George B. Benedek

Theory of transparency of the eye.

The present work relates the turbidity of the eye to microscopic spatial fluctuations in its index of refraction. Such fluctuations are indicated in electron microscope photographs. By examining the superposition of phases of waves scattered from each point in the medium, we provide a mathematical demonstration of the Bragg reflection principle which we have recently used in the interpretation of experimental investigations: namely, that the scattering of light is produced only by those fluctuations whose fourier components have a wavelength equal to or larger than one half the wavelength of light in the medium. This consideration is applied first to the scattering of light from collagen fibers in the normal cornea. We demonstrate physically and quantitatively that a limited correlation in the position of near neighbor collagen fibers leads to corneal transparency. Next, the theory is extended to predict the turbidity of swollen, pathologic corneas, wherein the normal distribution of collagen fibers is disturbed by the presence of numerous lakes-regions where collagen is absent. A quantitative expression for the turbidity of the swollen cornea is given in terms of the size and density of such lakes. Finally, the theory is applied to the case of the cataractous lens. We assume that the cataracts are produced by aggregation of the normal lens proteins into an albuminoid fraction and provide a formula for the lens turbidity in terms of the molecular weight and index of refraction of the individual albuminoid macromolecules. We provide a crude estimate of the mean albuminoid molecular weight required for lens opacity.

Spectrum of light scattered from a viscoelastic gel

We report measurements of the spectrum of light scattered from thermally excited displacement fluctuations in polyacrylamide gels. These measurements have been carried out on the polarized scattered light as a function of scattering angle and temperature for 5% and 2.5% polyacrylamide gels using the methods of optical mixing spectroscopy. We also present a theory for the amplitude and time dependence of the thermally excited longitudinal and transverse displacements of the gel fiber network. These displacements are responsible, respectively, for the polarized and depolarized scattered light. The correlation function for the displacements having wave vector q is predicted for these gels to have the form of an exponential decay: exp(− Γt). The decay rate is given by Γ = Glq2/f or Gtq2/f, where f is the frictional force per unit volume on the fiber network as it moves with unit velocity relative to the gel liquid. Gl is the longitudinal compressional modulus for longitudinal displacements and Gt is the shear...

Amyloid beta -protein (Abeta) assembly: Abeta 40 and Abeta 42 oligomerize through distinct pathways.

Amyloid β-protein (Aβ) is linked to neuronal injury and death in Alzheimers disease (AD). Of particular relevance for elucidating the role of Aβ in AD is new evidence that oligomeric forms of Aβ are potent neurotoxins that play a major role in neurodegeneration and the strong association of the 42-residue form of Aβ, Aβ42, with the disease. Detailed knowledge of the structure and assembly dynamics of Aβ thus is important for the development of properly targeted AD therapeutics. Recently, we have shown that Aβ oligomers can be cross-linked efficiently, and their relative abundances quantified, by using the technique of photo-induced cross-linking of unmodified proteins (PICUP). Here, PICUP, size-exclusion chromatography, dynamic light scattering, circular dichroism spectroscopy, and electron microscopy have been combined to elucidate fundamental features of the early assembly of Aβ40 and Aβ42. Carefully prepared aggregate-free Aβ40 existed as monomers, dimers, trimers, and tetramers, in rapid equilibrium. In contrast, Aβ42 preferentially formed pentamer/hexamer units (paranuclei) that assembled further to form beaded superstructures similar to early protofibrils. Addition of Ile-41 to Aβ40 was sufficient to induce formation of paranuclei, but the presence of Ala-42 was required for their further association. These data demonstrate that Aβ42 assembly involves formation of several distinct transient structures that gradually rearrange into protofibrils. The strong etiologic association of Aβ42 with AD may thus be a result of assemblies formed at the earliest stages of peptide oligomerization.

Amyloid β-protein (Aβ) assembly: Aβ40 and Aβ42 oligomerize through distinct pathways

Amyloid β-protein (Aβ) is linked to neuronal injury and death in Alzheimers disease (AD). Of particular relevance for elucidating the role of Aβ in AD is new evidence that oligomeric forms of Aβ are potent neurotoxins that play a major role in neurodegeneration and the strong association of the 42-residue form of Aβ, Aβ42, with the disease. Detailed knowledge of the structure and assembly dynamics of Aβ thus is important for the development of properly targeted AD therapeutics. Recently, we have shown that Aβ oligomers can be cross-linked efficiently, and their relative abundances quantified, by using the technique of photo-induced cross-linking of unmodified proteins (PICUP). Here, PICUP, size-exclusion chromatography, dynamic light scattering, circular dichroism spectroscopy, and electron microscopy have been combined to elucidate fundamental features of the early assembly of Aβ40 and Aβ42. Carefully prepared aggregate-free Aβ40 existed as monomers, dimers, trimers, and tetramers, in rapid equilibrium. In contrast, Aβ42 preferentially formed pentamer/hexamer units (paranuclei) that assembled further to form beaded superstructures similar to early protofibrils. Addition of Ile-41 to Aβ40 was sufficient to induce formation of paranuclei, but the presence of Ala-42 was required for their further association. These data demonstrate that Aβ42 assembly involves formation of several distinct transient structures that gradually rearrange into protofibrils. The strong etiologic association of Aβ42 with AD may thus be a result of assemblies formed at the earliest stages of peptide oligomerization.

Crystal cataracts: Human genetic cataract caused by protein crystallization

Several human genetic cataracts have been linked recently to point mutations in the γD crystallin gene. Here we provide a molecular basis for lens opacity in two genetic cataracts and suggest that the opacity occurs because of the spontaneous crystallization of the mutant proteins. Such crystallization of endogenous proteins leading to pathology is an unusual event. Measurements of the solubility curves of crystals of the Arg-58 to His and Arg-36 to Ser mutants of γD crystallin show that the mutations dramatically lower the solubility of the protein. Furthermore, the crystal nucleation rate of the mutants is enhanced considerably relative to that of the wild-type protein. It should be noted that, although there is a marked difference in phase behavior, there is no significant difference in protein conformation among the three proteins.

