Takaisin Ajatusvarikolle
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Back to the Thought Deposit
Dinoglyyfit
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Dinoglyfs
Footnotes and extra appendixes transferred away from the PhD manuscript for the lack of space
FUTURE PROSPECTS
No biochemistry has been carried out with
twinfilin in its tissue-extracted, possibly modified, physiological form. This
is a caveat in comparison to the AC- and drebrin studies, where the work
started from the native proteins. Surprises or enhancement for the recombinant forms
were seen e.g. in severing activity of cofilin (Ichetovkin et al 2000).
It remains to be studied, whether twinfilin is
expressed in the highly specialized cell types such as neutrophiles or
platelets. The differences in the PIP2-binding pattern of yeast
profilin and cofilin was a surprise. The differential nature had not been found
before and awaits clarification. The
main difference between AC and twf still is the ATP-binding detected by Beeler
et al (1994) and Rohwer et al (1997), although the affinity and the relevance
of the ATP/ADP-binding remain to be confirmed. Affinities with physiological
significance to ATP range from picomolar (myosin) to millimolar (glycolytic
enzymes) KDs, so a quantitative measure has a value as such.
Kinetic studies in (~7 %)
glycerol with viscosity as a parameter to follow collisions and intraprotein
substrate shuffling should be sought in order to confirm the conformational
change and the role of wt twinfilin in it. The linker and tail regions could be
classified as motifs in the databases, if they were studied in more detail. I
continued the study of the putative hinge region by NMR to get a definite
answer, whether the 9s-1 refers to actin or twinfilin. In biphasic
binding, what is calculated from the steady state (equilibrium) kinetics is an
apparent KD, which incorporates both of the steps. A real
equilibrium constant for the first binding step could also be calculated e.g.
from the double reciprocal plot – if reliable data only was available. {If 1/kobs
was plotted against 1/[twf], then intercept on y-axis = 1/k2; slope
= 1/(k1 x k2).}
The shortcoming in determining the rate for the
first step apart from the second one in the (at least) two-step interaction of
wt twf is mainly due to the experimental system that is limiting. To fit and
measure the exponential second order rate constant one would need to use very
low twinfilin concentrations (below 100 nM or so) and, consequently, much lower
NBD-actin concentrations to simplify data analysis. This would obviously limit
the amplitude of the fluorescence signal even in the case of an external
photomultiplier and our 500 nm cut-off filter. The major question would be the
physiological significance of this elaboration, given the high micromolar
concentrations of twinfilin from yeast. The only interest would be in
elucidation of the molecular mechanism and the precise role of the N-terminal
domain.
To get a fluorescence signal from the
NBD-label, the binding has to be direct (proximal) with a maximum distance of
some 20 Å, and many ACs cannot be measured by these means. To avoid the NBD-caveat,
FRET could be applied. FRET permits the accurate distance measurements of complexes over distances from ~10 to 100 Å
with an accuracy of ~1 nm, in real time (Deniz et al 2000).
The mammalian cofilin
pseudogene is considered nothing but an annoyance. When the human genome draft
was released, over 30 actin pseudogenes were found (Pollard 2001). Where are
they derived from? Could the intriguing question of the physiological role of
introns be answered by phylogenetics at a nucleotide level in the light of the
conserved actin? Are introns a method of keeping the genes in their place, away
from degenerating shuffling and cut'n paste errors? Mutations accumulate faster
on pseudogenes, so pseudogene databanks
should be self-evident in order for
the evolutionary claims to be verifiable. Copying of deliberate and trivial
errors in software is the most convincing evidence for piratism in the court.
What if the best way to evaluate the probabilities and barriers of variation,
adaptation and speciation in nature, would turn out to be pseudogene
statistics?