I understand the process behind why fluoro alcohols break up existing secondary structures, but for the alkaline conditions (NH4OH or NaOH) used in some protocols how are the basic conditions breaking up beta structures. Is it that the change in the charge state of the peptide, making it more soluble therefore shifting the equilibrium of monomer to fibril addition thus breaking it apart maybe slower. I feel this is a simple question but haven't found a clear answer in my initial literature searches, maybe cause it's that obvious?
My hunch is that at high pH the Hydrogen bonds stop "working" and Lysine residues (which are important to stabilise Ab fibrils) get deprotonated (Lys pK =10.8) so the salt bridge in the loop (if the model correct) stop working.
Ones upon a time I did time-laps imaging on Ab25-35 with AFM and at high pH (around 11, 12) the fibrils started falling apart, they lost full segments which suggested me that H bond system is compromised while the side chain interactions perpendicular to the longitudinal axis are fine even though they are much weaker.
Two answers, one practical and the other theoretical. The practical answer is that in the history of amyloid research, NaOH has played a prominent role in solubilization of these aggregates. I presume, although I have no evidence that, other solvents were tried and that NaOH was found to be particularly useful. Theoretically, alkaline pH conditions are most distant from the pI of Abeta, which means that the average deprotonation state of carboxylates is greatest relative to the average protonation states of amines or imines at low pH. In both cases, charge repulsion would be expected to interfere with intra- and intermolecular interactions leading to monomer folding and assembly. This would be particularly important at the Abeta C-terminus, where charging of the carboxylate could interfere with hydrophobic interactions that drive peptide assembly. Amyloid assemblies ARE in equilibrium with monomers (and other lower-order assemblies, so pH-based perturbation of this equilibrium will drive the dissociation process.
I agree with Arpad Karsai. Dis aggregation is also promoted by the charges generated in Asp and Glu residues in the peptide
Me too, I agree with Dr Teplow. In addition I suggest you this paper
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3646356/pdf/peerj-01-73.pdf
Thank you again Dr. Teplow for your detailed explanation, and also thank you to Nicola Acerra for the paper. I have read is article before and find it suspicious,in the ThT plots, that the untreated AB aggregates slower than that of the HFIP treated AB
I too read the article, and we just tried---once--- the ammonia method on some Aβ42 and examined ThT fluorescence. We also examined untreated Aβ42 and HFIP-treated Aβ42. HFIP and ammonia yielded hyperbolic curves with relatively low final fluorescence. In contrast, HFIP treatment produced a similar curve, but one that had a lower initial slope and plateaued at a much higher FU. One answer may be that the HFIP eliminated pre-existent assemblies that were "off pathway" for development of ThT-binding structures. If so, assembly could occur more efficiently because the fibril assembly-incompetent structures would not first have to dissociate or rearrange to allow fibril formation to proceed.
Sorry, I am very interested in protocols for NaOH stabilization of monomeric samples, can you please link some papers?
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