Biomedical Engineering Reference
In-Depth Information
SS
z
I
b
T
ðÞ
~SS
z
I
b
T
ðÞ
e
{(R
2
{gzR
ex
)t
ð
7
:
10
Þ
In eqn (7.10) t 5 4nt
cp
(n 5 integer) and g is the transverse
15
N CSA/
15
N-
1
H
dipole-dipole relaxation interference rate constant. As in the non-TROSY
version,
22
the CPMG period is divided in two halves, and separated with a U-
period. However, unlike the non-TROSY version the U period must
additionally selectively invert one N-H multiplet component,
51
which
effectively inverts the sign of the cross-relaxation rate of the NH doublet. To
achieve this the U-period in the TROSY CPMG utilises an S
3
CT element.
51
Following the second relaxation period, the TROSY signal is detected in the t
1
and t
2
domains. To obtain a dispersion curve the peak intensity is quantified at
several values of t, by varying n at a single t
cp
value. The monoexponential
decay of the resonance height as t increases gives R
2
(1/t
cp
). This procedure is
then repeated at multiple t
cp
values until sufficient sampling of the dispersion
curve is obtained. Subsequent fitting with either eqn (7.2) or (7.7) provides the
physical parameters describing the exchange process. This experiment hasbeen
utilised for characterising ms-ms motions in large enzymes such as arginine
kinase
52
(42 kDa) and in the integral membrane enzyme, PagP (y50 kDa).
53
The procedure for obtaining a TROSY-selected R
1r
(ref. 54) dispersion
curve is somewhat more complex and the relaxation period for this experiment
is shown in Figure 7.2(B). Prior to the relaxation period, an S
3
E filter
55
selects
the slowly relaxing H
b
N
z
component of the
1
H-
15
N spin system. Subsequently,
a spin-locking period of T/2, flanked by adiabatic rotations,
56
locks the
magnetisation in the rotating frame during which time it relaxes before being
returned to the z-axis. Between the two T/2 spin-locking periods, an S
3
CT
selective inversion
51
element refocuses cross-relaxation. Here, relaxation
dispersion curves are obtained by measuring monoexponential peak decay as
a function of spin-locking field strength, which can be varied by changing v
e
.
As above, TROSY detection follows the relaxation period.
7.3.2 Methyl-TROSY
Recently, Kay and co-workers have exploited the TROSY idea for
characterisation of methyl side-chains in large proteins.
25,57,58
Side-chain
methyl groups provide an attractive alternative probe to backbone amide
groups because the rapid three-fold rotation about the methyl axis produces
narrow lineshapes. Moreover, because methyl protons do not exchange with
the solvent, the available pH range for study can be more varied than for the
15
N-based experiments. In addition, methyl groups are also located throughout
most proteins, particularly in the hydrophobic core, making them relevant
probes of protein motion.
59
There are also considerably fewer methyl
resonances present in large enzymes than backbone amides, which reduces
the incidence of spectral overlap at the expense of full coverage of protein-wide
motions.
