Protein aggregation is important in food processing, and this work investigated the aggregation of food proteins as a source of amyloid fibrils for use in bionanotechnology. Both purified and crude mixtures of albumin proteins were denatured by heat, which caused aggregation to occur. Protein denaturation was measured by using circular dichroism spectrometry and by following thioflavin T fluorescence, which is widely used as a diagnostic test for amyloid formation. There was a good correlation between the increase in thioflavin T fluorescence and loss of helical structure as the temperature was increased. Formation of thioflavin T fluorescence was dependent on temperature, but less dependent on salt and protein concentration. X-ray fiber diffraction patterns of denatured bovine serum albumin suggested that the protein had a similar cross-beta structure to that of amyloid fibrils. These results are consistent with the aggregates seen during food processing, being amyloid-like in nature.

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During catalysis, all Rubisco (D-ribulose-1,5-bisphosphate carboxylase/oxygenase) enzymes produce traces of several by-products. Some of these by-products are released slowly from the active site of Rubisco from higher plants, thus progressively inhibiting turnover. Prompted by observations that Form I Rubisco enzymes from cyanobacteria and red algae, and the Form II Rubisco enzyme from bacteria, do not show inhibition over time, the production and binding of catalytic by-products was measured to ascertain the underlying differences. In the present study we show that the Form IB Rubisco from the cyanobacterium Synechococcus PCC6301, the Form ID enzyme from the red alga Galdieria sulfuraria and the low-specificity Form II type from the bacterium Rhodospirillum rubrum all catalyse formation of by-products to varying degrees; however, the by-products are not inhibitory under substrate-saturated conditions. Study of the binding and release of phosphorylated analogues of the substrate or reaction intermediates revealed diverse strategies for avoiding inhibition. Rubisco from Synechococcus and R. rubrum have an increased rate of inhibitor release. G. sulfuraria Rubisco releases inhibitors very slowly, but has an increased binding constant and maintains the enzyme in an activated state. These strategies may provide information about enzyme dynamics, and the degree of enzyme flexibility. Our observations also illustrate the phylogenetic diversity of mechanisms for regulating Rubisco and raise questions about whether an activase-like mechanism should be expected outside the green-algal/higher-plant lineage.

Significance CRISPR-Cas systems provide prokaryotic adaptive immunity against invading genetic elements. For immunity, fragments of invader DNA are integrated into CRISPR arrays by Cas1 and Cas2 proteins. Type I-F systems contain a unique fusion of Cas2 to Cas3, the enzyme responsible for destruction of invading DNA. Structural, biophysical, and biochemical analyses of Cas1 and Cas2-3 from Pectobacterium atrosepticum demonstrated that they form a 400-kDa complex with a Cas1 4 :Cas2-3 2 stoichiometry. Cas1–Cas2-3 binds, processes, and catalyzes the integration of DNA into CRISPR arrays independent of Cas3 activity. The arrangement of Cas3 in the complex, together with its redundant role in processing and integration, supports a scenario where Cas3 couples invader destruction with immunization—a process recently demonstrated in vivo.