Disability for Function: Loss of Ca²⁺-Binding Is Obligatory for Fitness of Mammalian βγ-Crystallins

Vertebrate βγ-crystallins belonging to the βγ-crystallin superfamily lack functional Ca²⁺-binding sites, while their microbial homologues do not; for example, three out of four sites in lens γ-crystallins are disabled. Such loss of Ca²⁺-binding function in non-lens βγ-crystallins from mammals (e.g., AIM1 and Crybg3) raises the possibility of a trade-off in the evolutionary extinction of Ca²⁺-binding. We test this hypothesis by reconstructing ancestral Ca²⁺-binding motifs (transforming disabled motifs into the canonical ones) in the lens γB-crystallin by introducing minimal sets of mutations. Upon incorporation of serine at the fifth position in the N/D-N/D-X-X-S/T(5)-S motif, which endowed a domain with microbial characteristics, a decreased domain stability was observed. Ca²⁺ further destabilized the N-terminal domain (NTD) and its serine mutants profoundly, while the incorporation of a C-terminal domain (CTD) nullified this destabilization. On the other hand, Ca²⁺-induced destabilization of the CTD was not rescued by the introduction of an NTD. Of note, only one out of four sites is functional in the NTD of γB-crystallins responsible for weak Ca²⁺ binding, but the deleterious effects of Ca²⁺ are overcome by introduction of a CTD. The rationale for the onset of cataracts by certain mutations, such as R77S, which have not been clarified by structural means, could be explained by this work. The findings presented here shed light on the evolutionary innovations in terms of the functional loss of Ca²⁺-binding and acquisition of a bilobed domain, besides imparting additional advantages (e.g., protection from light) required for specialized functions

Brightness and photostability of emerging red and near-IR fluorescent nanomaterials for bioimaging

Many novel fluorescent nanomaterials exhibit radically different optical properties compared to organic fluorophores that are still the most extensively used class of fluorophores in biology today. Assessing the practical impact of these optical differences for bioimaging experiments is challenging due to a lack of published quantitative benchmarking data. This study therefore directly and quantitatively compares the brightness and photostability of representatives from seven classes of fluorescent materials in spectroscopy and fluorescence microscopy experiments for the first time. These material classes are: organic dyes, semiconductor quantum dots, fluorescent beads, carbon dots, gold nanoclusters, nanodiamonds, and nanorubies. The relative brightness of each material is determined and the minimum material concentrations required to generate sufficient contrast in a fluorescence microscopy image are assessed. The influence of optical filters used for imaging is also discussed and suitable filter combinations are identified. The photostability of all materials is determined under typical imaging conditions and the number of images that can be acquired is inferred. The results are expected to facilitate the transition of novel fluorescent materials from physics and chemistry into biology laboratories.9 page(s