caa a22 4500 445325887 CHVBK 20180317142720.0 cr unu---uuuuu 170323e20110801xx s 000 0 eng 10.1007/s10858-011-9528-y doi (NATIONALLICENCE)springer-10.1007/s10858-011-9528-y Optimization of NMR spectroscopy of encapsulated proteins dissolved in low viscosity fluids [Elektronische Daten] [Nathaniel Nucci, Bryan Marques, Sabrina Bédard, Jakob Dogan, John Gledhill Jr., Veronica Moorman, Ronald Peterson, Kathleen Valentine, Alison Wand, A. Wand] Comprehensive application of solution NMR spectroscopy to studies of macromolecules remains fundamentally limited by the molecular rotational correlation time. For proteins, molecules larger than 30kDa 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 43kDa maltose binding protein from ~23 to ~10ns. These conditions enable, for example, acquisition of TOCSY-type data resolved on the adjacent amide NH for the 43kDa 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. Springer Science+Business Media B.V., 2011 Encapsulated proteins nationallicence Reverse miclles nationallicence Low viscosity fluids nationallicence Triple resonance NMR nationallicence Resonance assignment nationallicence Structure determination nationallicence Nucci Nathaniel Graduate Group in Biochemistry and Molecular Biophysics and Department of Biochemistry and Biophysics, 905 Stellar-Chance Laboratories, University of Pennsylvania, 422 Curie Blvd, 19104-6059, Philadelphia, PA, USA aut Marques Bryan Graduate Group in Biochemistry and Molecular Biophysics and Department of Biochemistry and Biophysics, 905 Stellar-Chance Laboratories, University of Pennsylvania, 422 Curie Blvd, 19104-6059, Philadelphia, PA, USA aut Bédard Sabrina Graduate Group in Biochemistry and Molecular Biophysics and Department of Biochemistry and Biophysics, 905 Stellar-Chance Laboratories, University of Pennsylvania, 422 Curie Blvd, 19104-6059, Philadelphia, PA, USA aut Dogan Jakob Graduate Group in Biochemistry and Molecular Biophysics and Department of Biochemistry and Biophysics, 905 Stellar-Chance Laboratories, University of Pennsylvania, 422 Curie Blvd, 19104-6059, Philadelphia, PA, USA aut Gledhill Jr. John Graduate Group in Biochemistry and Molecular Biophysics and Department of Biochemistry and Biophysics, 905 Stellar-Chance Laboratories, University of Pennsylvania, 422 Curie Blvd, 19104-6059, Philadelphia, PA, USA aut Moorman Veronica Graduate Group in Biochemistry and Molecular Biophysics and Department of Biochemistry and Biophysics, 905 Stellar-Chance Laboratories, University of Pennsylvania, 422 Curie Blvd, 19104-6059, Philadelphia, PA, USA aut Peterson Ronald Graduate Group in Biochemistry and Molecular Biophysics and Department of Biochemistry and Biophysics, 905 Stellar-Chance Laboratories, University of Pennsylvania, 422 Curie Blvd, 19104-6059, Philadelphia, PA, USA aut Valentine Kathleen Graduate Group in Biochemistry and Molecular Biophysics and Department of Biochemistry and Biophysics, 905 Stellar-Chance Laboratories, University of Pennsylvania, 422 Curie Blvd, 19104-6059, Philadelphia, PA, USA aut Wand Alison Graduate Group in Biochemistry and Molecular Biophysics and Department of Biochemistry and Biophysics, 905 Stellar-Chance Laboratories, University of Pennsylvania, 422 Curie Blvd, 19104-6059, Philadelphia, PA, USA aut Wand A. Graduate Group in Biochemistry and Molecular Biophysics and Department of Biochemistry and Biophysics, 905 Stellar-Chance Laboratories, University of Pennsylvania, 422 Curie Blvd, 19104-6059, Philadelphia, PA, USA aut Journal of Biomolecular NMR Springer Netherlands 50/4(2011-08-01), 421-430 0925-2738 50:4<421 2011 50 10858 https://doi.org/10.1007/s10858-011-9528-y text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10858-011-9528-y text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Nucci Nathaniel Graduate Group in Biochemistry and Molecular Biophysics and Department of Biochemistry and Biophysics, 905 Stellar-Chance Laboratories, University of Pennsylvania, 422 Curie Blvd, 19104-6059, Philadelphia, PA, USA aut NATIONALLICENCE 700 1- Marques Bryan Graduate Group in Biochemistry and Molecular Biophysics and Department of Biochemistry and Biophysics, 905 Stellar-Chance Laboratories, University of Pennsylvania, 422 Curie Blvd, 19104-6059, Philadelphia, PA, USA aut NATIONALLICENCE 700 1- Bédard Sabrina Graduate Group in Biochemistry and Molecular Biophysics and Department of Biochemistry and Biophysics, 905 Stellar-Chance Laboratories, University of Pennsylvania, 422 Curie Blvd, 19104-6059, Philadelphia, PA, USA aut NATIONALLICENCE 700 1- Dogan Jakob Graduate Group in Biochemistry and Molecular Biophysics and Department of Biochemistry and Biophysics, 905 Stellar-Chance Laboratories, University of Pennsylvania, 422 Curie Blvd, 19104-6059, Philadelphia, PA, USA aut NATIONALLICENCE 700 1- Gledhill Jr John Graduate Group in Biochemistry and Molecular Biophysics and Department of Biochemistry and Biophysics, 905 Stellar-Chance Laboratories, University of Pennsylvania, 422 Curie Blvd, 19104-6059, Philadelphia, PA, USA aut NATIONALLICENCE 700 1- Moorman Veronica Graduate Group in Biochemistry and Molecular Biophysics and Department of Biochemistry and Biophysics, 905 Stellar-Chance Laboratories, University of Pennsylvania, 422 Curie Blvd, 19104-6059, Philadelphia, PA, USA aut NATIONALLICENCE 700 1- Peterson Ronald Graduate Group in Biochemistry and Molecular Biophysics and Department of Biochemistry and Biophysics, 905 Stellar-Chance Laboratories, University of Pennsylvania, 422 Curie Blvd, 19104-6059, Philadelphia, PA, USA aut NATIONALLICENCE 700 1- Valentine Kathleen Graduate Group in Biochemistry and Molecular Biophysics and Department of Biochemistry and Biophysics, 905 Stellar-Chance Laboratories, University of Pennsylvania, 422 Curie Blvd, 19104-6059, Philadelphia, PA, USA aut NATIONALLICENCE 700 1- Wand Alison Graduate Group in Biochemistry and Molecular Biophysics and Department of Biochemistry and Biophysics, 905 Stellar-Chance Laboratories, University of Pennsylvania, 422 Curie Blvd, 19104-6059, Philadelphia, PA, USA aut NATIONALLICENCE 700 1- Wand A. Graduate Group in Biochemistry and Molecular Biophysics and Department of Biochemistry and Biophysics, 905 Stellar-Chance Laboratories, University of Pennsylvania, 422 Curie Blvd, 19104-6059, Philadelphia, PA, USA aut NATIONALLICENCE 773 0- Journal of Biomolecular NMR Springer Netherlands 50/4(2011-08-01), 421-430 0925-2738 50:4<421 2011 50 10858 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer