Members of the chloride intracellular channel (CLIC) family exist primarily as soluble proteins but can also auto-insert into cellular membranes to form ion channels. While little is known about the process of CLIC membrane insertion, a unique feature of mammalian CLIC1 is its ability to undergo a dramatic structural metamorphosis between a monomeric glutathione-S-transferase homolog and an all-helical dimer upon oxidation in solution. Whether this oxidation-induced metamorphosis facilitates CLIC1 membrane insertion is unclear. In this work, we have sought to characterise the role of oxidation in the process of CLIC1 membrane insertion. We examined how redox conditions modify the ability of CLIC1 to associate with and insert into the membrane using fluorescence quenching studies and a sucrose-loaded vesicle sedimentation assay to measure membrane binding. Our results suggest that oxidation of monomeric CLIC1, in the presence of membranes, promotes insertion into the bilayer more effectively than the oxidised CLIC1 dimer.

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.

Troponin C (TnC) is the calcium-binding subunit of the troponin complex responsible for initiating striated muscle contraction in response to calcium influx. In the skeletal TnC isoform, calcium binding induces a structural change in the regulatory N-domain of TnC that involves a transition from a closed to open structural state and accompanying exposure of a large hydrophobic patch for troponin I (TnI) to subsequently bind. However, little is understood about how calcium primes the N-domain of the cardiac isoform (cTnC) for interaction with the TnI subunit as the open conformation of the regulatory domain of cTnC has been observed only in the presence of bound TnI. Here we use paramagnetic relaxation enhancement (PRE) to characterize the closed to open transition of isolated cTnC in solution, a process that cannot be observed by traditional nuclear magnetic resonance methods. Our PRE data from four spin-labeled monocysteine constructs of isolated cTnC reveal that calcium binding triggers movement of the N-domain helices toward an open state. Fitting of the PRE data to a closed to open transition model reveals the presence of a small population of cTnC molecules in the absence of calcium that possess an open conformation, the level of which increases substantially upon Ca(2+) binding. These data support a model in which calcium binding creates a dynamic equilibrium between the closed and open structural states to prime cTnC for interaction with its target peptide. We also used PRE data to assess the structural effects of a familial hypertrophic cardiomyopathy point mutation located within the N-domain of cTnC (A8V). The PRE data show that the Ca(2+) switch mechanism is perturbed by the A8V mutation, resulting in a more open N-domain conformation in both the apo and holo states.

Cardiac troponin C (cTnC) is the calcium binding subunit of the troponin complex that triggers the thin filament response to calcium influx into the sarcomere. cTnC consists of two globular EF-hand domains (termed the N-and C-domains) connected by a flexible linker. While the conformation of each domain of cTnC has been thoroughly characterized through NMR studies involving either the isolated N-domain (N-cTnC) or C-domain (C-cTnC), little attention has been paid to the range of interdomain orientations possible in full-length cTnC that arises as a consequence of the flexibility of the domain linker. Flexibility in the domain linker of cTnC is essential for effective regulatory function of troponin. We have therefore utilized paramagnetic relaxation enhancement (PRE) NMR to assess the interdomain orientation of cTnC. Ensemble fitting of our interdomain PRE measurements reveals that isolated cTnC has considerable interdomain flexibility and preferentially adopts a bent conformation in solution, with a defined range of relative domain orientations.

Bio-imaging is a key technique in tracking and monitoring important biological processes and fundamental biomolecular interactions, however the interference of background autofluorescence with targeted fluorophores is problematic for many bio-imaging applications. This study reports on two novel methods for reducing interference with cellular autofluorescence for bio-imaging. The first method uses fluorescent nanodiamonds (FNDs), containing nitrogen vacancy centers. FNDs emit at near-infrared wavelengths typically higher than most cellular autofluorescence; and when appropriately functionalized, can be used for background-free imaging of targeted biomolecules. The second method uses europium-chelating tags with long fluorescence lifetimes. These europium-chelating tags enhance background-free imaging due to the short fluorescent lifetimes of cellular autofluorescence. In this study, we used both methods to target E-selectin, a transmembrane glycoprotein that is activated by inflammation, to demonstrate background-free fluorescent staining in fixed endothelial cells. Our findings indicate that both FND and Europium based staining can improve fluorescent bio-imaging capabilities by reducing competition with cellular autofluorescence. 30 nm nanodiamonds coated with the E-selectin antibody was found to enable the most sensitive detective of E-selectin in inflamed cells, with a 40-fold increase in intensity detected.