DYNAMICAL ANALYSIS OF WT M-PMV MATRIX PROTEIN AND ITS SINGLE POINT MUTANT R55F WITH RESPECT TO THEIR DIFFERENT OLIGOMERIZATION PROPERTIES
Pavel Srb1,2, Jan Prchal2,3,
Jiří Vlach2 Jan Lang 1,2, Marián Grocký1, Jan, Tomáš Ruml3
and Richard Hrabal2
1Department of Low Temperature Physics, Faculty of Mathematics and Physics, Charles University, V Holešovičkách 2, 180 00 Prague 8, Czech Republic
2Laboratory of NMR Spectroscopy and
3Department of Biochemistry and Microbiology
Institute of Chemical Technology, Technická 5, 166 28 Prague 6, Czech Republic
richard.hrabal@vscht.cz
Mason-Pfizer monkey virus
(M-PMV) belongs to the genus of betaretroviruses, in which the matrix protein
(MA) plays the essential role in certain stages of their life cycle (e.g. in
the assembly, transport and budding of new viral particles). Several single or
double point mutants are known to cause dramatic changes in the virus life
cycle. In particular, the single point mutation R55F in MA redirects the
assembly of the viral capsid to the plasma membrane instead of to cytoplasm,
which is the place of assembly of the wild type form (WT).
In our recent work [1] we
found that M-PMV WT MA exists in a monomer–dimer–trimer equilibrium in solution
with the corresponding dissociation constants of 2.3 mM and 0.24 mM,
respectively. Conversely, almost negligible and nonspecific oligomerization was
observed for the R55F mutant. Structural comparison of matrix proteins
explained their different behavior in solution, concluding that the key
residues involved in the intermonomeric interaction are exposed in the WT MA
while being buried in the mutant, which prevents the oligomerization of R55F
MA.
Here, we present the motional
analyses of the experimental 15N NMR relaxation and “in silico” molecular dynamic simulations (MD) for WT and R55F
MAs. Both approaches enlighten molecular motions from the microscopic point of
view. Analysis of experimental data provides information about global
reorientation of molecule, flexibility of each residue (through order
parameters, S2) and for most sites also the timescale
of local motion. Although MD simulations provide a wealth of information giving
almost complete picture of protein dynamics, careful analysis of results is
necessary to avoid possible artifacts coming from poor sampling of rare
dynamical processes during simulated trajectory. Two computational approaches
were used here; the first one was the isotropical reorientational eigenmode
dynamics (iRED) [2] which provided especially information about correlated
motions. The second one was the calculation of the timescale window-dependent
order parameters, S2(w), which provided
information inaccessible by experiment. Computational routine is implemented in
program PAIN [3]. Important changes in local dynamics caused by the single
point mutation will be demonstrated and discussed.
References
[1] J. Vlach et al. J. Mol. Biol., 390, 5, 967-980, (2009)
[2] J. Prompers, R. Brüschweiler, J.Am.Chem.Soc., 124, 4522-4534, (2002)
[3] P. Macek, et al., J.Phys.Chem.B, 111, 5731-5739, (2007)