Dynamics in unfolded polypeptide chains as model for elementary steps in protein folding

This thesis deals with the dynamics of unfolded polypeptide chains as model for the
earliest steps in protein folding. Starting from an ensemble of unfolded conformations a
folding polypeptide chain has to form specific backbone and side-chain interactions to
reach the native state. The rate at which two defined contacts are formed on a
polypeptide chain is limited by intrachain diffusion. The characterization of rate
constants of intrachain contact formation in polypeptides and their dependence on
length, sequence and solvent effects give new insights for an understanding of the
dynamics of the earliest steps in protein folding.
Until recently, little was known about absolute time scales of intramolecular contact
formation in polypeptide chains. Direct measurements of fast intramolecular diffusion
processes became possible with the development of fast diffusion-controlled electron
transfer processes. In the presented work triplet-triplet energy transfer was used to
characterize intrachain contact formation in homo-polypeptides and peptide fragments
derived from natural protein sequences.
The transfer of triplet electrons between the triplet donor xanthone to the triplet acceptor
naphthalene is diffusion-controlled as tested by measuring its temperature and viscosity
dependencies. The results suggest that triplet-triplet energy transfer from xanthone to
naphthalene provides the requirement to determine absolute intramolecular contact
formation rate constants in polypeptide chains.
Intrachain contact formation in unstructured polypeptides is well described as a single
exponential process. The loop-size dependence of the rate constants of intrachain
contact formation revealed that intrachain motions over short and long distances are
limited by different rate-limiting steps. In short peptide chains end-to-end contact
formation is with a minimum time constant of 5-10 ns virtually independent of chain length and limited by an activation barrier of 12-16 kJ/mol. In long flexible
poly(glycine-serine) peptide chains with more than twenty peptide bonds N the rate
constants decrease with N-1.7±0.1 and end-to-end contact formation becomes nearly
completely entropy-driven.
Glycine and proline residues significantly change local intrachain dynamics compared
to all other amino acids. Glycine accelerates contact formation whereas short proline
containing peptides reveal complex kinetics of contact formation. Local chain dynamics
are accelerated by a cis and slowed down by a trans peptidyl-prolyl bond. The effects
vanish in peptide chains if the sequence contains more than five amino acids on each
side of a single glycyine or a single proline residue.
The dynamics of loop formation are sensitive to the nature of the solvent. Good
solvents, such as denaturants slow down intrachain dynamics compared to water. The
effect of solvent composition on chain dynamics indicates that the chain properties of
polypeptides strongly depend on the surrounding conditions.
Natural protein sequences are more complex than homo-polypeptide chains because
they consist of 20 different amino acids. We determined the dynamics of loop formation
in sequences derived from two proteins, carp muscle β-parvalbumin and protein G B1
domain. Compared to homo-polypeptides the intrachain dynamics in natural loop
sequences are slowed down and higher activation barriers are determined. The results
suggest that the dynamics of the earliest steps in protein folding are limited by
significant activation barriers.
The results allow us to estimate an upper time scale for rates of contact formation in
unstructured peptide chains. In glycine-rich sequences, which are often found in β-
hairpins and turns a first contact over 3-4 peptide bonds will be formed within 10-15 ns.
For glycine-free sequences local contact formation is slowed down to 15-50 ns depending on the sequence. Due to the strong distance dependence of the rate constant
of the end-to-end contact formation long-range interactions on an unfolded polypeptide
chain over 50-60 peptide bonds will not be formed faster than in 500 ns.