Please wait while the transcript is being prepared...
This talk deals with the mechanisms of protein folding reactions.
The outline of my talk,
I will first give a brief introduction into the history of protein folding,
I will then talk about intrinsically slow reactions coupled to protein folding.
These are prolyl peptide bond isomerization,
non-prolyl peptide bond isomerization,
association reactions of oligomeric proteins.
In the second part of my talk,
I will address kinetic mechanisms of protein folding,
and I will specifically discuss how we can
place an intermediate on a kinetic folding pathway.
This will be shown in two case studies.
The first one is folding of Iysozyme,
and the second one is folding of Semliki Forest virus protease.
If you regard protein folding as a chemical reaction,
we start from the ensemble of different conformations representing
the unfolding state and end up in the biologically active native state,
which represents a single conformation with defined side chain and backbone interactions.
This is a simplification, of course,
because we know that there is conformation of fluctuations in the native state.
But the basic problem in protein folding is how the ensemble of
different states from the unfolded ensemble reach the native state.
And the questions we are addressing is,
are there intermediates located between the unfolded and the native state and what are
the rate limiting steps between the unfolded and the native state?
My talk deals with protein folding studies in vitro,
with purified proteins under well-defined conditions.
However, in vivo, protein folding occurs on the ribosome and it has been argued that
results from in vitro protein folding studies
may not be relevant for the situation in the cell.
Based on concept dating back to Van't Hoff's work in 1884,
I will try to show you that folding on the ribosome rather
represents a special case for protein folding reaction in the cell.
If you assume an equilibrium between
the ensemble of unfolded states and the native state,
with typical values for rate constants and stability,
we assume that delta G zero value of minus 17 kilojoules per mole for
the native state corresponding to an equilibrium constant of 10 to the three.
That means, we have a thousand times more native molecules
than unfolded molecules in equilibrium.
And we assume rate constants for folding of 10 per
second and the unfolding of point zero one per second.
These are also typical values formed for
small proteins or for domains of larger proteins.
We further assume a protein concentration of 10 micro molar.
We can then calculate that we have about 10 micro molar of molecules in the native state
and 10 to the minus two micro molar of molecules in the unfolded state in equilibrium.
Since the flux in the forward and
backward reaction are identical under equilibrium conditions,
we can calculate that, per second,
point one micro molar of molecules fold and unfold.
Now, let us apply these numbers to experimental systems.
If you perform experiments in vitro,
we typically have volumes of one milliliter in a cuvette.
That is, in this cuvette,
we have 10 to the 16 molecules in the native state,
and 10 to the 13 molecules in the unfolded state.
And every second, 10 to the 14 molecules fold and unfold.
If you do the same calculations for an E.
Coli cell with a volume of 10 to the minus 12 milliliter,
we see that we have 10 to the four molecules in the native state,
and 10 molecules in the unfolded state.
And every second, 100 molecules fold and unfold.
This means that without correct folding,
there would be less than one percent of native molecules after 500 seconds.
That means folding has to be able to occur in the absence of
the ribosome for protein to survive in a cell for longer times.
Let's take a short look at the history of protein folding experiments.