Two-dimensional IR spectroscopy and segmental 13C labeling reveals the domain structure of human γD-crystallin amyloid fibrils

The structural eye lens protein γD-crystallin is a major component of cataracts, but its conformation when aggregated is unknown. Using expressed protein ligation, we uniformly ¹³C labeled one of the two Greek key domains so that they are individually resolved in two-dimensional (2D) IR spectra for structural and kinetic analysis. Upon acid-induced amyloid fibril formation, the 2D IR spectra reveal that the C-terminal domain forms amyloid β-sheets, whereas the N-terminal domain becomes extremely disordered but lies in close proximity to the β-sheets. Two-dimensional IR kinetics experiments show that fibril nucleation and extension occur exclusively in the C-terminal domain. These results are unexpected because the N-terminal domain is less stable in the monomer form. Isotope dilution experiments reveal that each C-terminal domain contributes two or fewer adjacent β-strands to each β-sheet. From these observations, we propose an initial structural model for γD-crystallin amyloid fibrils. Because only 1 μg of protein is required for a 2D IR spectrum, even poorly expressing proteins can be studied under many conditions using this approach. Thus, we believe that 2D IR and protein ligation will be useful for structural and kinetic studies of many protein systems for which IR spectroscopy can be straightforwardly applied, such as membrane and amyloidogenic proteins.Cataracts are a protein misfolding disease caused by the aggregation of lens crystallin proteins into insoluble deposits that blur vision (1, 2). Because these proteins are not regenerated, damage from UV radiation, oxidative stress, and other chemical modifications accumulates with time (1, 2). As a result, over 50% of the population over 55 develops age-related cataracts (2). Additionally, numerous mutations that destabilize crystallin protein folds are linked to inherited and juvenile-onset cataracts (1). Although the causative factors associated with this disease are known, the structures of the aggregates and the mechanisms by which they form are unknown.Like other protein aggregation diseases such as type II diabetes mellitus and Alzheimer’s disease, the molecular structures of proteins in cataracts are difficult to determine. Atomic-level structures have been obtained for some amyloid aggregates of peptides using NMR spectroscopy (3, 4) and X-ray crystallography (5). However, the most widely used techniques for studying aggregate structures and aggregation mechanisms are circular dichroism spectroscopy, fluorescence spectroscopy, and transmission electron microscopy, which provide little detailed structural information. Two-dimensional (2D) IR spectroscopy is emerging as an important tool for studying protein aggregates such as amyloid fibrils (6–8) because it provides bond-by-bond structural resolution on kinetically evolving samples (6, 8–10). Two-dimensional IR spectroscopy probes secondary structure through cross peak couplings and solvent exposure through 2D lineshapes. Its bond-specific structural resolution comes from isotope labeling. Mechanistic information is obtained from rapid-scan methods that track the kinetics of aggregate formation (6). Spectra can be calculated from molecular dynamics simulations allowing structural models to be tested (11–15). Isotope-edited 2D IR spectroscopy using ¹³C = ¹⁸O labeled backbone amides has been used to study the fibril structure and mechanism of fibril formation for amyloid beta (Aβ) (8) and the human islet amyloid polypeptide (hIAPP) (6, 7), as well as many membrane-bound (13, 16) and soluble polypeptides (9, 10).Thus far, isotope-edited infrared spectroscopy has been limited to peptides that can be made by solid phase synthesis, which is how the ¹³C = ¹⁸O labels are incorporated (17). However, solid phase synthesis is limited to peptides of about 45–70 residues. Even if longer sequences could be synthesized, a single ¹³C = ¹⁸O label would eventually be obscured by Glu, Asp, and Arg side chains, as well as natural abundance ¹³C = ¹⁶O bonds, all of which absorb in the same frequency range (18). Nonnatural amino acids and deuterium labels are well-resolved in protein IR spectra, but only report on local environment not structure (19–21). In this article, we describe an alternative approach in which we isotope label an entire section of a protein rather than a single C═O bond. To do so, we express labeled and unlabeled segments of the protein separately, and then reconstitute the full-length protein using expressed protein ligation (22), which is a variant of native chemical ligation (23). We show that the unlabeled and ¹³C labeled regions are spectroscopically distinguishable, enabling the acquisition of structural and kinetic information on large proteins. We apply our technique to study amyloid fibril formation in the 173 amino acid eye lens protein γD-crystallin and learn that only one of its two structurally homologous domains forms the β-sheet core of the fibrils.γD-crystallin is a member of the γ-crystallin family of monomeric, soluble structural eye lens proteins which constitute about 20% of the total lens proteins and are a major component of insoluble cataract deposits (24). Native γD-crystallin consists of two Greek key domains that are each 80–90 residues long, connected by a short loop (Fig. 1) (25). Numerous modes of aggregation have been proposed for the two-domain structure, including amorphous precipitation, domain-swap oligomerization, and amyloid fibril formation (1, 26, 27). In vitro, fibrillar deposits of γ-crystallins are created by UV damage, low pH, and chemical and thermal denaturation, although it is unclear to what extent each form (or others) contribute to in vivo cataracts (1, 27–29). We utilize acid initiated aggregation because it reproducibly generates fibrillar aggregates of γ-crystallins and because low pH conditions encountered during lens development may be relevant to juvenile cataract formation (27). γD-crystallin, and each of its isolated domains, forms amyloid fibrils in vitro at pH 3 and 37 °C (27). Although there is no domain-specific structural information in the fibril state, fibrils formed from the isolated N-terminal domain (NTD) more closely resemble fibrils from full-length γD-crystallin than do fibrils from the C-terminal domain (CTD), as characterized by FTIR spectroscopy and transmission electron microscopy (TEM) imaging (27). Moreover, the NTD is less stable (30, 31) and has partially unfolded states at pH 7. Although the relationship between domain stability, partially unfolded states, and aggregation propensity is unclear (32), these results have led to the suggestion that the NTD is most likely to form the β-sheets of the amyloid fibril core (33). We show that it is actually the CTD of full-length human γD-crystallin that forms the β-sheets of the amyloid fibril core in low pH aggregates.Open in a separate windowFig. 1.Two-dimensional IR spectroscopy of native human γD-crystallin. (Top) Crystal structure of human γD-crystallin indicating the division of domains in the segmental labeling scheme. (Bottom, A–D) Two-dimensional IR spectra of full-length γD-crystallin at pD = 7. Frequencies and FWHM node slopes are summarized in Table S1.     

