Local conformational dynamics in α-helices measured by fast triplet transfer

Abstract

Coupling fast triplet-triplet energy transfer (TTET) between xanthone and naphthylalanine to the helix-coil equilibrium in alanine-based peptides allowed the observation of local equilibrium fluctuations in ␣-helices on the nanoseconds to microseconds time scale. The experiments revealed faster helix unfolding in the terminal regions compared with the central parts of the helix with time constants varying from 250 ns to 1.4 s at 5°C. Local helix formation occurs with a time constant of Ϸ400 ns, independent of the position in the helix. Comparing the experimental data with simulations using a kinetic Ising model showed that the experimentally observed dynamics can be explained by a 1-dimensional boundary diffusion with positionindependent elementary time constants of Ϸ50 ns for the addition and of Ϸ65 ns for the removal of an ␣-helical segment. The elementary time constant for helix growth agrees well with previously measured time constants for formation of short loops in unfolded polypeptide chains, suggesting that helix elongation is mainly limited by a conformational search.␣-helix-coil transition ͉ protein dynamics ͉ protein folding ͉ triplet-triplet energy transfer

Testing the diffusing boundary model for the helix–coil transition in peptides

“…The results revealed a position-independent helix elongation time constant (1/k f ) of about 400 ns at 5°C and a position-dependent helix unfolding rate constant (1/k u ) with faster unfolding at the termini (1/k u = 250 ns) compared with the center (1/k u = 1.4 μs). This behavior could be reproduced in simulations using a kinetic version of the linear Ising model (10,11), which suggested that helix elongation and unfolding mainly occur via a diffusing boundary mechanism (i.e., by the movement of the helix/coil boundary along the polypeptide chain). Helix nucleation in a completely unfolded chain and coil nucleation within a helical region are rare events in short model helices (10).…”

“…This behavior could be reproduced in simulations using a kinetic version of the linear Ising model (10,11), which suggested that helix elongation and unfolding mainly occur via a diffusing boundary mechanism (i.e., by the movement of the helix/coil boundary along the polypeptide chain). Helix nucleation in a completely unfolded chain and coil nucleation within a helical region are rare events in short model helices (10). The helix/coil boundary is statistically more likely located near the peptide ends than near the center (6,10).…”

“…Helix nucleation in a completely unfolded chain and coil nucleation within a helical region are rare events in short model helices (10). The helix/coil boundary is statistically more likely located near the peptide ends than near the center (6,10). Thus, it takes, on average, longer for the moving helix/coil boundary to reach the helix center than the helix ends, which leads to the observed position dependence of helix unfolding.…”

“…Temperature jump-induced unfolding experiments on short Ala-based helices showed that global unfolding occurs on the hundreds of nanoseconds time scale (8,9). In a previous study, we applied triplet-triplet energy transfer (TTET) to investigate local folding and unfolding dynamics at different positions in a 21-aa Ala-based α-helix at equilibrium (10). The results revealed a position-independent helix elongation time constant (1/k f ) of about 400 ns at 5°C and a position-dependent helix unfolding rate constant (1/k u ) with faster unfolding at the termini (1/k u = 250 ns) compared with the center (1/k u = 1.4 μs).…”

Making connections between ultrafast protein folding kinetics and molecular dynamics simulations

“…1-5). The idea of determining the kinetics of fast processes, other than intramolecular contact formation, by monitoring triplet-state populations was first applied to the helix-coil transition by Lapidus et al (35) and to protein folding by Buscaglia et al (38) using intrinsic probes (tryptophan and cysteine), and has subsequently been used by Kiefhaber and coworkers using extrinsic probes (xanthone and naphthylalanine) to further investigate the helix-coil transition (41), as well as submicrosecond processes in the folding of the villin subdomain (42).…”