Authors: Nathaniel V Nucci Bryan S Marques Sabrina Bédard Jakob Dogan John M Gledhill Veronica R Moorman Ronald W Peterson Kathleen G Valentine Alison L Wand A Joshua Wand
Publish Date: 2011/07/12
Volume: 50, Issue: 4, Pages: 421-
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 nanoscale water pool of a reverse micelle dissolved in ultralow viscosity nonpolar solvents overcomes the slow tumbling problem presented by large proteins Here we characterize the contributions from the various components of the proteincontaining 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 ultralow 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 TOCSYtype 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 effectSupported by NSF grants MCB0842814 and DMR05200020 and NIH grant GM 085120 and NIH postdoctoral fellowship GM087099 to NVN JD acknowledges financial support from the WennerGren Foundations AJW and RWP declare a financial conflict of interest as Members of Daeadalus Innovations LLC a manufacturer of highpressure and reverse micelle NMR apparatus
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