 100 solution=	total volume of the mixture) investi-gated, C  50 closely resembles the natural - and-crystallin concentrations found in the lens nucleus [9].Therefore, the model development is demonstrated on thissample, and its validity is tested by comparison with theremaining samples.First we determined parameters that provide a quantita-tive description of Iq for the individual components. For-crystallin we used a purely repulsive hard-sphere modeland a diameter of d  16:3 nm [13], which results inperfect agreement between experimental and simulatedIq [Fig. 1(a)]. For C  0 the strongly enhanced inten-sity at low q indicates that a hard-sphere model is notsufﬁcient to describe the interactions betweenB-crystallins (diameter d  3:6 nm) [Fig. 1(b)]. Thisis a direct consequence of interprotein attraction and prox-imity to the critical concentration Cc [14,15,17], whichleads to long-wavelength concentration ﬂuctuations thatare a potential source of lens turbidity in cataract [5]. Toaccount for this we added a square-well attractive potentialwith a range of 0.25 d [16] and a depth u  1kBT,where the temperature T is set to T  0:7875 (for kB  1)for all simulated mixtures. This temperature T has beenﬁxed to reproduce the -pure case and results in goodagreement between simulated and experimental Iq inthe low-q regime (q & 0:047 nm1) relevant for lenstransparency [Fig. 1(b)]. While square-well potentialshave unphysical shape, they have been widely used tostudy colloids with interaction ranges shorter than particlediameter, since the physics then depends only weakly onthe shape of the potential [23]. Figure 1 indeed demon-strates that simple colloid models are capable of reproduc-ing Iq for individual - and B-crystallin solutions.In a next step we investigated the mixtures. Figure 1(c)demonstrates the effect of adding alpha crystallin, fromC  0 to C  100. The forward scattering ﬁrst in-creases and reaches a maximum for C  25. For C 50 it becomes highly suppressed, similar to the situationfor pure -crystallin. In MD simulations we ﬁrst assumedhard-sphere =B interactions. The striking disagreementbetween the forward scattering of the corresponding simu-lation with experimental data for C  50 is shown inFig. 2. The low-q increase of simulated intensity likelyarises from depletion attractions known in hard-spheremixtures [24]. Such an effect would increase Tc forliquid-liquid phase separation and result in segregation ofthe proteins into large domains of -crystallin-rich andB-crystallin-rich regions (Fig. 2). As a consequence, lightscattering would have increased and transparency wouldhave been lost, in contrast to the experimental data and thevisual appearance of the sample. Such effects are in factmanifest in -B mixtures at temperatures lower thanthose investigated here [21]. However, the low forwardscattering intensity I0 of the present SANS data indicatesthat they are suppressed under the present conditions,closer to body temperature. Nature must have circum-vented long-wavelength ﬂuctuations and eye lens cloudingfrom hard-sphere -B interactions, and we can speculatethat  and B could have additional attraction.In a second attempt we thus added an attractive interac-tion between - and B-crystallins, assuming that theattractive part of the potential has the same range as thatused for the attraction between B-crystallins. The depthof this interspecies attractive potential, u, was chosen toreproduce Iq and is roughly one half of the B-Battraction. The simulated Iq (Fig. 3) indeed perfectlyreproduces the SANS data throughout the entire q range.Use of the novel attractive term leads to good agreementbetween simulations and SANS for all mixing ratios(Fig. 4). A snapshot of the C  50 simulation (Fig. 3)suggests a qualitative explanation: attractions between un-0.1 1q[nm -1]0.1110Cα=1000.1 1q [nm-1]0.1110Cα=0bac0.1 1q[nm-1]0.010.1110010I(q) [a.u.]I(q) [a.u.]I(q) [a.u.]1 2 3C  = 100α C  = 0αC  = 50αC  = 12.5αC  = 25αFIG. 1 (color online). Scattered intensities Iq of a230 mg=ml (C  100) and a 260 mg=mL (C  0) solutionand their mixtures. (a) Experimental Iq of C  100 (opensymbols) together with MD computer simulations (full curve).