title: Peptides and proteins in a confined environment: NMR spectra at natural isotopic abundance.
abstract: Confinement of proteins and peptides in a small inert space mimics the natural environment of the cell, allowing structural studies in conditions that stabilize folded conformations. We have previously shown that confinement in polyacrylamide gels (PAGs) is sufficient to induce a change in the viscosity of the aqueous solution without changing the composition and temperature of the solvent. The main limitation of a PAG to run NMR experiments in a confined environment is the need for labelling the peptides. Here we report the use of the agarose gel to run the NMR spectra of proteins and peptides. We show that agarose gels are completely transparent in NMR experiments, relieving the need for labelling. Although it is necessary to expose biomolecules to fairly high temperatures during sample preparation, we believe that this is not generally an obstacle to the study of peptides, and found that the method is also compatible with temperature-resistant proteins. The mesh of agarose gels is too wide for direct effects of confinement on the stability of proteins but confinement can be easily exploited to interact the proteins with other reagents, including crowding macromolecules that can eventually lead to fold stabilization. The use of these gels is ideally suited for low-temperature studies; we show that a very flexible peptide at subzero temperatures is stabilized into a well-folded conformation.
authors: National Institute for Medical Research, Medical Research Council, London, UK.
authors: Annalisa
authors: A
authors: Pastore
authors: Severo
authors: S
authors: Salvadori
authors: Piero Andrea
authors: PA
authors: Temussi
mesh_terms: D000818:Animals; D004587:Electrophoresis, Agar Gel; D004795:Enzyme Stability; D006801:Humans; D009113:Muramidase / Q000737:chemistry*; D009479:Neuropeptides / Q000138:chemical synthesis / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding
chemical_list: D009479:Neuropeptides; C506531:neuropeptide S, human; D009113:Muramidase