Using participatory video to challenge the stigma of mental illness: a case study

The diagnosis of mental ill health continues to attract substantial stigma in western society, with evidence suggesting public attitudes to be increasingly negative. Recent reviews have highlighted the extensive research on the nature of this stigma but with limited work on the development of strategies to challenge the stigma. The aim of this case study was to explore the potential of researchers and mental health service users (MHSUs) working collaboratively to identify the main problems the service users experience in their everyday lives and to produce a video challenging the negative image of mental ill health. Discussions were held with volunteers involved in a mental health media action group; all volunteers had been or were currently MHSU. These discussions identified a variety of problems including difficulties in everyday social interaction and negative portrayal of mental ill health in the media. A short video was developed with volunteers summarizing the issues they had raised: this was subsequently shown to a wider audience. The MHSUs reported considerable personal benefits of participation in the project. The paper discusses these findings and the process of producing the video.     

Measuring quality of life in aphasia: Results from two scales

Background: Although aphasia affects quality of life (QoL), the impact within specific domains (e.g., psychosocial, communication) is poorly understood. Moreover, the complex and multidimensional nature of QoL renders it difficult to measure accurately using a single global scale.

Aims: Using two recently developed QoL scales, the Stroke and Aphasia Quality of Life Scale‐39, (SAQOL; Hilari, Byng, Lamping, & Smith, 2003a Hilari, K., Byng, S., Lamping, D. L. and Smith, S. C. 2003a. Stroke and Quality of Life Scale‐39 (SAQOL‐39): Evaluation of acceptability, reliability and validity.. Stroke, 34: 1944–1950. [Crossref], [PubMed], [Web of Science ®] , [Google Scholar]) and the American Speech Language Hearing Association's Quality of Communication Life Scale (QCL; Paul et al., 2004 Paul, D. R., Frattali, C. M., Holland, A. L., Thompson, C. K., Caperton, C. J. and Slater, S. C. 2004. Quality of Communication Life Scale, Rockville, MD: The American Speech‐Language‐Hearing Association.  [Google Scholar]), this study aimed to document the domains of QoL that were most affected for participants with aphasia compared to control participants, as well as to determine the relationship between the two scales, their sub‐domains, and linguistic variables in aphasia.

Methods & Procedures: The two scales were administered to a group of 19 participants with aphasia (14 male, 5 female), ages ranging from 27 to 79 years, and 19 age‐ and gender‐matched control participants. Various types and severity of aphasia were represented in the aphasia group. The performances of aphasia and control groups were compared, and correlation analyses examined the relationship between the two scales and their sub‐domains in the aphasia group only.

Outcomes & Results: Compared to control participants, QoL was lower in participants with aphasia, with the communication sub‐domain of SAQOL and socialisation/activities sub‐domain of QCL being the most affected areas of functioning. Between the two scales, the communication sub‐domain of SAQOL correlated with the socialisation/activities sub‐domain and the QCL mean. Moreover, linguistic variables correlated strongly with psychosocial, communication and socialisation/activities sub‐domains of QoL.

Conclusions: Measuring QoL using the SAQOL and the QCL captures different but equally important aspects of experiences of living with aphasia. When interpreted together, they provide a holistic picture of functioning in aphasia that includes broad overviews of QoL from the SAQOL and a finer‐grained analysis of communication impairments on QoL from the QCL.     

Digital Promotion of Energy Drinks to Young Adults Is More Strongly Linked to Consumption Than Other Media

Objective

To examine whether digital marketing strategies are more strongly associated with energy drink use than other marketing and whether Theory of Planned Behavior (TPB) constructs mediated the effects of digital marketing on energy drink use.