biochemistry – Lightsources.org

Unravelling the molecular structure, self-assembly, and properties of a cephalopod protein variant

2021/04/062021/05/06

Cephalopods, such as the loliginid in Figure 1A, are known for their remarkable ability to rapidly change the color and appearance of their skin. These capabilities are enabled in part by unique structural proteins called reflectins, which play essential roles in optical behavior of cephalopod skin cells. Moreover, reflectins have demonstrated exciting potential as functional materials within the context of biophotonic and bioelectronic systems. Given reflectins’ demonstrated significance from both fundamental biology and applications perspectives, some research effort has been devoted to resolving their three-dimensional (3D) structures. However, the peculiar sequence composition of reflectins has made them extremely sensitive to subtle changes in environmental conditions and prone to aggregation, thus significantly complicating the study of their structure-function relationships and precluding their definitive molecular-level structural characterization. In this work, we have elucidated the structure of a reflectin variant at the molecular level, demonstrated a robust methodology for controlling its assembly and optical properties.

We began our studies by rationally selecting a prototypical reflectin variant (RfA1TV) by using a bioinformatics-guided approach (Figure 1B). Next, we not only produced the variant in high yield and purity but also optimized conditions for maintaining this protein in a monomeric state (Figure 1C). We then probed the protein with small angle X-ray scattering (SAXS) using the Austrian SAXS beamline at the Elettra Synchrotron Laboratory in Trieste, Italy. For this purpose, a well-dispersed solution of RfA1TV was prepared in a low-pH buffer and transferred into a glass capillary, which was positioned in the path of an incident X-ray beam. The X-rays scattered by the solution-borne RfA1TV molecules formed a 2-D pattern on a Pilatus3 1M detector (Figure 1D). Subsequently, radial averaging and image calibration of the two-dimensional data furnished corresponding one-dimensional curves, which were further processed, analyzed, and correlated with other experiments to obtain insight into the protein’s geometry (Figure 1E).

Read more on the Elettra website

Image: (A) A camera image of a Doryteuthis pealeii squid. (B) An illustration of the selection of the prototypical truncated reflectin variant (RfA1TV) from full-length Doryteuthis pealeii reflectin A1. (C) A digital camera image of a solution of primarily monomeric RfA1TV (Upper) and a corresponding cartoon of RfA1TV monomers (Lower Inset). (D) An illustration of the SAXS analysis of the reflectin variant, wherein incident X-rays are scattered by the solution-borne proteins to furnish a corresponding scattering pattern. (E)The 3D structure of RfA1TV (random coils – gray, helices – orange, β-strands – purple).

Credit: This figure has been adapted from M. J. Umerani*, P. Pratakshya* et al.Proc. Natl. Acad. Sci. U.S.A 117, 32891-32901 (2020).

Inorganic nanoparticles activity as artificial pro-enzymes

2018/09/112018/10/08

Research opens perspective for treatment of several diseases tailored to the needs of each patient

From the biochemical point of view, we are a complex set of interconnected chemical reactions. The molecules that make up our bodies are in constant transformation, and this is what makes it possible for us to get energy from food, to regenerate damage to our tissues, and to synthesize the compounds necessary for life.
These modifications usually occur with the aid of other molecules called enzymes, which promote and accelerate chemical reactions without being consumed during the process.

For the proper functioning of this complex system, the enzymes must act only at the necessary place and time. Hence, nature has developed an ingenious strategy for this to happen: inactive forms of enzymes, known as proenzymes, are continuously produced, but are activated only by specific stimuli.
The occurrence of a problem in the production of these enzymes can result in highly debilitating diseases. However, the treatment of patients by means of enzymatic replacement from natural sources is not always an adequate solution.
Therefore, researchers have been investigating synthetic systems to mimic the action of natural enzymes for biomedical applications and one of the most promising alternatives is the use of nanoparticles.

Image: Schematic figure of the action of the ultrafine cerium(III) hydroxide and cerium oxide CeO (2-x) nanoparticles . Back cover image from the Journal of Materials Chemistry B [1].

Probing tumour interiors

2018/06/192018/06/21

X-ray fluorescence mapping to measure tumour penetration by a novel anticancer agent.

A new anticancer agent developed by the University of Warwick has been studied using microfocus synchrotron X-ray fluorescence (SXRF) at I18 at Diamond Light Source. As described in The Journal of Inorganic Biochemistry, researchers saw that the drug penetrated ovarian cancer cell spheroids and the distribution of zinc and calcium was perturbed.

Platinum-based chemotherapy agents are used to treat many cancer patients, but some can develop resistance to them. To address this issue, scientists from the University of Warwick sought to employ alternative precious metals. They developed an osmium-based agent, known as FY26, which exhibits high potency against a range of cancer cell lines. To unlock the potential of this novel agent and to test its efficacy and safety in clinical trials, the team need to fully understand its mechanism of action.

