Enhanced Sensitivity to Local Dynamics in Peptides by Use of Temperature‐Jump IR Spectroscopy and Isotope Labeling

Abstract

Site‐specific isotopic labeling of molecules is a widely used approach in IR spectroscopy to resolve local contributions to vibrational modes. The induced frequency shift of the corresponding IR band depends on the substituted masses, as well as on hydrogen bonding and vibrational coupling. The impact of these different factors was analyzed with a designed three‐stranded β‐sheet peptide and by use of selected 13C isotope substitutions at multiple positions in the peptide backbone. Single‐strand labels give rise to isotopically shifted bands at different frequencies, depending on the specific sites; this demonstrates sensitivity to the local environment. Cross‐strand double‐ and triple‐labeled peptides exhibited two resolved bands that could be uniquely assigned to specific residues, the equilibrium IR spectra of which indicated only weak local‐mode coupling. Temperature‐jump IR laser spectroscopy was applied to monitor structural dynamics and revealed an impressive enhancement of the isotope sensitivity to both local positions and coupling between them, relative to that of equilibrium FTIR spectroscopy. Site‐specific relaxation rates were altered upon the introduction of additional cross‐strand isotopes. Likewise, the rates for the global β‐sheet dynamics were affected in a manner dependent on the distinct relaxation behavior of the labeled oscillator. This study reveals that isotope labels provide not only local structural probes, but rather sense the dynamic complexity of the molecular environment.

Temperature-Jump 2D IR Spectroscopy with Intensity-Modulated CW Optical Heating

“…Infrared (IR) spectroscopy is a structurally sensitive probe that has been employed to track T-jump responses from proteins, nucleic acids, and small molecules. ,,,− In DNA or RNA, each nucleobase exhibits a distinct mid-IR spectrum reporting on in-plane vibrational stretching motions that is reshaped upon changes in hydrogen bonding and base stacking. − The amide I vibrations of proteins are sensitive to hydrogen bonding, allowing them to report on the disruption of secondary structures such as β-sheets and α-helices. , IR spectroscopy can resolve individual residues among a large protein or nucleic acid through isotope labeling of certain atoms, and this approach has been coupled with T-jump spectroscopy to resolve detailed mechanisms of protein unfolding. ,− Relative to linear IR spectroscopy, two-dimensional IR (2D IR) spectroscopy offers greater insight into the structure of biomolecules and their interactions with the environment. The 2D IR spectrum reports on coupling between vibrational modes, reveals contributions to the line width of an IR transition, and can distinguish whether peaks arise from distinct chemical species. ,, Changes in line shape and cross-peaks between vibrational modes of DNA bases have been used to report on base pair formation and base stacking as well as the loss of β-sheet character in proteins, while the 2D IR line shape provides information on the distribution of environments the molecule experiences. ,,, Due to the added technical difficulties relative to T-jump linear IR spectroscopy, development of T-jump 2D IR (t-2D IR) spectrometers has been limited …”

Template-assisted design of monomeric polyQ models to unravel the unique role of glutamine side chains in disease-related aggregation

“…Time-resolved IR-detected temperature-jump dynamics T-jump IR measurements were performed using the quantum cascade laser-based spectrometer that has been described in detail previously. 38,41,44,46 Relaxation dynamics of the peptides were probed at selected wavenumbers with a quantum cascade laser (QCL), installed in a MIRcat-QT laser system (Daylight Solutions Inc., USA). The continuous-wave (cw) QCL used (M2062-PCX) has a tuning range from 1730 to 1480 cm À1 .…”

“…As template we chose the peptide Trpzip2 introduced by Cochran and coworkers. 33 We and others have extensively studied the dynamics of Trpzip2 variants by laser-excited temperature-jump (T-jump) spectroscopy, [34][35][36][37][38][39][40][41][42] including the effect of hydrophobic interactions 43 and the b-turn region 44 on the folding dynamics and stability. Thus, Trpzip2 is very well suited as b-hairpin template.…”

Application of tunable quantum cascade lasers to monitor dynamics of bacteriorhodopsin in the mid-IR spectral range

“…For further investigations of the interaction-induced dynamics of membrane proteins and lipid membranes, it is highly desirable that also minor changes in absorption are resolved. Since their introduction [2], the development of QCL proceeds and has become a suitable tool in various scientific branches [4,18,21]. The application in mid-IR spectroscopy succeeded with the opportunity of gapless tuning over broad spectral regions.…”