Beta Laktoglobulin

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Eur. Phys. J. E 29, 173182 (2009) DOI 10.1140/epje/i2009-10465-y

THE EUROPEAN PHYSICAL JOURNAL E

Regular Article

Investigating the inner structure of irregular -lactoglobulin spherulitesK.R. Domike1,4,a , E. Hardin1,2 , D.N. Armstead1,3 , and A.M. Donald41 2 3 4

Department of Physics, The College of Wooster, Wooster, Ohio 44691, USA Department of Physics, Slippery Rock University, Slippery Rock, PA, 16057, USA Department of Physics, Westminster College, New Wilmington, PA, 16172, USA Cavendish Laboratory, University of Cambridge, JJ Thomson Avenue, Cambridge CB3 0HE, UK Received 10 July 2008 and Received in nal form 31 December 2008 Published online: 19 June 2009 c EDP Sciences / Societ` Italiana di Fisica / Springer-Verlag 2009 a Abstract. When -lactoglobulin in low pH aqueous solutions is exposed to high temperature for extended time, spherulites composed of amyloid brils of the -lactoglobulin protein form. Many of these spherulites have brils that radiate out from a centre and, under crossed polarisers, exhibit a symmetric Maltese Cross structure. However, a signicant fraction (50 of the 101 observed spherulites) of -lactoglobulin spherulites formed under these conditions demonstrate various forms of irregularity in apparent structure. The irregularities of spherulites structures were qualitatively investigated by comparing optical microscopy images observed under crossed polarisers to computationally produced images of various internal structures. In this way, inner spherulite structures are inferred from microscopy images. Modelled structures that were found to produce computed images similar to some of the experimentally viewed images include brils curving as they radiate from a single nucleation point; multiple spherulites nucleating in close proximity to one another; and brils curving in opposite directions above and below a single nucleation point. PACS. 87.14.E- Proteins 87.15.ad Analytical theories 87.15.bk Structure of aggregates

1 IntroductionProtein aggregates from misfolded or partially unfolded proteins are thought to be the cause of amyloidoses; diseases which include Alzheimers, type II diabetes, and Parkinsons [1,2]. Aggregates such as amyloid brils, which are characterized by a cross-beta sheet quaternary structure, and spherulite aggregates, characterized by spherical or near-spherical shape, have been shown to be present in some humans suering from these diseases [3]. The exact role that the protein aggregates play in the diseases and details about the internal structure of these spherulites remains unknown [1,4]. The propensity to form spherulites is not unique to proteins but has also been found in crystallisable synthetic polymers [2,57] and crystallising metals [8,9]. The protein used in this study is bovine -lactoglobulin (BLG). BLG has been found to form amyloid brils and spherulites readily under low pH (most reproducibly at pH 1.52.0) and elevated temperature (up to 90 C) conditions [2,6,1012]. Over time, typically 1248 h depending on temperature, BLG spherulite aggregates can be viewed directly by optical microscopy [2,5,1114]. BLGa

e-mail: kdomike@wooster.edu

spherulites with diameters up to 200 m have been observed [3]. The spherulites have been shown to be composed of amyloid brils arranged radially outwards from a single centre [13]. The optical anisotropy of a spherulite (arising from the oriented amyloid brils within the spherulite) interacts with polarised light as it traverses the spherulite. The resulting modulation of light of dierent polarisations enables analysis of the image generated in a light microscope under crossed polarisers to provide insight into the internal structure of the spherulite. Specically, the electric eld of the light that exits a polariser can be computed from the electric eld of the entering light and the polarization [1517]. Optical microscopy with crossed polarisers has been used in a wide variety of biological and polymeric spherulite formation experiments to observe internal structure and orientation [3,13,17,18]. The simplest internal spherulite structure is that in which the amyloid brils point radially away from a central point as shown in g. 1. This symmetric structure produces an image under crossed polarisers with a clear Maltese Cross, as shown from experiment in g. 2 [3,13]. In this research, the structures of BLG spherulites that do not form a regular Maltese Cross when imaged under crossed polarisers are analyzed and mathematically

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The European Physical Journal E

Fig. 1. Two-dimensional visualisation of spherulite structure with amyloid brils or bril bundles (shown as lines) pointing radially from a central point.

Fig. 3. (Colour on-line) Example images of characteristic shapes seen for BLG spherulites viewed as optical microscopy images through crossed-polarisers: A) normal Maltese Cross; B) rotated Maltese Cross (compared to horizontal and vertical dashed lines which represent the orientation of the polariser and analyser); C) small wedge; D) extra lobes; E) other misshapen (two spherulites); F) other mis-shapen (two lobes). The solid scale bar in each image represents 50 m.

