Two mechanisms underlie the inhibitory/acceleratory action of chemical compounds on tau aggregation including the regulation of cellular kinases and phosphatases activity and direct binding to tau protein. Vitamin B12 is one of the tau polymerization inhibitors, and its deficiency is linked to inactivation of protein phosphatase 2A and subsequently hyperphosphorylation and aggregation of tau protein. Regarding the structure and function of vitamin B12 and tau protein, we assumed that vitamin B12 is also able to directly bind to tau protein. Hence, we investigated the interaction of vitamin B12 with tau protein in vitro using fluorometry and circular dichrosim. Interaction studies was followed by investigation into the effect of vitamin B12 on tau aggregation using ThT fluorescence, circular dichroism, transmission electron microscopy, and SDS-PAGE. The results indicated that vitamin B12 interacts with tau protein and prevents fibrillization of tau protein. Blocking the cysteine residues of tau confirmed the cysteine-mediated binding of vitamin B12 to tau and showed that binding to cysteine is essential for inhibitory effect of vitamin B12 on tau aggregation. SDS-PAGE analysis indicated that vitamin B12 inhibits tau aggregation and that tau oligomers formed in the presence of vitamin B12 are mostly SDS-soluble. We propose that direct binding of vitamin B12 is another mechanism underlying the inhibitory role of vitamin B12 on tau aggregation and neurodegeneration.

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Here, we introduce isatoic anhydride as a sensitive and commodious fluorescent prelabel for detection of proteins in one-dimensional polyacrylamide gels. High reactivity of isatoic anhydride with nucleophiles in mild alkaline environments makes it an appropriate tag for labeling of biomolecules. In this study, we show that preelectrophoresis labeling of proteins with isatoic anhydride for few minutes at room temperature allows detection of 2-4 ng of standard proteins, BSA and lysozyme, per band. Proteins were successfully labeled in the presence of a wide range of common biological reagents and in crude cell extract. The labeled proteins have the same electrophoretic migration in comparison to unlabeled proteins; however the application of saturation labeling method results in slight band broadening. Compatibility of the method with downstream processes was assessed by tryptic digestion of labeled proteins and study of peptide mixture using gel electrophoresis which revealed partial digestion of labeled proteins due to lysine modification. The present procedure is sensitive, rapid, and inexpensive and is a promising alternative for current protein staining procedures, where downstream processes are not desired.

G protein-coupled receptors are the largest family of integral membrane proteins in humans that have roles in almost all physiological processes. The binding of extracellular ligands allosterically modulates the intracellular interaction of the GPCR with transducer proteins such as G proteins and arrestins. This allosteric coupling operates via a network of conserved microswitches to adjust the equilibrium of active, intermediate and inactive states of the GPCR. Crystallography and cryo-electron microscopy have determined the structures of many active and inactive state GPCRs, while solution-state methods such as NMR spectroscopy inform on the dynamics of additional states and their role in signalling. In addition, solution NMR spectroscopy is providing insight into the pathways and mechanisms of ligand binding, including disordered peptides, to GPCRs. This chapter reviews the challenges in preparing GPCRs for solution NMR data collection, the knowledge gained about the conformational landscapes and ligand binding to GPCRs.