To explore how FY26 behaves in tumours, the team grew ovarian cancer spheroids and used SXRF at I18 to probe the depth of penetration of the drug. They noted that FY26 could enter the cores of the spheroids, which is critical for its activity and very encouraging for the future of the drug. SXRF also enabled them to probe other metals within the cells, which showed that the distribution of zinc and calcium was altered, providing new insights into the mechanism of FY26-induced cell death.

Figure: (extract) A) Structure of FY26and related complexes, [(ŋ⁶-p-cym)Os(Azpy-NMe₂)X]⁺. B) Bright field images and SXRF elemental maps of Os, Ca and Zn in A2780 human ovarian carcinoma spheroid sections (500 nm thick) treated with 0.7 µM FY26(½ IC₅₀) for 0 or 48 h. Raster scan: 2×2 µm² step size, 1 s dwell time. Scale bar 100 µm. Calibration bar in ng mm^-2. Yellow squares in bright field images indicate areas of the spheroid studied using SXRF. Red areas in SXRF elemental maps indicate the limits of the spheroids. C) Average Os content (in ng mm^-2) as a function of distance from A2780 3D spheroid surface, after treatment for 16 h (green), 24 h (blue) or 48 h (red) with 0.7 µM FY26.

Canadian researchers unlock how seaweed is digested

2018/06/142018/06/15

Cattle on the Prairies are hundreds of kilometres from the coast and yet it’s possible that seaweed could make its way into their diet as an additive.

“Seaweed is an incredible opportunity. It is a sustainable feedstock. It grows rapidly, it doesn’t require arable land or fresh water to grow,” said Wade Abbott, research scientist at Agriculture and Agi-Food Canada’s Lethbridge Research and Development Centre.

It may seem like a leap to go from the human gut to that of cattle, but Abbott explained that by understanding the human gut microbiome, or microorganisms, and the microbiome’s ability to use the sugars found in seaweed in its symbiotic relationship with the host, he sees potential to expand what is now a limited use of algae products.

Image: Culturing gut bacteria in the lab (shown in these test tubes) allows researchers ‎to determine which genes in the genomes of bacteria are activated and discover new enzymes that digest rare substrates like agarose.
Credit: Wade Abbott

Metallic drivers of Alzheimer’s disease

2018/05/172018/05/18

The detection of iron and calcium compounds in amyloid plaque cores

X-ray spectromicroscopy at the Scanning X-ray Microscopy beamline (I08), here at Diamond, has been utilised to pinpoint chemically reduced iron and calcium compounds within protein plaques derived from brains of Alzheimer’s disease patients. The study, published in Nanoscale, has shed light on the way in which metallic species contribute to the pathogenesis of Alzheimer’s disease and could help direct future therapies.

Alzheimer’s disease is a neurodegenerative disease that is associated with dementia and shortened life expectancy. The disease is characterised by the formation of protein plaques and tangles in the brain that impair function. As well as protein plaques, perturbed metal ion homeostasis is also linked with pathogenesis, and iron levels in particular are elevated in certain regions of the brain.

A team of scientists with a long history in exploring biomineralisation in Alzheimer’s brains set out to characterise the iron species that are associated with the amyloid protein plaques. They extracted samples from the brains of two deceased patients who had Alzheimer’s and applied synchrotron X-ray spectromicroscopy to differentiate the iron oxide phases in the samples.

They noted evidence that the chemical reduction of iron, and indeed the formation of a magnetic iron oxide called magnetite, which is not commonly found in the human brain, had occurred during amyloid plaque formation, a finding that could help inform the outcomes of future Alzheimer’s therapies.

Image: Synchrotron soft X-ray nano-imaging and spectromicroscopy reveals iron and calcium biomineralisation in Alzheimer’s disease amyloid plaques.

Scientists map important immune system enzyme for the first time

2018/03/292018/04/12

Biochemists from McGill University are getting a good look at just how a specific enzyme that is part of the human immune system interacts with a certain group of bacteria that are described as gram-negative.

Researchers around the world “have been studying the enzyme, known as AOAH, for more than 30 years. This is the first time anyone has been able to see exactly what it looks like,” according to Bhushan Nagar, an associate professor of biochemistry at McGill University in Montreal.

More than that, the 3D images captured a moment in time which shows just how AOAH inactivates a toxic molecule that is commonly part of various gram-negative bacteria. The research was conducted at the Canadian Light Source.

Numerous types of gram-negative bacteria exist throughout the environment. While some are harmless, many cause a variety of human illnesses, says Nagar. For example, several species such as E. coli and Salmonella, cause food borne illness. Others cause infections such as pneumonia, meningitis, bloodstream infections or gonorrhea.

>Read more on the Canadian Light Source

Image: Bhushan Nagar (principal investigator), Alexei Gorelik (first author of paper) and Katalin Illes (research assistant at Nagar lab) at their McGill University lab.
Credit: Bhushan Nagar.