Oligomerization and phase separation in globular protein solutions

Asherie, Neer; Pande, Jayanti; Lomakin, Aleksey; Ogun, Olutayo; Hanson, Stacy R.A.; Smith, Jean B.; Benedek, George B.

doi:10.1016/s0301-4622(98)00208-7

Cited by 38 publications

(39 citation statements)

References 41 publications

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“…A similar type of phenomenon, related to the onset of a liquid-liquid phase transition, has been reported for γ-crystallin aqueous solutions (24)(25)(26)(27)(28)(29)(30)(31). This phase transition has been associated with the existence of protein-protein attractive interactions and thus is directly relevant to the refractive index properties of the eye lens and cataract formation.…”

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confidence: 63%

Oligomerization and Phase Transitions in Aqueous Solutions of Native and Truncated Human βB1-Crystallin

Annunziata

Pande

et al. 2005

Biochemistry

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Human B1-crystallin is a major eye-lens protein that undergoes in vivo truncation at the N-terminus with aging. By studying native B1 and truncated B1∆N41, which mimics an age-related in vivo truncation, we have determined quantitatively the effect of truncation on the oligomerization and phase transition properties of B1 aqueous solutions. The oligomerization studies show that the energy of attraction between the B1∆N41 proteins is about 10% greater than that of the B1 proteins. We have found that B1∆N41 aqueous solutions undergo two distinct types of phase transitions. The first phase transition involves an initial formation of thin rodlike assemblies, which then evolve to form crystals. The induction time for the formation of rodlike assemblies is sensitive to oligomerization. The second phase transition can be described as liquid-liquid phase separation (LLPS) accompanied by gelation within the protein-rich phase. We refer to this process as heterogeneous gelation. These two phase transitions are not observed in the case of B1 aqueous solutions. However, upon the addition of poly(ethylene glycol) (PEG), we observe heterogeneous gelation also for B1. Our PEG experiments allow us to estimate the difference in phase separation temperatures between B1 and B1∆N41. This difference is consistent with the increase in energy of attraction found in our oligomerization studies. Our work suggests that truncation is a cataractogenic modification since it favors protein condensation and the consequent formation of light scattering elements, and highlights the importance of the N-terminus of B1 in maintaining lens transparency.

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confidence: 63%

Oligomerization and Phase Transitions in Aqueous Solutions of Native and Truncated Human βB1-Crystallin

Annunziata

Pande

et al. 2005

Biochemistry

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“…We showed earlier that intermolecular, disulfide-crosslinked aggregates (both directly S-S-linked and chemically crosslinked with a spacer) markedly affect another important property of the ␥-crystallins, namely liquid-liquid phase separation (8,19). In this phenomenon, the protein solution segregates spontaneously into protein-rich and protein-poor phases (20).…”

Section: Resultsmentioning

confidence: 99%

“…Proteins were eluted isocratically with 0.1 M sodium phosphate buffer (pH 7.1) containing 0.02% sodium azide (8). Phase Separation Measurements.…”

