Folding, stability and aggregation of the long-lived eye lens protein human gamma D crystallin

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2006.Vita.Includes bibliographical references (p. 179-194).Human [gamma]D crystallin (H[gamma]D-Crys) is a monomeric, two domain, primarily P-sheet protein found in high concentrations in the human eye lens. H[gamma]D-Crys and other crystallins are found in insoluble protein inclusions associated with the eye disease cataract. H[gamma]D-Crys is expressed in utero and does not regenerate during life, thus necessitating high stability and solubility. Covalent damage, including glutamine deamidation, of the lens crystallins increases with age and as a result of exposure to environmental insults. Such covalent damage may cause partial-unfolding into aggregation-prone confomations that cause cataract. The in vitro stability of H[gamma]D-Crys was analyzed in the denaturant guanidine h[gamma]Drochloride at pH 7.0 and 370C. An off-pathway aggregation reaction that competed with refolding was previously reported when H[gamma]D-Crys was refolded to less than 1 M GuHC1. Equilibrium transitions of H[gamma]D-Crys were best fit to a three-state model suggesting the presence of a partially-folded intermediate that likely had a structured C-terminal domain (C-td) and unstructured N-terminal domain (N-td). Similarly, previous analyses revealed a sequential domain refolding pathway where the C-td refolds first followed by the N-td.(cont.) These findings suggest that the inter-domain interface of H[gamma]D-Crys is important in both folding and stability. The domain interface of H[gamma]D-Crys contains a central h[gamma]Drophobic cluster of six residues and two pairs of peripheral interacting residues. To test this importance of these residues in folding and stability, site-directed alanine mutants were constructed at all ten positions and properties of the mutant proteins were analyzed. Single mutations of h[gamma]Drophobic domain interface residues caused a decrease in stability of the N-td, but did not affect stability of the C-td. Similarly, stability of the N-td but not the C-td was reduced as a result of single and double mutations of peripheral interface residues. Minimal to no interaction energy was observed for the peripheral residues suggesting they contribute to stability indirectly, perhaps by shielding the central h[gamma]Drophobic cluster from solvent. Both the h[gamma]Drophobic and peripheral domain interface alanine mutants also had reduced rates of productive refolding for the N-td while refolding rates for the C-td were unchanged. These results suggest a productive folding pathway where the C-td refolds first and domain interface residues of the structured C-td act as a nucleating center for refolding of the N-td.(cont.) Effects on N-td refolding rates were most prominent for the h[gamma]Drophobic residues indicating the importance of proper h[gamma]Drophobic burial during refolding. The peripheral domain interface residues of H[gamma]D-Crys include a pair of two glutamines that are targets for covalent damage during aging. Deamidation mimics at these sites were constructed by site directed mutagenesis of glutamine to glutamate. Properties of the mutants were analyzed to assess the affects of deamidation on stability and folding. Similar to the alanine mutants at these sites, the deamidation mutants had a destabilized N-td but not C-td at pH 7.0. In contrast, stabilities of the mutants were indistinguishable from wild type at pH 3.0. The N-td of the deamidation mutants also unfolded faster than that of wild type during kinetic unfolding. These results indicate that deamidation of domain interface glutamines destabilizes H[gamma]D-Crys and lowers the kinetic barrier to unfolding. A reduction in the thermodynamic and kinetic stability as a result of domain interface deamidation could result in the population of partially-unfolded conformations in the lens that may aggregate through mechanisms such as domain swapping or loop-sheet insertion.by Shannon L. Flaugh.Ph.D

Silver Iodate

InChI = 1S/Ag.HIO3/c;2-1(3)4/h;(H,2,3,4)/q+1;/p-1 InChIKey = YSVXTGDPTJIEIX-UHFFFAOYSA-M (reagent used as a versatile oxidative amidation and cyclization component) Physical Data: mp >200 °C; d 5.53 g cm^(−3). Solubility: soluble in aqueous ammonia; practically insoluble in water (0.3 g L^(−1) at 10 °C). Form Supplied in: white crystalline powder; commercially available. Handling, Storage, and Precautions: irritant; light sensitive; causes ignition with reducing agents or combustibles; store in cool and dry conditions in well-sealed containers; handle in fume hood

Cataract-Causing Defect of a Mutant γ-Crystallin Proceeds through an Aggregation Pathway Which Bypasses Recognition by the α-Crystallin Chaperone

