Probing local changes to α-helical structures with 2D IR spectroscopy and isotope labeling

Abstract

α-Helical secondary structures impart specific mechanical and physiochemical properties to peptides and proteins, enabling them to perform a vast array of molecular tasks ranging from membrane insertion to molecular allostery. Loss of α-helical content in specific regions can inhibit native protein function or induce new, potentially toxic, biological activities. Thus, identifying specific residues that exhibit loss or gain of helicity is critical for understanding the molecular basis of function. Two-dimensional infrared (2D IR) spectroscopy coupled with isotope labeling is capable of capturing detailed structural changes in polypeptides. Yet, questions remain regarding the inherent sensitivity of isotope-labeled modes to local changes in α-helicity, such as terminal fraying; the origin of spectral shifts (hydrogen-bonding versus vibrational coupling); and the ability to definitively detect coupled isotopic signals in the presence of overlapping side chains. Here, we address each of these points individually by characterizing a short, model α-helix (DPAEAAKAAAGR-NH₂) with 2D IR and isotope labeling. These results demonstrate that pairs of ¹³C¹⁸O probes placed three residues apart can detect subtle structural changes and variations along the length of the model peptide as the α-helicity is systematically tuned. Comparison of singly and doubly labeled peptides affirm that frequency shifts arise primarily from hydrogen-bonding, while vibrational coupling between paired isotopes leads to increased peak areas that can be clearly differentiated from underlying side-chain modes or uncoupled isotope labels not participating in helical structures. These results demonstrate that 2D IR in tandem with i,i+3 isotope-labeling schemes can capture residue-specific molecular interactions within a single turn of an α-helix.

Figure 1

Modulating MAHP α-helicity with solvent conditions. CD measurements revealed an increase in average α-helicity as pH was increased from 3 to 10 (from 8.13 to 25.13%). The addition of 40% TFE (v/v) further enhanced peptide helicity (up to 61.48% at pH 10). To see this figure in color, go online.

Figure 2

2D IR spectra of unlabeled MAHP. 2D IR spectra of MAHP adopting (A) a disordered structure at pH 3 without TFE, (B) a partially helical structure at pH 10 without TFE, and (C) a predominantly helical secondary structure at pH 10 with 40% TFE by volume. Amide I′ frequencies (red) and percent helicities quantified by CD experiments (black) are noted for each spectrum. The aspartate side chain presents as a shoulder near ∼1610 cm⁻¹ at pH 3 (A) and as a well-defined peak pair at 1580 cm⁻¹ at pH 10 (B and C). Average frequencies and standard deviations (SDs) over three replicates are summarized in Table S1. To see this figure in color, go online.

Figure 3

Double ¹³C¹⁸O-labeled MAHP variants exhibit red shifted isotope modes at increased α-helicity. 2D IR spectra of MAHP labeled with two ¹³C¹⁸O probes at (A–C) A3 and A6, (D–F) A5 and A8, and (G–I) A8 and G11. The dual isotopic probes exhibit varying red shifts upon α-helix formation, with frequencies noted in red. Average frequencies and SDs over three replicates are summarized in Table S2. To see this figure in color, go online.

Figure 4

Frequency shifts of A5 MAHP reveal hydrogen-bonding interactions. 2D IR spectra of A5 MAHP at (A) pH 3 without TFE, (B) pH 10 without TFE, and (C) pH 10 with 40% TFE by volume. The single isotopic probe exhibits a red shift similar to the doubly labeled A3A6 and A5A8 upon increasing sample α-helicity. To see this figure in color, go online.

Figure 5

Average ratio of integrated peak areas (low-frequency mode to amide I′ mode) for unlabeled, single, and double ¹³C¹⁸O-labeled MAHP as a function of solvent condition. The unlabeled (red) ratio increases at higher pH due to side-chain ionization. Incorporation of a single ¹³C¹⁸O isotope at A5 (orange) yields a higher peak ratio under all conditions due to the presence of both the isotope-labeled amide I′ mode and the side-chain mode within the low-frequency region. Double ¹³C¹⁸O labels within the C-terminal region (A8G11, blue) yield peak ratios that follow a similar trend to the single label at A5, suggesting that coupling between the A8 and G11 labels is minimal. Double ¹³C¹⁸O labels within the N-terminal (A3A6, yellow) and central (A5A8, green) regions exhibit enhanced peak ratios at high pH conditions compared, indicating significantly greater coupling between paired isotope labels within these regions compared with those at the C-terminus. The error bars indicate SDs over three replicates. To see this figure in color, go online.