Biomedical Engineering Reference
In-Depth Information
12.7
Functional Information
12.7.1 Dynamics
One of the main advantages of NMR spectroscopy is the ability to analyse
molecular dynamics on a per-atom basis potentially providing insights into the
mechanism of action of proteins. This is of particular value for membrane
proteins, many of which are important drug targets or are involved in disease
processes. NMR spectroscopy can investigate molecular dynamics on a range
of timescales from ps-to-ns motions through to ms-to-ms processes and even
slower conformational exchange events. In particular, for solution NMR, the
ability to view the protein in a dynamic configuration in solution, rather than
'frozen', means that multiple conformations of a protein may be observed in
contrast to other techniques which provide a 'snapshot'. More detailed
discussion of relaxation phenomena and their analysis is given by a number of
excellent reviews
270-272
and a number of solid-state relaxation studies have also
been reported recently.
273,274
These techniques have been successfully applied to elucidate functional
information for a number of membrane proteins (reviewed in Chill and
Naider
275
).
1
H-
15
NNOE,
15
N T
1
and
15
N T
2
values were recorded for PagP,
which palmitoylates lipid A in the bacterial outer membrane. With the longer
rotational correlation times of larger protein-detergent complexes, T
1
values are
shown to be highly sensitive to faster ns motions, demonstrating the existence of
highly flexible extracellular loop regions, as well as rapid motions at the
cytoplasmic end of the b-barrel.
14
On lowering the temperature, doubling of peaks
was observed identifying a smaller population with an alternative conformation.
An equilibrium between the R-form, observed at higher temperatures, and the T-
form which involves rearrangements in the b-bulge region and ordering of the L1
loop was observed. Rate constants for the exchange could be calculated and
further analysis of CPMG exchange experiments
272
demonstrated considerably
enhanced flexibility in the R-state which was suggested as an important factor in
facilitating substrate entry, whilst rigidification in the T-state may define the
position of the active site.
14
Analysis of a similar nature was also carried out for
the outer membrane protein, OmpA, another b-barrel protein.
276
Order
parameters, which are used to describe internal motions, were calculated using
the Lipari-Szabo 'model-free' approach and extensions of this method.
277,278
A
rigidity gradient was identified, decreasing away from the centre of the bilayer,
opposite to that of the phospholipid molecules.
276
Another protein subjected to dynamics analysis is phospholamban (PLN),
which binds to the sarcoplasmic reticulum Ca
2+
ATPase (SERCA) and is thus
directly involved in modulating Ca
2+
influx into the heart. The presence of
both
15
N backbone and
13
C
d1
side-chain conformational exchange dynamics
on a ms-to-ms timescale was investigated to understand the T-R equilibrium
for this protein. T
1
, T
2
and heteronuclear NOE measurements suggested that
helix II consists of two domains; domain Ib and II.
279
Conformational
exchange measurements suggest that a simple T-R exchange model is not
