Abstract
Nuclear magnetic resonance (NMR) spectroscopy is a powerful experimental tool for obtaining information on three-dimensional (3D) structures of proteins at atomic resolution. In inherited forms of prion diseases, misfolding of cellular prion protein, PrPC, into its pathological form, PrPSc, is caused by mutations in the human prion protein gene (PRNP). Understanding of the earliest stages of the conformational changes leading to spontaneous generation of prions in inherited forms of prion diseases may benefit from detailed structural analysis of different human (Hu) PrP variants. Here, we describe the protocol for structure determination of HuPrP variants by NMR spectroscopy in solution that consists of preparation of NMR samples, acquisition of NMR data, NMR resonance assignments, and structure calculation.
Keywords:
Mutants; NMR structure determination; Prion diseases; Prion protein; Protein structure.
MeSH terms
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Carbon Isotopes
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Cloning, Molecular / methods*
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DNA Restriction Enzymes / chemistry
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Endopeptidases / chemistry
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Freeze Drying / methods
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Gene Expression
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Guanidine / chemistry
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Humans
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Inclusion Bodies / chemistry*
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Isotope Labeling / methods
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Kinetics
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Magnetic Resonance Spectroscopy / methods*
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Models, Molecular
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Mutation
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Nitrogen Isotopes
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Plasmids / chemistry
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Plasmids / metabolism
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Prion Proteins / biosynthesis
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Prion Proteins / genetics
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Prion Proteins / isolation & purification*
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Protein Conformation, alpha-Helical
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Protein Conformation, beta-Strand
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Protein Refolding
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Recombinant Fusion Proteins / biosynthesis
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / isolation & purification*
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Thermodynamics
Substances
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Carbon Isotopes
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Nitrogen Isotopes
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Prion Proteins
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Recombinant Fusion Proteins
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DNA Restriction Enzymes
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Endopeptidases
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TEV protease
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Guanidine