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

Abstract

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.