Effect
of Hydrophobic Interactions on the Folding
Mechanism of β‑Hairpins
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Abstract
Hydrophobic interactions are essential
in stabilizing protein structures.
How they affect the folding pathway and kinetics, however, is less
clear. We used time-resolved infrared spectroscopy to study the dynamics
of hydrophobic interactions of β-hairpin variants of the sequence
Trpzip2 (SWTWENGKWTWK-NH2) that is stabilized by two cross-strand
Trp–Trp pairs. The hydrophobicity strength was varied by substituting
the tryptophans pairwise by either tyrosines or valines. Relaxation
dynamics were induced by a laser-excited temperature jump, which separately
probed for the loss of the cross-strand β-hairpin interaction
and the rise of the disordered structure. All substitutions tested
result in reduced thermal stability, lower transition temperatures,
and faster dynamics compared to Trpzip2. However, the changes in folding
dynamics depend on the amino acid substituted for Trp. The aromatic
substitution of Tyr for Trp results in the same kinetics for the unfolding
of sheet and growth of disorder, with similar activation energies,
independent of the substitution position. Substitution of Trp with
a solely hydrophobic Val results in even faster kinetics than substitution
with Tyr but is additionally site-dependent. If the hairpin has a
Val pair close to its termini, the rate constants for loss of sheet
and gain of disorder are the same, but if the pair is close to the
turn, the sheet and disorder components show different relaxation
kinetics. The Trp → Val substitutions reveal that hydrophobic
interactions alone weakly stabilize the hairpin structure, but adding
edge-to-face aromatic interaction strengthens it, and both modify
the complex folding process