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

摘要

This thesis deals with the dynamics of unfolded polypeptide chains as model for theudearliest steps in protein folding. Starting from an ensemble of unfolded conformations audfolding polypeptide chain has to form specific backbone and side-chain interactions toudreach the native state. The rate at which two defined contacts are formed on audpolypeptide chain is limited by intrachain diffusion. The characterization of rateudconstants of intrachain contact formation in polypeptides and their dependence onudlength, sequence and solvent effects give new insights for an understanding of theuddynamics of the earliest steps in protein folding.udUntil recently, little was known about absolute time scales of intramolecular contactudformation in polypeptide chains. Direct measurements of fast intramolecular diffusionudprocesses became possible with the development of fast diffusion-controlled electronudtransfer processes. In the presented work triplet-triplet energy transfer was used toudcharacterize intrachain contact formation in homo-polypeptides and peptide fragmentsudderived from natural protein sequences.udThe transfer of triplet electrons between the triplet donor xanthone to the triplet acceptorudnaphthalene is diffusion-controlled as tested by measuring its temperature and viscosityuddependencies. The results suggest that triplet-triplet energy transfer from xanthone toudnaphthalene provides the requirement to determine absolute intramolecular contactudformation rate constants in polypeptide chains.udIntrachain contact formation in unstructured polypeptides is well described as a singleudexponential process. The loop-size dependence of the rate constants of intrachainudcontact formation revealed that intrachain motions over short and long distances areudlimited by different rate-limiting steps. In short peptide chains end-to-end contactudformation 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 flexibleudpoly(glycine-serine) peptide chains with more than twenty peptide bonds N the rateudconstants decrease with N-1.7±0.1 and end-to-end contact formation becomes nearlyudcompletely entropy-driven.udGlycine and proline residues significantly change local intrachain dynamics comparedudto all other amino acids. Glycine accelerates contact formation whereas short prolineudcontaining peptides reveal complex kinetics of contact formation. Local chain dynamicsudare accelerated by a cis and slowed down by a trans peptidyl-prolyl bond. The effectsudvanish in peptide chains if the sequence contains more than five amino acids on eachudside of a single glycyine or a single proline residue.udThe dynamics of loop formation are sensitive to the nature of the solvent. Goodudsolvents, such as denaturants slow down intrachain dynamics compared to water. Theudeffect of solvent composition on chain dynamics indicates that the chain properties ofudpolypeptides strongly depend on the surrounding conditions.udNatural protein sequences are more complex than homo-polypeptide chains becauseudthey consist of 20 different amino acids. We determined the dynamics of loop formationudin sequences derived from two proteins, carp muscle β-parvalbumin and protein G B1uddomain. Compared to homo-polypeptides the intrachain dynamics in natural loopudsequences are slowed down and higher activation barriers are determined. The resultsudsuggest that the dynamics of the earliest steps in protein folding are limited byudsignificant activation barriers.udThe results allow us to estimate an upper time scale for rates of contact formation inudunstructured peptide chains. In glycine-rich sequences, which are often found in β-udhairpins and turns a first contact over 3-4 peptide bonds will be formed within 10-15 ns.udFor 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 constantudof the end-to-end contact formation long-range interactions on an unfolded polypeptideudchain over 50-60 peptide bonds will not be formed faster than in 500 ns.