Comparison of Isotopic Substitution Methods for Equilibrium and T-Jump Infrared Studies of β-Hairpin Peptide Conformation

Abstract

Laser induced temperature jump (T-jump) relaxation kinetics were measured with infrared absorbance (IR) detection for a set of beta-hairpin peptides, related to the Trpzip2 hairpin, but containing single isotopic labels, (13)C on the amide C horizontal lineO of selected residues both in the center of the strands and at the terminal regions of the hairpin. Variations in the behavior of single labeled peptides are compared to those previously reported for double labeled variants. Although single labels do not result in spectral intensity enhancement, as seen for cross-strand labeling, the IR frequency shifts are still diagnostic of hairpin unfolding. If C horizontal lineO's in the beta-strand portion of the hairpin (between the Trp residues) are labeled, the dynamic behavior of the local modes is similar to the results obtained with double labels in terms of relaxation time and activation energy and closely tracks the kinetics of the beta-strand components. This implies that either property, local secondary structure (change of varphi,psi), or cross-strand coupling enabled by strand formation and H-bonding relaxes with the same kinetic mechanism. Single labeled residues on the terminal positions have a different behavior and are less able to be detected due to overlap with the (12)C components, in contrast to double labels involving these positions, which are enhanced due to coupling. DFT-based spectral simulations that use the NMR structure of Trpzip2C indicate that the single labeled peptides should have roughly equivalent (12)C bands but the (13)C mode frequencies will vary with sequence position. Effective solvent corrections using COSMO yield significant changes in the frequencies but not in the relative isotope shifts obtained in our calculated spectra. Sequence positional dependence of labels is shown to be more discriminatory for kinetics changes than for thermodynamic variations.

Impact of β‐Turn Sequence on β‐Hairpin Dynamics Studied with Infrared‐Detected Temperature Jump

“…The decreased peptide relaxation time of TZ2-TG with increasing temperature is consistent with normal Arrhenius behavior and parallels the data that we have observed on several other (strandcentered) variants of TZ2-NG [17,27] and helical peptides [31]. Unlike for TZ2-NG and its isotopic variants, the relaxation rates of the β-structure (cross-strand H-bonding) and the disordered structure did not show a clear differentiation for TZ2-TG.…”

“…Overall, the observed relaxation times of TZ2-TG are slower compared with TZ2-NG [17,27], although the turn mutation destabilizes the peptide secondary structure [25]. While it is difficult to draw conclusions about Arrhenius behavior from just a few high-temperature data points, the slopes for the relaxation rates obtained when probing at the two wavenumbers are in fact similar and suggest activation energies 2-3 times higher than seen for TZ2-NG, which is consistent with the slower rates we observe.…”

“…We have separately reported equilibrium studies of various modifications of the Trpzip2 (TZ2-NG) hairpin sequence Ser-Trp-Thr-Trp-GluAsn-Gly-Lys-Trp-Thr-Trp-Lys (SWTWENGKWTWK) [8], varying both the Trps and the turn sequence residues [13,14,16,17,25,26]. We and others have also used laser-initiated T-jump spectroscopy to study dynamics of the TZ2-NG sequence and have incorporated isotopic labels to specify sequence dependence of the folding mechanism [17,27,28]. In this paper, we compare dynamics for this type of hairpin by modifying the turn sequence (Figure 1), changing from Asn-Gly (TZ2-NG) to Thr-Gly (TZ2-TG), which lowers the hairpin stability, as we have shown separately [25] and slows its relaxation dynamics, as will be shown here.…”

Folding of a heterogeneous β-hairpin peptide from temperature-jump 2D IR spectroscopy

“…1) is a 12-residue peptide designed to form a type I′ β-turn due to stabilization by cross-strand H-bonds and packing of two pairs of tryptophan (Trp) indole rings (15). TZ2's small size and fast folding rates have made it an attractive system to study both experimentally (9,10,(15)(16)(17)(18)(19)(20)(21)(22)(23) and computationally (3,16,18,(24)(25)(26)(27), which has fostered comparison and enriched interpretation of results. In general, recent work has focused on discriminating between two folding processes: (i) hydrophobic collapse (25,28), in which H-bond contacts are formed after hydrophobic contacts create a favorable collapsed state, and (ii) zipping (18,26,29), where native H-bonds (nHBs) form rapidly following a nucleation event.…”

“…However, recent experiments interpreted probe-dependent melting points measured with circular dichroism, fluorescence, FTIR, and 2D IR as signs of multistate behavior (10,16,20). Heterogeneous folding was also proposed based on frequency-dependent differences in isotope-labeled 2D IR experiments and rates in T-jump measurements (10,17,19,21,22). Despite many studies, there has been no consensus on the folding mechanism or the specific assignment of states.…”