title: De novo 3D structure determination from sub-milligram protein samples by solid-state 100 kHz MAS NMR spectroscopy.
abstract: Solid-state NMR spectroscopy is an emerging tool for structural studies of crystalline, membrane-associated, sedimented, and fibrillar proteins. A major limitation for many studies is still the large amount of sample needed for the experiments, typically several isotopically labeled samples of 10-20 mg each. Here we show that a new NMR probe, pushing magic-angle sample rotation to frequencies around 100 kHz, makes it possible to narrow the proton resonance lines sufficiently to provide the necessary sensitivity and spectral resolution for efficient and sensitive proton detection. Using restraints from such spectra, a well-defined de novo structure of the model protein ubiquitin was obtained from two samples of roughly 500 μg protein each. This proof of principle opens new avenues for structural studies of proteins available in microgram, or tens of nanomoles, quantities that are, for example, typically achieved for eukaryotic membrane proteins by in-cell or cell-free expression.
authors.affiliation: Laboratory of Physical Chemistry, ETH Zurich, 8093 Zurich (Switzerland).
authors.forename: Vipin
authors.initials: V
authors.lastname: Agarwal
authors.forename: Susanne
authors.initials: S
authors.lastname: Penzel
authors.forename: Kathrin
authors.initials: K
authors.lastname: Szekely
authors.forename: Riccardo
authors.initials: R
authors.lastname: Cadalbert
authors.forename: Emilie
authors.initials: E
authors.lastname: Testori
authors.forename: Andres
authors.initials: A
authors.lastname: Oss
authors.forename: Jaan
authors.initials: J
authors.lastname: Past
authors.forename: Ago
authors.initials: A
authors.lastname: Samoson
authors.forename: Matthias
authors.initials: M
authors.lastname: Ernst
authors.forename: Anja
authors.initials: A
authors.lastname: Böckmann
authors.forename: Beat H
authors.initials: BH
authors.lastname: Meier
mesh_terms: D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*
chemical_list: D011506:Proteins
keywords: magic-angle spinning; protein structures; proton detection; solid-state NMR spectroscopy