• Membrane Proteins are Oriented with respect to their environment • Orientational Restraints are Absolute Restraints, while Isotropic Restraints are Relative Restraints • Through Unique High Resolution Structure & Dynamics detailed Functional Understanding of Biological Systems can be Achieved • Taking Advantage of Anisotropy!! Wang et al., 2000 JMR 144:162 - 167. Marassi & Opella , 2000 JMR 144: 150 - 155. From Membrane Peptide & Protein Structures to Biological Understanding: Using Oriented Sample & MAS ssNMR
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• Membrane Proteins are Oriented
with respect to their
environment
• Orientational Restraints are
Absolute Restraints, while
Isotropic Restraints are Relative
Restraints
• Through Unique High Resolution
Structure & Dynamics detailed
Functional Understanding of
Biological Systems can be
Achieved
• Taking Advantage of Anisotropy!!
Wang et al., 2000 JMR 144:162-167.
Marassi & Opella, 2000 JMR 144: 150-155.
From Membrane Peptide
& Protein Structures to
Biological Understanding:
Using Oriented Sample &
MAS ssNMR
Tensor Characterization:
Chemical Shift Tensor
Element Magnitudes
Mai et al., Prot. Sci. JMR 1992
11 39 22 36 ppm
22 63 51 63 ppm
33 213 197 194 ppm
Val1 Gly2 Trp11
Tensor Characterization:
Tensor Element Magnitudes:
Sample Preparation
Lazo et al., BBRC 1993
Powder Pattern:15N Ala3 gA
MAS:15N Gly215N Ala3 gA
263K
123K
203K
233K
Span = 172 ppm
Span = 172
Span = 168
Span = 159
Tensor Characterization:
Tensor Element Magnitudes
Extracted from 3D
ROCSA spectra
Wylie et al., JPC B 2006
Tensor Characterization:Tensor Element Magnitudes
Wylie et al., JPC B 2006
d = dZZ -diso
h =dyy -dxx
dzz -diso
Motional Averaging of Tensors:
Local Sidechain Dihedral
(or torsional) Motions
M. Frey Dissertation, 1984
Motional Averaging of Tensors:
u||
a = u||
s 3cos2 q -1
2
æ
èç
ö
ø÷ = 0.34u||
s
Motional Averaging of Tensors:
Librational Motions
Hu et al., Biochem 1995
Note that 11 and 33 are
effected much more than 22
11 = -75 ppm
33 = 66 ppm22 = 9 ppm
Motional Averaging of Tensors:
Librational MotionsThree models for the Trp11 gA librational motions:
A. motion about 1 only
B. motion about 2 only
C. motion about 1 and 2
The model with motion
about 2 fits best
Hu et al.,
Biochem
1995
Tensor Characterization:
Tensor Element Magnitudes – definitions:
cc - perpendicular to the CNC plane
aa - in the C1NH bond angle
bb - completes the right-handed coordinate frame
Oas et al., JACS 1987Teng et al., JACS 1992
33 ≥ 22 ≥ 11 or
|zz-iso| ≥ |xx-iso| ≥ |yy-iso|
or:
33 1122
Tensor Characterization:
Stark et al., JMR 1983
This traditional approach: crystal mounted on
a goniometer that can rotate a sample
about three orthogonal axes.
From a knowledge of:
1) the orientation of the molecular frame
in the crystalline frame of reference
(from x-ray diffraction).
2) the orientation of the crystal with
respect to the goniometer
3) the observed resonance frequencies as
a function of rotations about the
three axes.
4) It is possible to determine the tensor
element magnitudes and tensor
orientation to the molecular frame.
H2NCH2-13CO-15NHCH2CO2H•H2O•HCl
Rotation about the Z axis
Rotation about the X axis
Rotation about the Y axis
Tensor Element Orientation
Tensor Characterization:
Tensor Element Orientation
Rotation patterns are a lot of work and
require a sizeable crystal – hence -
restricted to model compounds.
It is also possible to determine the
tensor orientations from powder
pattern spectra.
The addition of the 13C-15N dipolar
interaction causes a very
significant change in shape of the
powder pattern.
Teng et al., JMR 1989
Tensor Characterization:
Tensor Element Orientation A B C
39 63 213 ppm
22 51 197
36 63 194
28±5 0±5 0±5°
106±2 98±2 106±2°
Mai et al., Prot. Sci. 1993
[13C1]formyl-[15N]Val1 gA [13C1]Val1-[15N]Gly2 gA
[13C1]Leu10-[15N]Trpl11 gA
aD
N =
bDN =
s11 =
s 33 =
s 22 =
Gramicidin A: Chemical Shift Tensor
Element Magnitudes and Tensor Orientations
Mai et al., Prot. Sci. 1992
• Tensor Element Magnitudes vary
in part because of differences in
dynamics, but also from the structure and
environment. Glycine is a clear outlier.
• Amide tensor element
orientations orientations
are remarkably constant
with the exception of
Glycine and the formyl-Val
Uniformly Aligned Samples
• Hydrated lipid bilayer preparations of
membrane proteins and polypeptides
~ 50% by weight water
~ 1 to 5 mg protein per sample
~38 Slides
~5000 Lipid
Bilayers
~ 1:50 to 1:80 Molar Ratio of Protein:Lipid
6 mm
Oriented Sample Solid State NMR:
n
n =
=
=
s obs =s ||
Nicholson et al., Biochem 1987
obs = ||(cos2q11-1) + (cos2q33-1)
Oriented Sample Solid State NMR:
Photomicrograph of a single glass
slide under crossed polarizers
40x and 40°C – bar is 100µm
A) Uniformly aligned bilayers
B) Parabolic Focal Conics
C) Oily streaks
Moll et al., Biophys. J. 1990
62%
38%
52%
48%
Mosaic spread 8°
Mosaic spread 12°
37% 16%
63% 84%
Das et al., Nature Protocols, 2013
Oriented Sample Solid State NMR: 15N Gramicidin
Mai et al., Protein Sci 1993
Oriented Sample Solid State NMR: More Backbone Data
Ketchem et al., JBN 1997
• With the anisotropic 15N chemical shift and the 1H-15N and 13C-15N dipolar interactions the
peptide plane orientations can be characterized. • Separated Local Field Spectra
Oriented Sample Solid State NMR: Additional Restraints
Ketchem Dissertation 1995
High Resolution Gramicidin Structure: All Atom Structure
Ketchem et al., Science 1993
Ketchem et al., Structure 1997
8+ Restraints/Residue
Validation of the All-Atom Gramicidin Structure
Kim et al., JACS 2001
• Stereoview of the refined
structure of gramicidin A.
• 40 simulated annealing
structures superimposed.
• The root mean square
deviation of the backbone
coordinates from the
refined ensembles of
structures is 0.11Å
Quiz: Where is the error in the PNAS article?
Nicholson et al., Biochem 1987
Uniformly 15N Labeled
Gramicidin A
Powder
Pattern
Oriented
Sample
A-B
Burkhardt, Li, Langs, Pangborn, Duax, PNAS 1998
Quiz: Where is the error in the PNAS article?
Nicholson et al., Biochem 1987
Uniformly 15N Labeled
Gramicidin A
Powder
Pattern
Oriented
Sample
A-B
a) “The qNH scale was aligned with the ppm scale by using
the calibration: 198 ppm ~15°, 180 ppm ~25°, and
146 ppm ~39°. Although there is not a linear
relationship between the two scales, the appropriate
alignment using this calibration demonstrates the
excellent agreement between the ensemble of NH
angles measured in the helices (of the X-ray structure)