Distinct growth regimes of α-synuclein amyloid elongation.

Addition of amyloid seeds to aggregation-prone monomers allows for amyloid fiber growth (elongation) omitting slow nucleation. We here combine Thioflavin T fluorescence (probing formation of amyloids) and solution-state NMR spectroscopy (probing disappearance of monomers) to assess elongation kinetics of the amyloidogenic protein, α-synuclein, for which aggregation is linked to Parkinson's disease. We found that both spectroscopic detection methods give similar kinetic results, which can be fitted by applying double exponential decay functions.

Fluorine NMR Spectroscopy Enables to Quantify the Affinity Between DNA and Proteins in Cell Lysate.

The determination of the binding affinity quantifying the interaction between proteins and nucleic acids is of crucial interest in biological and chemical research. Here, we have made use of site-specific fluorine labeling of the cold shock protein from Bacillus subtilis, BsCspB, enabling to directly monitor the interaction with single stranded DNA molecules in cell lysate. High-resolution F NMR spectroscopy has been applied to exclusively report on resonance signals arising from the protein under study.

Insights into Protein Stability in Cell Lysate by F NMR Spectroscopy.

In living organisms, protein folding and function take place in an inhomogeneous, highly crowded environment possessing a concentration of diverse macromolecules of up to 400 g/L. It has been shown that the intracellular environment has a pronounced effect on the stability, dynamics and function of the protein under study, and has for this reason to be considered. However, most protein studies neglect the presence of these macromolecules.

What does fluorine do to a protein? Thermodynamic, and highly-resolved structural insights into fluorine-labelled variants of the cold shock protein.

Fluorine labelling represents one promising approach to study proteins in their native environment due to efficient suppressing of background signals. Here, we systematically probe inherent thermodynamic and structural characteristics of the Cold shock protein B from Bacillus subtilis (BsCspB) upon fluorine labelling. A sophisticated combination of fluorescence and NMR experiments has been applied to elucidate potential perturbations due to insertion of fluorine into the protein.

Targeted expression and purification of fluorine labelled cold shock protein B by using an auxotrophic strategy.

High resolution NMR spectroscopy is a seminal method in modern structural biology to obtain insights into proteins' structure, dynamics and function at dilute condition as well as in a cell-like environment or even intracellularly. Usually, H, N or C nuclei are predominantly used for the characterization of the protein of interest. These measurements are limited due to the wealth of chemical shifts and background signals arising from all molecules present in the NMR test tube.