Biomedical Engineering Reference
In-Depth Information
partially aligned media, such as the liquid crystal matrix formed by
filamentous bacteriophage Pf1.
12,13
These solution-phase studies are largely
consistent with results from slow-MAS experiments
10
and confirm that, in
addition to setting new challenges for ab inito quantum chemistry techniques
and improving the analysis of relaxation data, CST measurements have great
potential for characterising backbone dihedral angles and hydrogen bond
strengths in proteins.
3.3 Referencing, Databases and Re-referencing
The extent of nuclear shielding at a particular site i is measured by comparison
with the resonance frequency of an internal reference compound, which is
assigned a chemical shift of zero:
d
i
~10
6
ð
u
i
{u
ref
Þ=
u
ref
ð
3
:
3
Þ
where u
i
and u
ref
correspond to the frequencies of the signal of interest and the
internal standard, respectively, and d
i
has units of parts per million (ppm). The
choice of reference compound is arbitrary, but the IUPAC/IUBMB standard
for
1
H and
13
C chemical shift referencing of aqueous solutions is the soluble,
inert compound 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS; 0 ppm).
14
Unfortunately, deuterated DSS is not commercially available, so additional
multiplet signals appear in the spectrum, rather than the single peak produced
by tetramethylsilane (TMS) in organic solvents (0 ppm). As a consequence,
several other reference compounds are in common use, including trimethylsi-
lylpropionic acid (TSP; 0 ppm), dioxane (3.75 ppm) and the residual H
2
O
solvent signal (y4.8 ppm), although these alternatives must be treated
carefully. For example, the frequency of the water signal is highly sensitive to
changes in hydrogen bonding, varying with temperature, pH and the
concentration of co-solutes such as urea. The water signal can even be used
as an internal thermometer,
15
with a chemical shift (in ppm) that varies linearly
with absolute temperature (T) between 0 C and 52 C at pH 7.0 according to:
d
H
2
O
~7
:
83{T
=
96
:
9
:
ð
3
:
4
Þ
Internal reference compounds should always be assessed to ensure that theydo
not interact with the protein under study; for example, DSS and TSP are
known to bind weakly to cationic peptides and partially folded molten globule
states, inducing unexpected apparent chemical shift changes.
16,17
TSP signals
are
also
pH-dependent
with
a
pK
a
of
5.0,
which
can
cause
particular
13
C shifts.
18,19
referencing problems for
15
N and many
13
C shifts are referenced indirectly, fixing
In practice, most
the
origin
of
the
chemical
shift
scale
by
multiplying
the
experimentally
1
H signal by a ratio of 'j value' conversion
determined frequency of a DSS
factors.
14,20
1
H frequency of 100.000000 MHz, the j values for
Relative to a
