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Fig.:
The structure of the
protein CI2 consists of an a-helix packed against a ß-sheet. CI2
is a ‘two-state protein’ that folds, from the unfolded state into
the folded state, by crossing just a single transition state
barrier.
Image: MPI for Colloids and Interfaces
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Proteins are chain molecules
assembled from amino acids. The precise sequence of the twenty
different types of amino acids in a protein chain is what determines
which structure a protein folds into. The three-dimensional structures
in turn specify the functions of proteins, which range from the
transport of oxygen in our blood, to the conversion of energy in our
muscles, and the strengthening of our hair. During evolution, the
protein sequences encoded in our DNA have been optimised for these
functions.
The reliable folding of proteins is a prerequisite
for them to function robustly. Mis-folding can lead to protein
aggregates that cause severe diseases, such as Alzheimer's,
Parkinson's, or the variant Creutzfeldt-Jakob disease. To understand
protein folding, research has long focused on metastable folding
intermediates, which were thought to guide the unfolded protein chain
into its folded structure. It came as a surprise about a decade ago
that certain small proteins fold without any detectable intermediates.
This astonishingly direct folding from the unfolded state into the
folded state has been termed ‘two-state folding’. In the past few
years, scientists have shown that the majority of small single-domain
proteins are ‘two-state folders’, which are now a new paradigm in
protein folding.
The characteristic event of two-state folding is the
crossing of a barrier between the unfolded and folded state. This
folding barrier is thought to consist of a large number of extremely
short-lived transition state structures. Each of these structures is
partially folded and will either complete the folding process, or will
unfold again, with equal probability. Transition state structures are
thus similar to a ball on a saddle point, which has the same
probability, 0.5, of rolling to either side of the saddle.
Since transition state structures are highly instable,
they cannot be observed directly. To explore two-state folding,
experimentalists instead create mutants of a protein. The mutants
typically differ from the original protein -- the wild type -- in just
a single amino acid. The majority of these mutants still fold into the
same structure, however the mutations may slightly change the
transition state barrier and, thus the folding time; that is, the time
an unfolding protein chain on average needs to cross the folding
barrier.
The central question is: can we reconstruct the
transition state from the observed changes in the folding times? Such
a reconstruction clearly requires experimental data on a large number
of mutants. In the traditional interpretation, the structural
information is extracted for each mutation, independent of the other
mutations. If a mutation does not change the folding time, then the
mutated amino acid traditionally is interpreted to be still
unstructured in the transition state. In contrast, if a mutation
changes the folding time, the mutated amino acid is interpreted to be
partially or fully structured in the transition state, depending on
the magnitude of the change.
This traditional interpretation is however often not
consistent. For example, twenty single-residue mutations in the
a-helix of the protein Chymotrypsin Inhibitor 2 (CI2) have very
different effects on the folding time. Naïvely interpreted, these
differences seem to indicate that some of the helical residues are
unstructured in the transition state, while other residues, often
direct neighbours, are highly structured. This naïve interpretation
contradicts the fact that the folding of helices is co-operative, and
can only occur if several consecutive helical turns are structured,
stabilizing each other.
In a recent article in PNAS, a research team from the
Max Planck Institute of Colloids and Interfaces and the University of
California, San Francisco has suggested a novel interpretation of the
mutational data. Instead of considering each mutation on its own, the
new interpretation collectively considers all mutations within a
cooperative substructure, such as a helix. In case of the a-helix of
the protein CI2, this leads to a structurally consistent picture, in
which the helix is fully formed in the transition state, but has not
yet formed significant interactions with the ß-sheet.
In the future, the Max Planck researchers hope to
construct complete transition states from mutational data. An
important step is to identify the cooperative subunits of proteins,
which requires molecular modelling. In a similar way to how a mountain
pass shows us how to cross the landscape, the transition states
eventually may help us to understand how proteins navigate from the
unfolded into the folded structure.
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