Folding and Unfolding of the Short Light-Triggered β-Hairpin Peptide AzoChignolin Occurs within 100 ns

Abstract

To map the underlying molecular mechanisms of folding dynamics in proteins, light-operated peptides have emerged as promising tools. In this study, we reveal the complete sequence of light-induced structural changes of AzoChignolin, a short β-hairpin peptide containing an azobenzene photoswitch in its loop region. Light-triggered structural changes were monitored by time-resolved IR spectroscopy. Formation and destruction of the hairpin structure is very fast and occurs within 100 ns for AzoChignolin in methanol. Atomistic molecular dynamics simulations using two explicit solvents, methanol and water, revealed the underlying molecular processes and allowed us to gain further insight into the reaction mechanism. Despite its rapid reaction time, hairpin formation in these solvents is not force-driven by the molecular switch but proceeded via formation of interstrand hydrogen bonds and contacts between aromatic residues. Moreover, the combined experimental and theoretical study demonstrates that the solvent (methanol vs water) does not dictate the velocity of β-hairpin formation in the AzoChignolin peptide comprising only a few hydrophobic residues in the strands.

Mechanism of β‐hairpin formation in AzoChignolin and Chignolin

“…The next two eigenvectors are more related to the folding process, as they involve mainly transitions from and to the hairpin state 3 (eigenvector 2) with a timescale of 210 ns and mainly transitions from and to the disordered hub state 4 (eigenvector 1) with a timescale of 185.5 ns, matching autocorrelation timescales of a previous MD study. 2 The fully folded β-hairpin of AzoChignolin is stabilized by hydrophobic interactions between mainly Tyr2-Trp9 and to a lesser extent Tyr2-Pro4, similar to native Chignolin. While additional hydrophobic interactions would be possible with the introduced AMPP turn as well, they are only observed within the other states.…”

“…The speed at which AzoChignolin folds (and unfolds) in MeOH was measured in the experimental study of Hofmann et al 2 Note that these mean arrival times, particularly the latter, would be much higher if all simulations were included in the average by prolonging the simulations beyond 1000 ns until all have reached the low-RMSD region. The Markov model estimated on the longer ensemble of simulations allows us to also quantify the transition with the mean first passage time (MFPT).…”

“…Cis-AzoChignolin is able to form a β-hairpin structure similar to native Chignolin, while trans-AzoChignolin mostly exhibits disordered elongated structures, as trans-AMPP has a slightly longer end-to-end distance and thus hinders interaction between the two strands of trans-AzoChignolin. 1,2 The ability to induce synchronized folding of AzoChignolin by sudden illumination allows one to directly follow the process using techniques such as time resolved IR spectroscopy. Since the folding time scale of the structural motif is also accessible using atomistic molecular dynamics (MD) simulations it is an ideal system to study the thermodynamics and kinetics of hairpin loop formation.…”

“…The AMPP chromophore exhibits cis‐trans isomerism and allows, upon illumination, a change of the global structure of AzoChignolin due to its center position in the loop. Cis‐AzoChignolin is able to form a β ‐hairpin structure similar to native Chignolin, while trans‐AzoChignolin mostly exhibits disordered elongated structures, as trans‐AMPP has a slightly longer end‐to‐end distance and thus hinders interaction between the two strands of trans‐AzoChignolin 1,2 . The ability to induce synchronized folding of AzoChignolin by sudden illumination allows one to directly follow the process using techniques such as time resolved IR spectroscopy.…”

“…The helical intermediates of Chignolin, particularly encountered in MeOH, are also not observable for AzoChignolin in water and barely in MeOH, as its central azobenzene has a helix-breaking effect. With the help of Markov modeling we were able to extract timescales from the slowest eigenprocesses which match previous shorter MD simulations2 and can be compared to experiment 38. Both general MSMs and miniMSMs showed that the kinetics of AzoChignolin are generally significantly faster compared to Chignolin, especially for turn formation, likely due to the turn-promoting shape of cis-azobenzene, but also for the hydrophobic collapse involving Tyr and Trp.…”