Characterization of the interaction between bovine pancreatic trypsin inhibitor and thiocyanate by NMR.

International audienceThe interaction between Bovine Pancreatic Trypsin Inhibitor and thiocyanate was studied using NMR spectroscopy following several experimental approaches. The chemical shift variations of the BPTI protons in the absence and in the presence of increasing thiocyanate concentrations (up to 0.2 M) were significant (> 0.05 ppm) for 30 protein protons belonging to 20 residues. The largest deviation, 0.2 ppm, was observed for the amide backbone proton of Arg42 in the absence of thiocyanate and in the presence of 40 molar equivalents of thiocyanate. The influence of the presence of thiocyanate on the electrostatic potential surrounding the protein was demonstrated by NOESY spectra selective at the water frequency: the presence of SCN- favours acid catalysed exchange and disfavours base catalysis. However, a specific effect of thiocyanate was pointed out since the comparison of the chemical shifts in the presence of 40 molar equivalents of KSCN and KCl, respectively, showed much more as well as larger deviations compared to measurements in the absence of salt. A dissociation constant, KD, for a 1/1 complex between BPTI and thiocyanate was calculated from chemical shifts measurements: KD = 89 +/- 8 mM. A second value, KD = 99 +/- 10 mM, was extracted from SC15N relaxation time measurements.The interaction between Bovine Pancreatic Trypsin Inhibitor and thiocyanate was studied using NMR spectroscopy following several experimental approaches. The chemical shift variations of the BPTI protons in the absence and in the presence of increasing thiocyanate concentrations (up to 0.2 M) were significant (> 0.05 ppm) for 30 protein protons belonging to 20 residues. The largest deviation, 0.2 ppm, was observed for the amide backbone proton of Arg42 in the absence of thiocyanate and in the presence of 40 molar equivalents of thiocyanate. The influence of the presence of thiocyanate on the electrostatic potential surrounding the protein was demonstrated by NOESY spectra selective at the water frequency: the presence of SCN- favours acid catalysed exchange and disfavours base catalysis. However, a specific effect of thiocyanate was pointed out since the comparison of the chemical shifts in the presence of 40 molar equivalents of KSCN and KCl, respectively, showed much more as well as larger deviations compared to measurements in the absence of salt. A dissociation constant, KD, for a 1/1 complex between BPTI and thiocyanate was calculated from chemical shifts measurements: KD = 89 +/- 8 mM. A second value, KD = 99 +/- 10 mM, was extracted from SC15N relaxation time measurements

New isolated or recombinant or engineered variant beta-2-microglobulin polypeptide which is amyloidogenic under essentially physiological conditions in vitro, used e.g. to study amyloid fibrillogenesis to treat e.g. Alzheimer's disease

NOVELTY - Isolated or recombinant or engineered variant beta -2-microglobulin ( beta 2M) polypeptide (P1) which is amyloidogenic under essentially physiological conditions in vitro, is new. USE - The isolated or recombinant or engineered variant beta 2M polypeptide (P1) is useful for: forming variant beta 2M amyloid fibrils in vitro (claimed); and studying amyloid fibrillogenesis, including diagnostic and therapeutic applications which is useful for treating Alzheimer's disease, type 2 diabetes, Parkinson's disease and Huntington's disease. No biological data given. DETAILED DESCRIPTION - INDEPENDENT CLAIMS are also included for: (1) a polypeptide having at least 95% identity with the polypeptide (P1); (2) a nucleic acid molecule encoding the polypeptide (P1); (3) a recombinant vector expressing the nucleic acid molecules; (4) a host cell expressing the vector; (5) a host cell expressing the plasmid; (6) a composition comprising the polypeptide (P1); (7) a method (M1) of forming variant beta 2M amyloid fibrils in vitro, comprising: adding an isolated or recombinant or engineered variant beta 2M polypeptide (P1) to a solution under essentially physiological conditions; incubating the solution at 37 degrees C, where variant beta 2M amyloid fibrils are formed; and determining that the variant beta 2M amyloid fibrils formed specifically bind Congo red from an alkaline alcoholic solution and then show red/green birefringence when viewed under crossed polarized light; and (8) a method (M2) of testing whether a compound or composition inhibits variant beta 2M amyloid formation, comprising: adding an isolated or recombinant or engineered variant beta 2M polypeptide (P1) to a solution under essentially physiological conditions; incubating the solution at 4-37 degrees C where variant beta 2M amyloid fibrils are formed; adding the compound or composition to the variant beta 2M amyloid fibrils; and determining whether the compound or composition inhibits the formation of variant beta 2M amyloid fibrils