Length Dependent Helix-Coil Transition Kinetics of Nine Alanine-Based Peptides

Abstract

It is well-known that end caps and the peptide length can dramatically influence the thermodynamics of the helix-coil transition. However, their roles in determining the kinetics of the helix-coil transition have not been studied extensively and are less well understood. Kinetic Ising models and sequential kinetic models involving barrier crossing via diffusion all predict that the helix formation time depends monotonically on the peptide length with the relaxation time increasing with respect to increasing chain length. Here, we have studied the helix-coil transition kinetics of a series of Ala-based α-helical peptides of different length (19-39 residues), with and without end caps, using time-resolved infrared spectroscopy coupled with laser-induced temperature jump (T-jump) initiation method. The helical content of these peptides was kinetically monitored by probing the amide carbonyl stretching frequencies (i.e., the amide I' band) of the peptide backbone. We found that the relaxation rates for peptides with efficient end caps are more rapid than those of the corresponding peptides without good end caps. These results indicate that efficient end-capping sequences can not only stabilize preexisting helices but also promote helix formation through initiation. Furthermore, we found that the relaxation times of these peptides, following a T-jump of 1-11 °C, show rather complex behaviors as a function of the peptide length, in disagreement with theoretical predications. Theses results are not readily explained by theories in which Ala is taken to have a single helical propensity (s). However, recent studies have suggested that s depends on chain length; when this factor is considered, the mean first-passage times of the coil-to-helix transition show similar dependence on the peptide length as those observed experimentally.

Figure 1

Equilibrium helix–coil transitions of SPE₁ (□), SPE₂ (×), SPE₃ (△), SPE₄ (+), and SPE₅ (○) peptides as measured by the mean residue ellipticitiy at 222 nm as a function of temperature. As expected, the longest peptide in this series, SPE₅, is the most stable and its thermal transition is more cooperative.

Figure 2

(a) Temperature-dependent equilibrium and (b) difference FTIR spectra of the SPE₄ peptide. These spectra were collected roughly every 7 °C, from 5 to 75 °C. Difference spectra were generated by subtracting the spectrum collected at 5 °C from the spectra collected at other temperatures. Arrows indicate the direction of changes when temperature was increased. The negative-going feature at ~1630 cm^–1 corresponds to loss of helical conformations, whereas the positive-going signal ~1655 cm^–1 is due to the formation of disordered structures.

Figure 3

(a) Relaxation kinetics of AKA₂ and AKA₄ peptides and (b) relaxation kinetics of SPE₂ and SPE₄ peptides, as indicated. Note that the signals have been scaled to the same amplitude in each case. The T-jump was ~10 ± 1 °C, from ~1 to ~11 °C. The probing frequency was 1630 cm^–1. The smooth lines are fits to the following function, △OD(t) = A*[1 –B* exp(–t/τ)^β], convolved with the instrument response function that was determined by fitting the rise time of the D₂O signal. The fitting parameters are listed in Table 1.

Figure 4

Relaxation times of AKA_n (△) and SPE_n (○) peptides following a T-jump from 1 to 11 °C. It is evident that peptides of the same length but containing end caps take less time to relax to the new equilibrium point. Inset: Relaxation times of AKA_n (△) and SPE_n (○) peptides following a T-jump from 25 to 35 °C.

Figure 5

Arrhenius plots of (a) AKA₂ (△) and SPE₂ (○), (b) AKA₃ (△) and SPE₃ (○), (c) AKA₄ (△) and SPE₄ (○), and (d) AKA₅ (△) and SPE₅ (○) peptides. The T-jump amplitude for each point was roughly 10 °C. The straight lines are linear fits to the data, and the corresponding Arrhenius activation energies are listed in Table 1.

Figure 6

Mean first-passage time of the transition from all-coil state to all-helical state as a function of peptide length. (a) Our results that were generated by using values of s described by curve c. (b) The dashed line is the result of Straub et al., using σ = 0.002, and s = 1.5 (note: other Ising-like models yield similar results under the same conditions).