Fig. 2. Experimentally observed Maltese Cross pattern produced by a BLG protein spherulite when imaged under crossed polarisers in an optical microscope.

modelled. The sections of this paper include: the method of obtaining the spherulite images, a description of the observations of abnormal spherulites, the method of mathematical modelling, discussion of the results of relating the modelling to the observed spherulites, and relevant conclusions.

2 Technique of monitoring BLG spherulitesThe results in this paper are based on temperature experiments published previously [14]. The novelty of this research is in developing and applying a mathematical model of the spherulite inner structure to relate its structure to the resulting microscopy images and thereby obtaining insight into the structural sources of irregularities in the observed images. The protein used in this research was analytical grade bovine -lactoglobulin (BLG) (product number L0130; mixture of types A and B) obtained from Sigma-Aldrich (Gillingham, UK). Protein solutions were made by dissolving the protein in distilled and de-ionized water. The desired pH was achieved by adding 1 M HCl (Sigma-Aldrich) in 550 l increments. Glass slides with wells were lled with 100 l of protein solution, covered with a glass coverslip, and heated to the required temperature on a heating stage placed within the Zeiss Axioplan optical microscope (Carl Zeiss Ltd., Welwyn Garden City, UK). Total magnication of either 50, 100, 200, or 500 was used for the optical imaging. A polariser and analyzer were put in xed positions, orthogonal to one another for the

crossed-polariser imaging. The size of spherulites seen in the images was determined from calibration with a scale bar of known dimension, and subsequent quantication of the spatial distance per image pixel. For example, the images of BLG spherulites taken at 50 magnication had a resolution of 1.56 m/pixel; at this magnication spherulites with radii less than 10 m could not be clearly resolved. The time required to bring all of the BLG protein solution to the temperature of the heating stage was not more than three minutes, as determined via nite element simulation published previously [14], and this oset is ignored from here on as the incubation times were of the order of hours and days. The solution temperature was not measured experimentally.

3 Observations of BLG spherulitesWhen viewed through the crossed-polarisers, it was observed that -lactoglobulin (BLG) spherulites did not always have a Maltese Cross composed of four nearly identical lobes without any rotation of the lobes or noticeable defects as depicted in g. 3A and described here as a normal cross. Across the experiments performed, many instances of abnormal spherulite images were observed. Four types of abnormal crosses and shapes that were seen regularly in BLG incubation experiments are presented in g. 3B-E. In some spherulites, the Maltese Cross is significantly rotated from the orthogonal axes as exemplied in g. 3B with the orthogonal axes depicted as dashed yellow lines. In all experiments and modelling, the polarisers were strictly horizontal and vertical relative to the optical microscope and mounted camera. Maltese Crosses with a deviation of 10 or greater from the orthogonal axes were noted as abnormal. Within some of the images, such as g. 3B, continuous strand shapes can be seen.

K.R. Domike et al.: Investigating the inner structure of irregular -lactoglobulin spherulites

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Table 1. Summary of Maltese Cross shapes in BLG incubation experiments at pH 1.6 and 4 wt%. Example shapes are shown in g. 3. Aggregates demonstrating multiple abnormalities were counted in multiple columns. Percentages relative to the total number of aggregates in the experiment are shown in parentheses. Solution temperature 60 C 60 C 60 C 70 C 70 C 70 C 80 C 80 C 80 C 85 C 85 C 85 C TOTAL Total number of aggregates 6 4 4 15 4 7 12 7 6 12 10 14 101 Normal Maltese Cross shape 3 (50%) 1 (25%) 2 (50%) 6 (40%) 2 (50%) 3 (43%) 5 (42%) 5 (71%) 4 (67%) 9 (75%) 8 (80%) 2 (14%) 50 (50%) Maltese Cross rotated Small wedge 1 (17%) Extra lobes Mis-shapen 2 (33%) 3 (75%) 2 (50%) 1 (7%) 4 (57%) 5 (42%) 2 (33%) 1 (8%) 1 (10%) 6 (43%) 17 (17%)

3 (20%) 2 (50%) 4 (33%) 2 (33%) 2 (17%) 2 (20%) 4 (29%) 16 (16%)

6 (40%)

2 (17%) 2 (29%)

12 (86%) 16 (16%)

20 (20%)

Based on the large apparent resolved width of the strand shapes relative to the width of a bril (8.5 1.4 nm) [19], the shapes seen are not likely to be individual brils but may be bril bundles. In many of the images that demonstrate Maltese Cross rotation, the ap