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Colloquia with Prof. Joshua Wand, University of Pennsylvania, Perelman School of Medicine

"Recent advances in NMR spectroscopy of encapsulated proteins & nucleic acids dissolved in low viscosity fluids"

Solution NMR spectroscopy is a powerful technique to study protein structure
and dynamics on multiple timescales and in many contexts. Sample preparation is
often the key ingredient that enables otherwise very difficult studies of
complex macromolecular systems. Some time ago we introduced the idea of using
solutions of proteins encapsulated within the protective aqueous core of a reverse or
inverted micelle and dissolved in low viscosity fluids as a means to overcome the
“slow tumbling” problem presented by large, soluble proteins. Since then
several advantageous properties of the reverse micelle particle have been used to
promote studies of integral and anchored membrane proteins, soluble proteins and
nucleic acids of marginal stability as well as investigations of various aspects of
protein biophysics such as cold denaturation, protein hydration and protein motion.
Despite this, the approach has not been generally adopted by the NMR community.
To make this reverse micelle encapsulation approach more accessible, we have
developed an optimized reverse micelle surfactant system. Comprised of the nonionic
1-decanoyl-rac-glycerol and the zwitterionic lauryldimethylamine-N-oxide (10MAG/LDAO),
this mixture is found to faithfully encapsulate a diverse set of proteins ranging up
to 80 kDa in size and having a broad spectrum of electrostatic properties.
Extensive chemical shift analyses indicate that encapsulation conditions that maintain
high structural fidelity can be directly found. A clear advantage of 10MAG/LDAO
is the active decrease of molecular reorientation time for encapsulated
macromolecules larger than ~20 kDa leading to improved signal-to-noise. The properties
of 10MAG/LDAO are also found to be very favorable for solution NMR studies of
lipidated proteins. New and efficient strategies for optimization of encapsulation
conditions have also been developed. 10MAG/LDAO performs well in both
the low viscosity pentane and ultra-low viscosity liquid ethane and should serve as
a general platform for initiating solution NMR studies of proteins and nucleic acids. In a
parallel effort, it has been realized that reverse micelle solutions potentially offer a
route to implement dynamic nuclear polarization enhancement of protein resonances
by avoiding the dielectric heating generally associated with standard aqueous
samples. Initial results will be presented. Supported by NSF and the NIH.

When
Mon Nov 11, 2013 4:15pm – 5:30pm Central Time
Where
5211 SC (map)