Chemistry Reference
In-Depth Information
RCSA, which is on the order of parts per billion (ppb), may be greatly affected. The
solvent effect can be removed by measuring several aligned samples at increasing
alignment orders and extrapolating the RCSA values to zero concentration [
32
]. The
methods were applied to backbone
15
N[
32
]and
13
C
0
[
45
] RCSA measurements.
Another way to overcome this is to measure isotropic sample under the same
unaligned medium. Liu and Prestegard [
33
] developed a two-stage NMR tube with
two different internal diameters (I.D.) at upper and lower parts of an open-ended
NMR tube. For isotropic condition the protein sample was soaked into polyacryl-
amide gel in 5 mm I.D. part, then the same piece of gel was pulled and stretched into
the 3 mm I.D. part by vacuum created using a syringe at the other end of the two-
stage tube, resulting anisotropic condition. The method allows for higher accuracy
measurements for both RDC and RCSA.
Aside from using both aligned and unaligned samples for RDC and RCSA, one
may also keep a single sample that contains alignment media for both isotropic and
anisotropic measurements. Upon the application of magic angle spinning, similar to
what is used in solid state NMR, the alignment effect for bicelles [
46
] or phage [
47
]
was removed. In this way the measurement will not contain any solvent effects.
4
Interpretation and Implementation
Measured RDC values are representative averages of the whole ensemble of dipolar
interactions within protein molecules in solution. Such an ensemble should include
all protein conformers interconverting at time scales faster than the inverse of RDC
values (1/
D
). For instance, the observed dipolar coupling is affected by the
internuclei or bond vectors that stretch and vibrate on a femtosecond to nanosecond
(fs-ns) time scale, the protein domain reorientation on a nanosecond to microsec-
ond (ns-
m
s) time scale, and conformational change that ranges from nanoseconds,
e.g., unstructured terminus, to milliseconds. It is nearly impossible to describe
protein structure and dynamics using RDC values without any assumptions. Some
approximations have to be made in interpreting RDC measurements.
4.1 Approximations
Most RDCs except for
D
H-H
[
48
] are measured on fixed internuclei distances such as
bond vectors or geometries within the peptide plane so that in Eqs (1) and (2) a
constant internuclei distance
r
AB
in
D
max
is assumed. This in itself is an approximation
because the effective bond lengths vary due to dynamic processes. For instance, Yao
et al. [
49
] determined the average protein backbone H-N bond length to be
1.01-1.02
˚
. However, for deriving the true alignment order an effective value of
1.04
˚
was proven proper and used extensively to account for H-N bond libration
dynamics [
50
]. A slight increase in effective bond length for RDC analysis also applies