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confidence: 99%

Molecular basis of a progressive juvenile-onset hereditary cataract

Pande

Asherie

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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We have expressed recombinant wild-type human ␥D crystallin (HGD) and its Arg-14 to Cys mutant (R14C) in Escherichia coli and show that R14C forms disulfide-linked oligomers, which markedly raise the phase separation temperature of the protein solution. Eventually, R14C precipitates. In contrast, HGD slowly forms only disulfide-linked dimers and no oligomers. These data strongly suggest that the observed cataract is triggered by the thiolmediated aggregation of R14C. The aggregation profiles of HGD and R14C are consistent with our homology modeling studies that reveal that R14C contains two exposed cysteine residues, whereas HGD has only one. Our CD, fluorescence, and differential scanning calorimetric studies show that HGD and R14C have nearly identical secondary and tertiary structures and stabilities. Thus, contrary to current views, unfolding or destabilization of the protein is not necessary for cataractogenesis.I n the hereditary, juvenile-onset cataract described by Stefan et al. (1), the lens, which is clear at birth, develops punctate opacities progressively, such that by two years of age the cataract is readily detectable, and matures by early childhood or adolescence. The punctate opacities seen in this cataract are in the nucleus and inner cortex, regions of the lens that are enriched in the ␥-crystallins. In the human lens, only two members of the ␥-crystallin family, ␥C and ␥D, are expressed in appreciable amounts, and only ␥D crystallin continues to be expressed until late childhood (2, 3). In affected individuals, a single point mutation has been identified in the ␥D crystallin gene that corresponds to the substitution of Arg-14 by a Cys. The identification of this mutation and the parallel between the time course of the pathology and the physiological expression of human ␥D crystallin strongly implicate the Arg-14 3 Cys mutant of ␥D in the development of this cataract. However, the molecular mechanism invoked to explain the observed opacity has been speculative (1).In the past, it has not been possible to conduct detailed studies on human ␥D crystallin because of the difficulty of obtaining sufficient quantities of pure protein from young, normal human lenses (4). Therefore, to characterize the normal protein thoroughly and investigate the mechanism by which the Arg-14 3 Cys mutation in ␥D could lead to cataract, we cloned and expressed human ␥D crystallin and its Arg-14 3 Cys mutant in Escherichia coli. Both the wild-type recombinant ␥D crystallin (HGD) and its Arg-14 3 Cys mutant (R14C) folded efficiently in E. coli and accumulated as soluble proteins. We isolated and purified the HGD and R14C proteins and determined their solution properties. Our results suggest that the disulfidecrosslinked oligomerization of R14C is responsible for the observed cataract. Furthermore, such oligomerization occurs without significant change in protein structure, conformation, and stability. Materials and MethodsCloning, Expression, and Isolation of Proteins. The human ␥D crystallin coding sequence was amplif...

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“…The presence of protein aggregates has been previously shown to increase T ph , but we find no evidence that this is the source of the increase in T ph in our case. 60,61 To probe this further, we induced LLPS in a sample and allowed the concentrated and dilute phases to fully phase separate. 62 We then measured both the total protein volume fraction and the fluorescence intensity in each of the two phases (Fig.…”

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confidence: 99%

How fluorescent labelling alters the solution behaviour of proteins

Quinn

Gnan²,

James

et al. 2015

Phys. Chem. Chem. Phys.

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A complete understanding of the role of molecular anisotropy in directing the self assembly of colloids and proteins remains a challenge for soft matter science and biophysics. For proteins in particular, the complexity of the surface at a molecular level poses a challenge for any theoretical and numerical description. A soft matter approach, based on patchy models, has been useful in describing protein phase behaviour. In this work we examine how chemical modification of the protein surface, by addition of a fluorophore, affects the physical properties of protein solutions. By using a carefully controlled experimental protein model (human gamma-D crystallin) and numerical simulations, we demonstrate that protein solution behaviour defined by anisotropic surface effects can be captured by a custom patchy particle model. In particular, the chemical modification is found to be equivalent to the addition of a large hydrophobic surface patch with a large attractive potential energy well, which produces a significant increase in the temperature at which liquid-liquid phase separation occurs, even for very low fractions of fluorescently labelled proteins. These results are therefore directly relevant to all applications based on the use of fluorescent labelling by chemical modification, which have become increasingly important in the understanding of biological processes and biophysical interactions.

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Oligomerization and phase separation in globular protein solutions

Cited by 38 publications

References 41 publications

Oligomerization and Phase Transitions in Aqueous Solutions of Native and Truncated Human βB1-Crystallin

Oligomerization and Phase Transitions in Aqueous Solutions of Native and Truncated Human βB1-Crystallin

Molecular basis of a progressive juvenile-onset hereditary cataract

How fluorescent labelling alters the solution behaviour of proteins

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