Protein refractive index increment is determined by conformation as well as composition.

The refractive index gradient of the eye lens is controlled by the concentration and distribution of its component crystallin proteins, which are highly enriched in polarizable amino acids. The current understanding of the refractive index increment ([Formula: see text]) of proteins is described using an additive model wherein the refractivity and specific volume of each amino acid type contributes according to abundance in the primary sequence. Here we present experimental measurements of [Formula: see text] for crystallins from the human lens and those of aquatic animals under uniform solvent conditions. In all cases, the measured values are much higher than those predicted from primary sequence alone, suggesting that structural factors also contribute to protein refractive index

Human αB-crystallin discriminates between aggregation-prone and function-preserving variants of a client protein.

BackgroundThe eye lens crystallins are highly soluble proteins that are required to last the lifespan of an organism due to low protein turnover in the lens. Crystallin aggregation leads to formation of light-scattering aggregates known as cataract. The G18V mutation of human γS-crystallin (γS-G18V), which is associated with childhood-onset cataract, causes structural changes throughout the N-terminal domain and increases aggregation propensity. The holdase chaperone protein αB-crystallin does not interact with wild-type γS-crystallin, but does bind its G18V variant. The specific molecular determinants of αB-crystallin binding to client proteins is incompletely charcterized. Here, a new variant of γS, γS-G18A, was created to test the limits of αB-crystallin selectivity.MethodsMolecular dynamics simulations were used to investigate the structure and dynamics of γS-G18A. The overall fold of γS-G18A was assessed by circular dichroism (CD) spectroscopy and intrinsic tryptophan fluorescence. Its thermal unfolding temperature and aggregation propensity were characterized by CD and DLS, respectively. Solution-state NMR was used to characterize interactions between αB-crystallin and γS-G18A.ResultsγS-G18A exhibits minimal structural changes, but has compromised thermal stability relative to γS-WT. The placement of alanine, rather than valine, at this highly conserved glycine position produces minor changes in hydrophobic surface exposure. However, human αB-crystallin does not bind the G18A variant, in contrast to previous observations for γS-G18V, which aggregates at physiological temperature.ConclusionsαB-crystallin is capable of distinguishing between aggregation-prone and function-preserving variants, and recognizing the transient unfolding or minor conformers that lead to aggregation in the disease-related variant.General significanceHuman αB-crystallin distinguishes between highly similar variants of a structural crystallin, binding the cataract-related γS-G18V variant, but not the function-preserving γS-G18A variant, which is monomeric at physiological temperature

Sequence comparison, molecular modeling, and network analysis predict structural diversity in cysteine proteases from the Cape sundew, Drosera capensis

Carnivorous plants represent a so far underexploited reservoir of novel proteases with potentially useful activities. Here we investigate 44 cysteine proteases from the Cape sundew, Drosera capensis, predicted from genomic DNA sequences. D. capensis has a large number of cysteine protease genes; analysis of their sequences reveals homologs of known plant proteases, some of which are predicted to have novel properties. Many functionally significant sequence and structural features are observed, including targeting signals and occluding loops. Several of the proteases contain a new type of granulin domain. Although active site residues are conserved, the sequence identity of these proteases to known proteins is moderate to low; therefore, comparative modeling with all-atom refinement and subsequent atomistic MD-simulation is used to predict their 3D structures. The structure prediction data, as well as analysis of protein structure networks, suggest multifarious variations on the papain-like cysteine protease structural theme. This in silico methodology provides a general framework for investigating a large pool of sequences that are potentially useful for biotechnology applications, enabling informed choices about which proteins to investigate in the laboratory

Sequence comparison, molecular modeling, and network analysis predict structural diversity in cysteine proteases from the Cape sundew, Drosera capensis.

Carnivorous plants represent a so far underexploited reservoir of novel proteases with potentially useful activities. Here we investigate 44 cysteine proteases from the Cape sundew, Drosera capensis, predicted from genomic DNA sequences. D. capensis has a large number of cysteine protease genes; analysis of their sequences reveals homologs of known plant proteases, some of which are predicted to have novel properties. Many functionally significant sequence and structural features are observed, including targeting signals and occluding loops. Several of the proteases contain a new type of granulin domain. Although active site residues are conserved, the sequence identity of these proteases to known proteins is moderate to low; therefore, comparative modeling with all-atom refinement and subsequent atomistic MD-simulation is used to predict their 3D structures. The structure prediction data, as well as analysis of protein structure networks, suggest multifarious variations on the papain-like cysteine protease structural theme. This in silico methodology provides a general framework for investigating a large pool of sequences that are potentially useful for biotechnology applications, enabling informed choices about which proteins to investigate in the laboratory

Deamidation of the human eye lens protein γS-crystallin accelerates oxidative aging

Cataract, a clouding of the eye lens from protein precipitation, affects millions of people every year. The lens proteins, the crystallins, show extensive post-translational modifications (PTMs) in cataractous lenses. The most common PTMs, deamidation and oxidation, promote crystallin aggregation; however, it is not clear precisely how these PTMs contribute to crystallin insolubilization. Here, we report six crystal structures of the lens protein γS-crystallin (γS): one of the wild-type and five of deamidated γS variants, from three to nine deamidation sites, after sample aging. The deamidation mutations do not change the overall fold of γS; however, increasing deamidation leads to accelerated disulfide-bond formation. Addition of deamidated sites progressively destabilized protein structure, and the deamidated variants display an increased propensity for aggregation. These results suggest that the deamidated variants are useful as models for accelerated aging; the structural changes observed provide support for redox activity of γS-crystallin in the lens

Stability of Protein-Specific Hydration Shell on Crowding

We demonstrate that the effect of protein crowding is critically dependent on the stability of the protein’s hydration shell, which can dramatically vary between different proteins. In the human eye lens, γS-crystallin (γS-WT) forms a densely packed transparent hydrogel with a high refractive index, making it an ideal system for studying the effects of protein crowding. A single point mutation generates the cataract-related variant γS-G18V, dramatically altering the optical properties of the eye lens. This system offers an opportunity to explore fundamental questions regarding the effect of protein crowding, using γS-WT and γS-G18V: (i) how do the diffusion dynamics of hydration water change as a function of protein crowding?; and (ii) upon hydrogel formation of γS-WT, has a dynamic transition occurred generating a single population of hydration water, or do populations of bulk and hydration water coexist? Using localized spin probes, we separately probe the local translational diffusivity of both surface hydration and interstitial water of γS-WT and γS-G18V in solution. Surprisingly, we find that under the influence of hydrogel formation at highly crowded γS-WT concentrations up to 500 mg/mL, the protein hydration shell remains remarkably dynamic, slowing by less than a factor of 2, if at all, compared to that in dilute protein solutions of ∼5 mg/mL. Upon self-crowding, the population of this robust surface hydration water increases, while a significant bulk-like water population coexists even at ∼500 mg/mL protein concentrations. In contrast, surface water of γS-G18V irreversibly dehydrates with moderate concentration increases or subtle alterations to the solution conditions, demonstrating that the effect of protein crowding is highly dependent on the stability of the protein-specific hydration shell. The core function of γS-crystallin in the eye lens may be precisely its capacity to preserve a robust hydration shell, whose stability is abolished by a single G18V mutation