Optimization of NMR spectroscopy of encapsulated proteins dissolved in low viscosity fluids |
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Authors: | Nathaniel?V?Nucci Bryan?S?Marques Sabrina?Bédard Jakob?Dogan Jr" target="_blank">John?M?GledhillJr Veronica?R?Moorman Ronald?W?Peterson Kathleen?G?Valentine Alison?L?Wand Email author" target="_blank">A?Joshua?WandEmail author |
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Institution: | (1) Graduate Group in Biochemistry and Molecular Biophysics and Department of Biochemistry and Biophysics, 905 Stellar-Chance Laboratories, University of Pennsylvania, 422 Curie Blvd, Philadelphia, PA 19104-6059, USA;(2) Present address: Daedalus Innovations, LLC, 3624 Market St., Philadelphia, PA 19104, USA |
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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. |
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