(b) Experimental Iq of C  0 (open symbols) together withMD computer simulations (full curve). (c) Iq of mixturescontaining different amounts of the crystallin solutions shownin (a) and (b). Shown are C  100 (), C  50 (), C  25( ? ), C  12:5 (), and C  0 (). Numbers indicate qvalues for composition dependence in Fig. 4.2http://doc.rero.chlike proteins counterbalance and suppress segregationof the two proteins into large domains [25]. Thus trans-parency of the mixture is enhanced by a weak short-ranged interspecies attraction that considerably decreasesTc and the corresponding critical ﬂuctuations at a giventemperature.We also performed simulations in which the attractionbetween  and Bwas further increased, to above 100% ofthe B-B attraction. Such stronger attractions again re-sulted in enhanced instability (inset of Fig. 3). Thus thestability of these high concentration crystallin mixturesdepends on -B attraction in a manner that is bothextremely sensitive and nonmonotonic. Such nonmono-tonic effects are a common feature of ternary liquid mix-ture phase separation [21,26].There are studies suggesting that -crystallins inhibitthe age-related aggregation of -crystallins [27]. On theother hand, numerous investigations have suggested thatinhibition of age- or heat-induced aggregation of - and-crystallins is associated with the chaperone activity of-crystallins [19,20,28]. In contrast to these stabilizingeffects of - interactions, it has recently been suggestedthat the observed increase in the noncovalent associa-tion of -crystallins to -crystallins in aging bovine lensesmight adversely affect the optical properties of the agedand/or cataractogenic lens [29], since strong attractionscould lead to larger aggregates and increased scatteringof light. While such a mechanism of increased protein-protein association is a possible route towards cataract[29], our experiments and simulations provide evidencethat a weak and short-range attraction between - andB-crystallins can potentially enhance transparency inhealthy lenses.A variety of mechanisms could underlie this ‘‘stabiliza-tion due to mutual attraction’’, as a simple colloidal -Battraction can represent our observations. The molecularorigins of the weak, short-range attraction remain a chal-lenging, open question, as for many proteins includingB-crystallin and lysozyme [2,30].Uniform, high concentration packing of crystallins,termed short-range order, suppresses refractive index ﬂuc-tuations and reduces light scattering [7]. The chemically11.0q [nm-1]0.1110I(q) [a.u.]0.5 1 1.5uαγ110100I(q ) [a.u.]Unstable UnstableStable* 0.55q*FIG. 3 (color online). Iq of an - andB-crystallin mixture (C  50) andsimulations incorporating mutual attrac-tion. (Left) SANS data from Fig. 2 (opensymbols) and simulations without(dashed curve) and with added short-range attractions (full curve; u 0:55kBT) between unlike proteins.Inset: Iq  0:0467 nm1 from simu-lations as a function of u. The mix-tures become unstable for u & 0:5kBTand * 1kBT. (Right) Snapshot and slabrepresentation of the box for this situ-ation as in Fig. 2. The solution is homo-geneous and does not give rise toincreased light scattering.11.0q [nm-1]0.1110I(q) [a.u.]FIG. 2 (color online). Scattered in-tensity Iq of a mixture of - andB-crystallin (C  50) and MD com-puter simulation assuming mutual repul-sion between unlike proteins. (Left) Iqof C  50 (open symbols) togetherwith MD simulation of the system (fullcurve). (Right) Snapshot from MDsimulation shown on the left. Assum-ing repulsive hard-sphere interactionsonly between - (gray spheres) andB-crystallins (black dots) leads to astrong segregation by protein type andthe corresponding large density ﬂuctua-tions would lead to a loss of transparencyfor visible light. Also shown is a slab ofthe box for this snapshot.3http://doc.rero.chspeciﬁc origins of short-range order are key to understand-ing lens transparency. Not only do crystallins vary widelyin short-range order properties, but sensitivity to intercrys-tallin interactions makes short-range order not a simplecombination of individual crystallin properties, as shownhere for - and B-crystallins. To understand transparencyof crystallin mixtures, high concentration short-range orderstudy, using liquid-state and colloid physics, remains aneeded complement