title: Optimization of NMR spectroscopy of encapsulated proteins dissolved in low viscosity fluids.
abstract: Comprehensive application of solution NMR spectroscopy to studies of macromolecules remains fundamentally limited by the molecular rotational correlation time. For proteins, molecules larger than 30 kDa require complex experimental methods, such as TROSY in conjunction with isotopic labeling schemes that are often expensive and generally reduce the potential information available. We have developed the reverse micelle encapsulation strategy as an alternative approach. Encapsulation of proteins within the protective nano-scale water pool of a reverse micelle dissolved in ultra-low viscosity nonpolar solvents overcomes the slow tumbling problem presented by large proteins. Here, we characterize the contributions from the various components of the protein-containing reverse micelle system to the rotational correlation time of the encapsulated protein. Importantly, we demonstrate that the protein encapsulated in the reverse micelle maintains a hydration shell comparable in size to that seen in bulk solution. Using moderate pressures, encapsulation in ultra-low viscosity propane or ethane can be used to magnify this advantage. We show that encapsulation in liquid ethane can be used to reduce the tumbling time of the 43 kDa maltose binding protein from ~23 to ~10 ns. These conditions enable, for example, acquisition of TOCSY-type data resolved on the adjacent amide NH for the 43 kDa encapsulated maltose binding protein dissolved in liquid ethane, which is typically impossible for proteins of such size without use of extensive deuteration or the TROSY effect.
authors: Graduate Group in Biochemistry and Molecular Biophysics and Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104-6059, USA.
authors: Nathaniel V
authors: NV
authors: Nucci
authors: Bryan S
authors: BS
authors: Marques
authors: Sabrina
authors: S
authors: Bédard
authors: Jakob
authors: J
authors: Dogan
authors: John M
authors: JM
authors: Gledhill
authors: Veronica R
authors: VR
authors: Moorman
authors: Ronald W
authors: RW
authors: Peterson
authors: Kathleen G
authors: KG
authors: Valentine
authors: Alison L
authors: AL
authors: Wand
authors: A Joshua
authors: AJ
authors: Wand
mesh_terms: D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry; D004980:Ethane / Q000737:chemistry; D000441:Hexanols / Q000737:chemistry; D006801:Humans; D058619:Maltose-Binding Proteins / Q000737:chemistry; D008823:Micelles; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D013501:Surface-Active Agents / Q000737:chemistry; D014783:Viscosity; D014867:Water / Q000737:chemistry
chemical_list: D002593:Cetrimonium Compounds; D029968:Escherichia coli Proteins; D000441:Hexanols; D058619:Maltose-Binding Proteins; D008823:Micelles; D011506:Proteins; D013501:Surface-Active Agents; D014867:Water; D004980:Ethane; D000077286:Cetrimonium