A scattering study of concentrated lens protein solutions and mixtures - Towards understanding the molecular origin of presbyopia

Abstract: Popular Abstract in English The eye is a complex device in charge of producing clear and sharp images of the outside world. While some people need glasses early on in their lives, others have nearly perfect vision until, all of a sudden, they have to hold books or documents further and further away from their eyes to be able to read them and eventually have to resort to reading glasses. This visual impairment, known as presbyopia, usually starts around the age of 40. But why? What happens? To be able to answer those questions, one has to have a closer look at how the eye is build. To ensure sharp, unblurred visual images, all light entering the eye through the pupil, no matter how far away its source, has to be focused onto the retina where the image is formed. If the focal point is shifted away from the retina, the image appears blurred. From optics, it is known that the focal point of a simple lens depends on its curvature and on the distance between the light source and the lens. For an unflexible, rigid lens with fixed curvature, light emitted by close objects thus has a different focal point than light coming from far away objects. For the eye, this means that the lens has to adjust shape to be able to focus light from close, as well as far away sources onto the same focal point (the retina). In order to do so, the lens has of course to be flexible, as is the case in a healthy eye. With age, however, the lens loses flexibility and becomes increasingly stiff, resulting in the inability to focus light emitted by close-by objects onto the retina, thus making those objects appear blurred. But what causes this gradual hardening of the lens? It is the aim of this thesis to answer this question by studying the proteins that make up the eye lens. The lens is formed of fibre cells that are filled with very concentrated solutions of proteins, known as crystallins that can be divided into three major classes: alpha-, beta- and gamma-crystallins. The sizes of these proteins range from 3.6 nm (gamma-crystallin) to 16 nm (alpha-crystallin), where 1 nm = 0.000000001 m (a strand of human hair has a diameter of about 180 000 nm). So-called scattering techniques are ideally suited to study particles as small as the crystallins. They are based on irradiating a sample (with either laser light, X-rays or neutrons) and analyzing the radiation that is scattered by the sample. I used a variety of different scattering techniques, allowing me to analyze the interactions, stability and diffusion (movement) of crystallins in solution. Proteins are rather complex structures, but to analyze the results obtained from scattering experiments, they can be simplified and described as either hard spheres (comparable to tiny billiard balls that do not interact with each other, except for the fact that they cannot occupy the same space at the same time) or short-range attractive hard spheres (comparable to tiny billiard balls that attract each other/stick together when they come close, but do not feel each other at larger separations). This approach was applied to solutions of crystallins at varying concentration, from very dilute to very concentrated. These proteins were purified from calf eye lenses purchased as a by-product from a local slaughterhouse. In a first step, only solutions of the individual proteins were studied and the main focus was put on their dynamic behavior. It was found that at concentrations comparable to those found in the eye lens, these solutions undergo a so-called dynamical arrest, leaving the solution in a glassy or gel-like, rather than fluid, state. In an attempt to further mimic conditions found in the eye, binary mixtures of different proteins were studied and while this study is far from complete, it does point in the same direction as the trend found for individual protein solutions, namely a dynamical arrest at physiologically relevant concentrations. While a lens filled with a fluid-like concentrated protein solution is able to adapt its shape to adjust focus, a lens with a dynamically arrested mixture of proteins becomes stiff and can no longer accommodate for close vision. The study presented here thus provides very compelling evidence for the hypothesis that age-related dynamical slowing down and eventual arrest of the concentrated protein mixture in the eye lens fibre cells leads to the gradual hardening of the eye lens that is at the origin of presbyopia.