Phenomenological theory of equilibrium thermodynamic properties and phase separation of micellar solutions

A detailed description and generalization of a recently developed theory, which provides analytic representations of the distribution of micellar species and the equilibrium thermodynamic properties of amphiphile–water solutions that exhibit phase separation and critical phenomena, is presented. We propose a form for the structure of the Gibbs free energy which accurately describes the essential physical factors responsible for micellization and phase separation. These are: the free‐energy advantage associated with the formation of individual micellar species,the entropy of mixing of the extended micelles and the water molecules, and the free energy of interaction between each member of the micellar size distribution. By applying to this Gibbs free energy the conditions of multiple chemical equilibrium and thermodynamic stability, all the relevant statistical and thermodynamic equilibrium properties of the micellar solution can be calculated. These properties include the location of the critical concentra...

On the presence and mechanism of formation of heavy molecular weight aggregates in human normal and cataractous lenses

It has been suggested that the presence of high molecular weight protein aggregates in the lens can lead to light scattering and a consequent loss of transparency. We have measured the concentration of aggregates having a molecular weight greater than approximately 150 × 106 g/mole present in the soluble fraction of both aging normal and cataractous human lenses. This protein population is approximately 5% of the total soluble protein in lenses up to age 75 and increases to 10–15% in lenses aged greater than 75 years and in cataracts. The amino acid composition of the heavy molecular weight soluble aggregates of normal lenses is different in leucine content from alpha, beta and gamma crystallins and different in tyrosine content from beta and gamma crystallins. The aggregates from cataractous lenses show a greater increase in leucine and greater decrease in tyrosine. An unidentified component is present in the cataractous aggregates. Furthermore, calcium, an ion which increases in concentration in cataractogenesis, induces alpha crystallin to aggregate and induces the heavy molecular weight fraction to aggregate. This aggregation is irreversible by dialysis or chelation.

Measurement of the Rotational Diffusion Coefficient of Lysozyme by Depolarized Light Scattering: Configuration of Lysozyme in Solution

We have determined the rotational diffusion coefficient (DR) of lysozyme from the spectrum of the depolarized light scattered by this protein. The sample was illuminated by a single‐frequency He–Ne laser and the spectrum of the depolarized scattered light was analyzed using a high‐resolution spherical Fabry–Perot interferometer. We have determined D20,wR to be (16.7 ± 0.8) × 106/sec at 15% protein concentration. We have, in addition, measured the translational diffusion coefficient (DT) of lysozyme by employing the now‐familiar techniques of optical mixing spectroscopy to observe the spectrum of the polarized light scattered by the protein. We find D20,wT to be (10.6 ± 0.1) × 10−7 cm2/sec, independent of protein concentration between 1% and 15%. Our results for DR and DT, combined with Perrins expressions for the rotational and translational diffusion coefficients of ellipsoids, indicate that lysozyme in solution is hydrodynamically equivalent to a prolate ellipsoid of revolution with major and minor axe...

Lysine-specific molecular tweezers are broad-spectrum inhibitors of assembly and toxicity of amyloid proteins

Amyloidoses are diseases characterized by abnormal protein folding and self-assembly, for which no cure is available. Inhibition or modulation of abnormal protein self-assembly, therefore, is an attractive strategy for prevention and treatment of amyloidoses. We examined Lys-specific molecular tweezers and discovered a lead compound termed CLR01, which is capable of inhibiting the aggregation and toxicity of multiple amyloidogenic proteins by binding to Lys residues and disrupting hydrophobic and electrostatic interactions important for nucleation, oligomerization, and fibril elongation. Importantly, CLR01 shows no toxicity at concentrations substantially higher than those needed for inhibition. We used amyloid β-protein (Aβ) to further explore the binding site(s) of CLR01 and the impact of its binding on the assembly process. Mass spectrometry and solution-state NMR demonstrated binding of CLR01 to the Lys residues in Aβ at the earliest stages of assembly. The resulting complexes were indistinguishable in size and morphology from Aβ oligomers but were nontoxic and were not recognized by the oligomer-specific antibody A11. Thus, CLR01 binds already at the monomer stage and modulates the assembly reaction into formation of nontoxic structures. The data suggest that molecular tweezers are unique, process-specific inhibitors of aberrant protein aggregation and toxicity, which hold promise for developing disease-modifying therapy for amyloidoses.

Structure determination of micelle-like intermediates in amyloid β-protein fibril assembly by using small angle neutron scattering

Increasing evidence supports the hypothesis that amyloid β-protein (Aβ) assembly is a key pathogenic feature of Alzheimers disease. Thus, understanding the assembly process offers opportunities for the development of strategies for treating this devastating disease. In prior studies, Aβ was found to form micelle-like aggregates under acidic conditions. These structures exhibited an average observed hydrodynamic radius of 7 nm. They were found to be in rapid equilibrium with Aβ monomers or low molecular weight oligomers, and were centers of fibril nucleation. Here the technique of small angle neutron scattering has been used to determine the structure of these Aβ micelles. The data reveal that the micellar assemblies comprise 30–50 Aβ monomers and have elongated geometries. The best fit of the data to a uniform spherocylinder yields a radius ≈2.4 nm and cylinder length ≈11 nm. These structure parameters remain constant over more than a decade in concentration range. The concentration independence of the length of the cylindrical aggregate indicates the presence of an internal nonrepetitive structure that spans the entire length of the Aβ assembly.