Biomedical Engineering Reference
In-Depth Information
appropriate since domain Ia and Ib unfold with different dynamics to lead to
the disordered R-state, implying a three-state model with initial unfolding of
domain Ib. Therefore, the authors suggest that the inhibitory process involves
a shift of the equilibrium rather than discrete transitions.
280
This suggests the
equilibrium may be tunable, and proposes potential future targets for gene
therapy approaches currently being attempted on PLN.
281
It may be
interesting to note that, in both studies discussed above, it was necessaryto
lower the recording temperatures to 25 uC and 22 uC respectively, in order to
successfully observe the conformational exchange phenomena.
While many studies involve investigations of backbone amide dynamics,
further information can be gained using experiments to measure side-chain
methyl dynamics.
282,283
Studies typically require highly deuterated ILV
methyl-protonated samples that are preferably only
13
C-labelled at the methyl
position. Useful information can be gained since the side-chains are crucial in
inter- and intramolecular interactions.
282
In contrast, due to the closer position
to the backbone, methyl-protonated Ala residues tend to report on the
backbone dynamics.
284 2
H relaxation studies may also be carried out on
samples labelled using the method of Otten et al.
181
12.7.2
Variation of Experimental Conditions
Whilst many studies of functionally relevant dynamic phenomena involve the
measurement of relaxation data, the flexibility of NMR in allowing experimental
conditions to be altered may also allow useful, functional data to be obtained as
demonstrated by a recent study on the potassium channel KcsA;
285
pH titrations
were carried out and chemical shift differences were measured. Strong
correlations were observed between the shift differences and previously recorded
functional pH titration data for a number of residues. Further studies to
measure the
3
J(H
a
,HN) scalar coupling enabled calculation of the backbone w
angles for Tyr78, involved in the K
+
selectivity filter. A change in the mean w
angle from 270u to 250u was observed implying a functional stereochemical
shift. Analysis of line broadening in the presence and absence of K
+
indicated
that at pH 4, significant exchange occurs between the closed and open
conformations in the absence of K
+
but that the exchange slows from 500 to 100
Hz on addition of calcium, suggesting the presence of a dynamic equilibrium
modified by the presence of the permeating ion. Transition to the open stateis
pH dependent, and C-terminal residues showed a similar pH dependence to that
observed for Tyr78, implying a conformational link between the selectivity filter
and the C-terminus. These data enabled a detailed model of the gating process in
KcsA to be developed.
285
12.7.3 Ligand-Binding Studies
The goal of many NMR investigations of membrane proteins is to achieve
comprehensive 3D structure determination. In cases where this is not feasible,
