Thermus thermophilus transcriptional factor TtCarH belongs to a newly discovered class of photoreceptors that use 5'-deoxyadenosylcobalamin (AdoB12) as the light-sensing chromophore. Photoregulation relies on the repressor activity of AdoB12-bound oligomers in the dark, which light counteracts by oligomer disruption due to AdoB12 photolysis. In this study, we investigated TtCarH self-association and binding to DNA in the dark and in the light using analytical ultracentrifugation (AUC) methods, both sedimentation velocity (SV) as well as equilibrium (SE). From a methodological point of view, this study shows that AUC can provide hydrodynamic insights in cases where light is a crucial determinant of solution properties. For the light-sensitive TtCarH, absorbance as well as interference AUC data yielded comparable results. Sedimentation coefficients and whole-body hydrodynamic analysis from SV experiments indicate that in solution apo-TtCarH and light-exposed AdoB12-TtCarH are predominantly aspherical, ellipsoidal monomers, in accord with SE data. By comparison, AdoB12-TtCarH exists as a more compact tetramer in the dark, with smaller forms such as dimers or monomers remaining undetected and low levels of larger oligomers appearing at higher protein concentrations. AUC analyses indicate that in the dark AdoB12-TtCarH associates as a tetramer with DNA but forms smaller complexes in the apo form or if exposed to light. The self-association and DNA-binding properties of TtCarH deduced from AUC are consistent with data from size-exclusion and DNA-binding gel-shift assays. AUC analyses together with hydrodynamic modeling provide insights into the AdoB12- and light-dependent self-association and DNA-binding of TtCarH.

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00249-013-0897-xDOI Listing

Publication Analysis

Top Keywords

analytical ultracentrifugation
8
dna-binding ttcarh
8
thermus thermophilus
8
dark light
8
auc analyses
8
self-association dna-binding
8
ttcarh
6
auc
6
ultracentrifugation studies
4
studies oligomerization
4

Similar Publications

Knowledge of the structure-property relationships of functional nanomaterials, including, for example, their size- and composition-dependent photoluminescence (PL) and particle-to-particle variations, is crucial for their design and reproducibility. Herein, the Angstrom-resolution capability of an analytical ultracentrifuge combined with an in-line multiwavelength emission detection system (MWE-AUC) for measuring the sedimentation coefficient-resolved spectrally corrected PL spectra of dispersed nanoparticles is demonstrated. The capabilities of this technique are shown for giant-shell CdSe/CdS quantum dots (g-QDs) with a PL quantum yield (PL QY) close to unity capped with oleic acid and oleylamine ligands.

View Article and Find Full Text PDF

The aim of this study was to assess the critical quality attributes of parenteral nanoemulsion formulations by measuring several physicochemical parameters and linking them to their in vitro performance, illustrating how simplistic and routinely used approaches are insufficient for understanding a potential nanomedicine. Physicochemical characterization should encompass size and size distribution through at least two orthogonal techniques, such as dynamic light scattering (DLS) and electron microscopy, with added value from analytical ultracentrifugation. In vitro toxicity assessment was performed using three different assays to determine mitochondrial activity (WST-1), membrane integrity (lactate dehydrogenase release (LDH) assay), and cell viability (propidium iodide (PI) staining).

View Article and Find Full Text PDF

Lipid nanoparticles (LNPs) are the most advanced delivery system currently available for RNA therapeutics. Their development has accelerated since the success of Patisiran, the first siRNA-LNP therapeutic, and the mRNA vaccines that emerged during the COVID-19 pandemic. Designing LNPs with specific targeting, high potency, and minimal side effects is crucial for their successful clinical use.

View Article and Find Full Text PDF

Recombinant adeno-associated virus (AAV) is one of the main viral vector-based gene therapy platforms. AAV is a virus consisting of a ≈25 nm diameter capsid with a ≈4.7 kb cargo capacity.

View Article and Find Full Text PDF

Background: Grossly lipaemic samples are a significant cause of analytical errors, potentially impacting patient care. The causes of lipaemia are varied and often unavoidable, while methods to reduce lipaemia through gold-standard ultracentrifugation are limited by availability, transportation and cost. Benchtop centrifugation has been proposed as an alternative method to reduce lipaemia.

View Article and Find Full Text PDF

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!