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Doty Scientific, Columbia, SC
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Progress on a TXI-HR-SAS-PFG
Probe for High-field Narrow-Bore Magnets

George Entzminger, S. Kini, J. Staab, G. Novak,
M. Stringer, J. R. Doty, J. Gravel, and F. D. Doty



Doty Scientific, Columbia, SC
Poster presented at the 44th ENC, Savannah, Georgia, 2003
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Abstract:

One of the more promising recent additions to the arsenal of NMR tools
has been the use of long-range constraints from residual dipolar couplings in partially
aligned solutions. Partial macromolecule alignment has been obtained by using dilute
liquid-crystal solutions of disc-shaped bicelles, but this alone is not sufficient for the
needed dynamical control over the alignment.

Very recent analyses and experiments indicate novel Switched Angle Spinning
(SAS) techniques should provide the needed dynamic control over the bicelle align-
ment
1-3
. When a sample containing discoidal bicelles of negative magnetic anisotropy is
spun at the Magic Angle, their interaction with B
0
vanishes and their orientation becomes
random. For sample spinning at angles less than 54.7
0
, they align with their normals
perpendicular to the spinning axis, while spinning at greater angles causes their normals
to align with the spinning axis. The dynamic control over the spinning axis provided by a
SAS probe may provide the protein alignment control needed for more effective utiliza-
tion of the bond angle information inherent in the residual dipolar coupling.

The goal of this project is to enable a new NMR spectroscopy method, Triple-
Resonance Indirect High-Resolution (TXI-HR) SAS in narrow-bore (NB) NMR magnets
up to 800 MHz. The NMR probe must be capable of multinuclear triple-resonance MAS
with highly sensitive indirect (
1
H) detection with high resolution (~0.01 ppm), at fields up
to 19 T. In addition, rapid (<30 ms) reorientation of the spinning axis is required without
adversely affecting spinning stability or rf tuning. Other requirements include pulsed
field gradients, stable temperature control, low
1
H background signals, and compatibility
with narrow-bore (NB) high-field magnets. The instrument will also facilitate several
other solids NMR techniques, both for the study of quadrupolar nuclides and for the
study of insoluble proteins. Here, we report our instrumentation development progress.
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Introduction. The probe described in this poster uses elements of several
probe technologies that we have been working on over the past 5 years for a
combined capability never before attempted in a single probe. Our XC technol-
ogy provides the necessary spinning robustness along with an RF circuit that
allows the required efficiency with long flexible leads. B
0
field homogeneity is
maintained by selection of materials and special symmetries included in the
spinner design. Angle positioning is accomplished using an upgraded, existing
SAS control system. This controller incorporates a small high torque servo mo-
tor and optical encoder for fast, accurate angle change. We have included a
PFG coil to allow a Z field gradient in the sample synchronous with SAS. All of
these capabilities have been included individually in other NMR probes but
combining them into a single probe is the biggest challenge in this project.
NMR Justification. The traditional NMR structure techniques are based
primarily on proton nuclear Overhauser effects (NOEs), where magnetization
transfer rates between pairs of protons are used to determine distances be-
tween the pairs. Assignments to specific sites are then required, based on a
complex array of experiments, most of which use indirect detection.

One problem with the common techniques is that distance errors between
sites along the backbone are cumulative. Longer range distance and angular
information is possible from dipolar couplings, but they average to zero in iso-
tropic solutions; and in rigid solids they cause severe line broadening. A recent
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approach, PALMO (Partial Alignment of Molecules), that looks quite promising
utilizes phospholipid "bicelles" to impart partial alignment. These discoidal
membrane-like structures align in solutions with their normals perpendicular to
B
0
because of their negative magnetic anisotropy, and their alignment imparts a
partial alignment on proteins in the solution around them. The degree of align-
ment may be adjusted by controlling temperature and concentration so as to
provide an optimal amount (several percent) of alignment that allows dipolar
distance and angular constraints to be determined from the residual dipolar
couplings without severe line broadening or spectral complexities [2]. These
and other advanced NMR liquids techniques promise to extend structural and
dynamical information to helical membrane proteins, protein dimers, higher oli-
gomers, and complexes with molecular weights up to ~10
6
.

Considerable improvement in residual dipolar coupling techniques should
be possible if one could achieve dynamic control over the direction and amount
of alignment. Very recent analysis and experiments by several foremost NMR
research groups indicate novel SAS techniques should provide the needed dy-
namic control over the bicelle (and hence, the protein) alignment. When a
sample containing bicelles of negative magnetic anisotropy is spun at the Magic
Angle (54.7
0
with respect to B
0
), their interaction with B
0
vanishes and their ori-
entation becomes random. For sample spinning at angles less than 54.7
0
, they
align with their normals perpendicular to the spinning axis, while spinning at
greater angles causes their normals to align with the spinning axis. Both solu-
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ble and integral membrane proteins and peptides with partial isotopic labeling
may be incorporated into the bicelle without disrupting the orientational proper-
ties of the bicellar system. Experiments may then be performed under SAS in
which the macromolecule is sometimes parallel, sometimes perpendicular, and
sometimes isotropically oriented with respect to the spinning axis. This should
provide more bond direction information and permit spectral simplifications.

A potentially important application is high-field TXI-HR-MAS with gradients
and low
1
H backgrounds Another use for HR-MAS is the elimination of distant
(bulk) dipolar field (DDF) effects in standard liquids NMR techniques.

The Silicon-nitride XC Spinner Design. While radial gas bearings are
relatively easy to stabilize, our experience with axial thrust micro-bearings of all
types and sizes has convinced us that it is unrealistic to expect them to perform
adequately over a wide range of loading conditions. The most effective ap-
proach is to design with axial symmetry and eliminate axial loads as much as
possible. Single-ended-drive designs are not generally capable of handling the
axial-force transients that are unavoidable in SAS, nor are they capable of sta-
ble spinning over as wide a range of conditions (in temperature, sample
density, and spinning speeds). Perhaps more importantly for SAS, axial
symmetry (reflection about the center) seems essential for minimizing axial-
force transients during rapid angle re-orientation.
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Figure 1: XC spinner cross section view.

Figure 1 depicts a longitudinal sectional view of the Doty XC5, our 4th
generation, 5 mm spinner design,
compatible with high resolution on
semi-solids, and capable of 19 kHz
MAS. Maximum spinning rates require
the drive turbines to have smaller
outside diameter than the rotor, and it
is this unique feature of Doty NMR
MAS spinner designs which enables
them to achieve 30% higher surface
speeds than other designs.
Material strength considerations
are normally a secondary reason to
reduce blade diameter; but for the
1
H
MAS case, it becomes a primary issue,
as it is desirable to be able to use a polymeric material for the turbine and the
best hydrogen-free option (Kel-F) has limited yield strength (20 MPa).

To maintain sufficient bearing stiffness for SAS, the radial gas bearings at
each end must exhaust axially in both directions, which means care must be
taken to ensure the drive turbines operate efficiently under transonic flow condi-
tions with rather low inlet static pressure. This complicates the design some-
what, and additional manufacturing constraints are imposed by the need to be
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able to precisely position and secure the rf coils without spoiling B
0
homogene-
ity and using a minimum of hydrogenous materials.

We use the best available rotor material (silicon nitride) even though it im-
poses a cost penalty on the rotors and stators compared to zirconia. The mod-
erately high-speed touchdowns ("crashes") that may occur when the spinning
angle is rapidly switched in SAS require the ultimate in wear resistance. The
wear resistance of silicon nitride is an order of magnitude better than that of zir-
conia under severe conditions.

High-resolution