to low concentration investigations.This investigation provides new insight into the stabilityand optical properties of lens protein mixtures relevant tocataract. The results also suggest mechanisms to tunecolloid mixture stability, a topic of industrial importance.This demonstrates the importance of a colloid-orientedapproach to proteins as a means of obtaining insight intoquestions of biological, medical, as well as fundamentalsoft matter physics relevance.This work is based on experiments performed at theSwiss spallation neutron source SINQ, Paul ScherrerInstitute, Villigen, Switzerland. We are grateful for theneutron beam time and we acknowledge the help of ourlocal contact J. Kohlbrecher. We thank A. Baldareschi forsupport and discussions. This work was supported by theSwiss National Science Foundation (Grant No. 99200021-105382/1), the State Secretariat for Education andResearch (SER) of Switzerland, the Marie Curie Networkon Dynamical Arrest of Soft Matter and Colloids(No. MCRTN-CT-2003504712), and NIH (US) GrantNo. EY11840.*giuseppe.fofﬁ@epﬂ.ch†george.thurston@rit.edu‡peter.schurtenberger@unifr.ch[1] G. B. Benedek, Invest. Ophthalmol. Vis. Sci. 38, 1911(1997).[2] R. Piazza, Curr. Opin. Colloid Interface Sci. 5, 38 (2000).[3] A. Stradner et al., Nature (London) 432, 492 (2004).[4] F. Cardinaux et al., Phys. Rev. Lett. 99, 118301 (2007).[5] G. B. Benedek, Appl. Opt. 10, 459 (1971).[6] G. B. Benedek et al., Progress in Retinal and Eye Research18, 391 (1999).[7] M. Delaye and A. Tardieu, Nature (London) 302, 415(1983).[8] H. Bloemendal et al., Prog. Biophys. Molec. Biol. 86, 407(2004).[9] R. J. Siezen et al., Proc. Natl. Acad. Sci. U.S.A. 82, 1701(1985).[10] F. Ve´re´tout, M. Delaye, and A. Tardieu, J. Mol. Biol. 205,713 (1989).[11] P. Schurtenberger et al., Phys. Rev. Lett. 63, 2064 (1989).[12] A. Tardieu, International Journal of BiologicalMacromolecules 22, 211 (1998).[13] S. Finet and A. Tardieu, J. Cryst. Growth 232, 40 (2001).[14] J. A. Thomson et al., Proc. Natl. Acad. Sci. U.S.A. 84,7079 (1987).[15] M. L. Broide et al., Proc. Natl. Acad. Sci. U.S.A. 88, 5660(1991).[16] A. Lomakin, N. Asherie, and G. B. Benedek, J. Chem.Phys. 104, 1646 (1996).[17] M. Malfois et al., J. Chem. Phys. 105, 3290 (1996).[18] A. Stradner, G.M. Thurston, and P. Schurtenberger,J. Phys. Condens. Matter 17, S2805 (2005).[19] J. Horwitz, Proc. Natl. Acad. Sci. U.S.A. 89, 10 449(1992).[20] T. Putilina et al., J. Biol. Chem. 278, 13 747 (2003).[21] G.M. Thurston, J. Chem. Phys. 124, 134909 (2006).[22] N. Dorsaz et al. (to be published).[23] G. Fofﬁ et al., Phys. Rev. E 65, 031407 (2002).[24] Mendez-Alcaraz and R. Klein, Phys. Rev. E 61, 4095(2000).[25] A.A. Louis et al., Phys. Rev. E 65, 061407 (2002).[26] J. L. Meijering, Philips Res. Rep. 5, 333 (1950); 6, 183(1951).[27] H. Mach et al., J. Biol. Chem. 265, 4844 (1990).[28] V. Pigaga and R.A. Quinlan, Exp. Cell Res. 312, 51(2006).[29] J. Peterson, G. Radke, and L. Takemoto, Exp. Eye Res. 81,680 (2005).[30] R. Piazza, Curr. Opin. Colloid Interface Sci. 8, 515 (2004).0 20 40 60 80 1000510150 20 40 60 80 1000510150 20 40 60 80 100Cα051015q=0.07 nm-1q=0.20 nm-1q=0.40 nm-1I(q) [a.u.]123ExperimentalMutually repulsiveMutually attractiveFIG. 4 (color online). Comparison of Iq obtained from simu-lations and SANS as a function of -crystallin mixing ratio (C)in the -B mixtures. The predicted low-q portion of Iq isvery sensitive to alpha-gamma mutual attraction, unlike thehigher-q portions. Iq at different C are from SANS () andsimulations without () and with () mutual attractions. Shownare data at three scattering vectors, q  0:07, 0.2, and 0:4 nm1[the numbered labels refer to the arrows in Fig. 1(c)]. Unlessexplicitly plotted, error bars are equal or smaller than thesymbols.4

New insight into cataract formation: enhanced stability through mutual attraction

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New Insight into Cataract Formation: Enhanced Stability through Mutual Attraction

New insight into cataract formation -- enhanced stability through mutual
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New insight into cataract formation -- enhanced stability through mutual attraction

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