title: A hypothesis to reconcile the physical and chemical unfolding of proteins.
abstract: High pressure (HP) or urea is commonly used to disturb folding species. Pressure favors the reversible unfolding of proteins by causing changes in the volumetric properties of the protein-solvent system. However, no mechanistic model has fully elucidated the effects of urea on structure unfolding, even though protein-urea interactions are considered to be crucial. Here, we provide NMR spectroscopy and 3D reconstructions from X-ray scattering to develop the "push-and-pull" hypothesis, which helps to explain the initial mechanism of chemical unfolding in light of the physical events triggered by HP. In studying MpNep2 from Moniliophthora perniciosa, we tracked two cooperative units using HP-NMR as MpNep2 moved uphill in the energy landscape; this process contrasts with the overall structural unfolding that occurs upon reaching a threshold concentration of urea. At subdenaturing concentrations of urea, we were able to trap a state in which urea is preferentially bound to the protein (as determined by NMR intensities and chemical shifts); this state is still folded and not additionally exposed to solvent [fluorescence and small-angle X-ray scattering (SAXS)]. This state has a higher susceptibility to pressure denaturation (lower p1/2 and larger ΔVu); thus, urea and HP share concomitant effects of urea binding and pulling and water-inducing pushing, respectively. These observations explain the differences between the molecular mechanisms that control the physical and chemical unfolding of proteins, thus opening up new possibilities for the study of protein folding and providing an interpretation of the nature of cooperativity in the folding and unfolding processes.
authors.affiliation: Programa de Biologia Estrutural, Instituto de Bioquímica Médica Leopoldo de Meis, Instituto Nacional de Biologia Estrutural e Bioimagem, Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, Brazil jerson@bioqmed.ufrj.br gaugusto@bioqmed.ufrj.br.
authors.forename: Guilherme A P
authors.initials: GA
authors.lastname: de Oliveira
authors.affiliation: Programa de Biologia Estrutural, Instituto de Bioquímica Médica Leopoldo de Meis, Instituto Nacional de Biologia Estrutural e Bioimagem, Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, Brazil jerson@bioqmed.ufrj.br gaugusto@bioqmed.ufrj.br.
authors.forename: Jerson L
authors.initials: JL
authors.lastname: Silva
mesh_terms: D000363:Agaricales / Q000737:chemistry; D000818:Animals; D055592:Biophysical Phenomena; D005656:Fungal Proteins / Q000737:chemistry; D006801:Humans; D006874:Hydrostatic Pressure; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D016315:Proto-Oncogene Proteins c-abl / Q000737:chemistry; D053838:Scattering, Small Angle; D013050:Spectrometry, Fluorescence; D055672:Static Electricity; D013816:Thermodynamics; D056811:Unfolded Protein Response / Q000502:physiology*; D014508:Urea; D014961:X-Ray Diffraction
chemical_list: D005656:Fungal Proteins; D014508:Urea; D016315:Proto-Oncogene Proteins c-abl
keywords: NMR; SAXS; hydrostatic pressure; protein folding; urea