Background: The transparency of the eye lens depends upon maintenance of the native state of the γ- and β-crystallins, which is aided by the abundant chaperones αA- and αB-crystallin. Mature onset cataract, the leading cause of blindness worldwide, involves the polymerization of covalently damaged or partially unfolded crystallins into light-scattering aggregates. A number of single amino acid substitutions and truncations of γ-crystallins result in congenital cataract in both humans and mice, though in many cases the coupling between the protein alterations and the accumulation of aggregates is poorly defined. Methodology/Principal Findings: We have studied the aggregation properties and chaperone interactions of human γD-crystallin carrying substitutions of two buried core mutants, I90F and V75D, which cause congenital cataract in mice. The in vitro aggregation pathway competing with productive refolding was not altered by either substitution. Furthermore, this aggregation pathway for both mutant proteins–originating from a partially folded intermediate–was efficiently suppressed by αB-crystallin. Thus the cataract pathology was unlikely to be associated with a direct folding defect. The native state of wild-type human γD-crystallin exhibited no tendency to aggregate under physiological conditions. However both I90F and V75D native-like proteins exhibited slow (days) aggregation to high molecular weight aggregates under physiological conditions. The perturbed conformation of I90F was recognized and bound by both αA and αB chaperones. In contrast, the aggregation derived from the perturbed state of V75D was not suppressed by either chaperone, and the aggregating species were not bound by the chaperone. Conclusions/Significance: The cataract phenotype of I90F in mice may be due to premature saturation of the finite α- crystallin pool. The V75D aggregation pathway and its escape from chaperone surveillance and aggregation suppression can account for the congenital cataract pathology of this mutant. Failure of chaperone recognition may be an important source of pathology for many other protein folding defects.National Eye Institute (Grant no. EY015834 )National Institutes of Health (U.S.) (Grant no. GM17980

The Congenital Cataract-Linked G61C Mutation Destabilizes γD-Crystallin and Promotes Non-Native Aggregation

γD-crystallin is one of the major structural proteins in human eye lens. The solubility and stability of γD-crystallin play a crucial role in maintaining the optical properties of the lens during the life span of an individual. Previous study has shown that the inherited mutation G61C results in autosomal dominant congenital cataract. In this research, we studied the effects of the G61C mutation on γD-crystallin structure, stability and aggregation via biophysical methods. CD, intrinsic and extrinsic fluorescence spectroscopy indicated that the G61C mutation did not affect the native structure of γD-crystallin. The stability of γD-crystallin against heat- or GdnHCl-induced denaturation was significantly decreased by the mutation, while no influence was observed on the acid-induced unfolding. The mutation mainly affected the transition from the native state to the intermediate but not that from the intermediate to the unfolded or aggregated states. At high temperatures, both proteins were able to form aggregates, and the aggregation of the mutant was much more serious than the wild type protein at the same temperature. At body temperature and acidic conditions, the mutant was more prone to form amyloid-like fibrils. The aggregation-prone property of the mutant was not altered by the addition of reductive reagent. These results suggested that the decrease in protein stability followed by aggregation-prone property might be the major cause in the hereditary cataract induced by the G61C mutation

Studies on Power Hop Using a Nonlinear Model of an Agricultural Tractor

Power hop is an unstable dynamic phenomenon that is observed on agricultural tractors operating at field working speeds under typical draft loads. This dynamic instability can be identified as a bounce motion preceded by an increasing oscillation in either the pitch or bounce modes. A definite and comprehensive correction or prevention strategy for the control of power hop does not exist. In this work a linear and a nonlinear model of an agricultural tractor were developed. The method of multiple time scales has been employed in the analysis of the nonlinear model. Numerical results have been obtained using Mathematica to verify that power hop can be modeled as a nonlinear oscillator. Solutions to the nonlinear model were obtained for several sets of system parameters. Parametric studies and frequency response curves were performed and investigated

The psychological reactance dilemma: Effects of restricting workers' personal social media use

U.S. organizations often employ deterrence mechanisms to regulate workers’ technology use, but such approaches are not always effective (Sommestad, Hallberg, Lundholm, & Bengtsson, 2014). The theory of psychological reactance (Brehm & Brehm, 1981) was explored as a potential explanation for the inconsistencies in the effectiveness of deterrence mechanisms. It was postulated that workers expect the freedom to use certain types of technology within the workplace and that restricting such use would result in opposition. This possibility was investigated in the context of intentions to use social media, a technology popular among U.S. workers. A 3 (personal social media use restriction) x 3 (sanctions) between-subject experimental design was used to test the effect of restrictive personal social media use guidelines and sanctions on workers’ compliance and use intentions. U.S. workers (N = 715) recruited through MTurk completed an online survey in which they were randomly assigned to one of nine conditions. The IVs were manipulated through the use of vignettes. The DVs were measured using both scales modified from previous studies and newly constructed scales. The results suggest that the average worker does not engage in freedom restoration when social media is restricted. Overall, participants were more compliant, had lower social media intentions, and social media’s valence was lowest when social media was restricted and sanctions were used. Explanations for the findings are provided with extensions to conservation of resource, deterrence, and justice theories