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

Abstract

We provide a time-and structure-resolved characterization of the folding of the heterogeneous β-hairpin peptide Tryptophan Zipper 2 (Trpzip2) using 2D IR spectroscopy. The amide I′ vibrations of three Trpzip2 isotopologues are used as a local probe of the midstrand contacts, β-turn, and overall β-sheet content. Our experiments distinguish between a folded state with a type I′ β-turn and a misfolded state with a bulged turn, providing evidence for distinct conformations of the peptide backbone. Transient 2D IR spectroscopy at 45°C following a laser temperature jump tracks the nanosecond and microsecond kinetics of unfolding and the exchange between conformers. Hydrogen bonds to the peptide backbone are loosened rapidly compared with the 5-ns temperature jump. Subsequently, all relaxation kinetics are characterized by an observed 1.2 ± 0.2-μs exponential. Our time-dependent 2D IR spectra are explained in terms of folding of either native or nonnative contacts from a common compact disordered state. Conversion from the disordered state to the folded state is consistent with a zip-out folding mechanism.protein folding | time-resolved spectroscopy | multidimensional | ultrafast E ven with technical advances in protein folding simulations and experiments, it remains difficult to compare them at a molecular level. As a result, the pictures that emerge from these studies differ. Experiments are commonly interpreted using two or three states separated along a reaction coordinate by transition states that are difficult to interpret. Molecular dynamics (MD) simulations provide richly detailed information on the conformational dynamics, often involving more configurational states than can be resolved in experiments (1-3). These heterogeneous folding scenarios reinforce funnel pictures of the folding to a native state (4). Although the connection between these pictures has been articulated (2), experimental validation of these concepts is scarce (5-7).Bridging the gap between theory and experiment requires experiments that are both sensitive to different conformational states of a protein and have time resolution to characterize their interconversion. Toward this goal, we have studied the folding of the β-hairpin peptide Tryptophan Zipper 2 (Trpzip2, TZ2) using 2D IR spectroscopy. Our experiments probe the amide I′ vibration, which is primarily C = O stretching of the peptide backbone. By spectroscopically isolating peptide units through isotope labeling, they become reporters of hydrogen bonding (H-bonding) to the amide oxygen. To connect amide I′ spectral observations with structures from MD simulations, models of amide I′ IR spectroscopy have recently become available (8). With these tools, we have identified multiple conformations for TZ2 (9, 10), including at least two turn structures. Transient 2D IR (t-2D IR) spectroscopy of peptides has proven powerful for following peptide conformational dynamics (5, 11-13). Here, t-2D IR spectroscopy of three TZ2 isotopologues is used to follow the conformational changes induced by...

Sequence, structure, and cooperativity in folding of elementary protein structural motifs

“…Muñoz and coworkers pioneered the multiprobe equilibrium approach for studies of "downhill" folding (5,7,8), where cooperativity is minimal, but subsequently demonstrated its applicability to other fast-folding proteins (9)(10)(11)(12)(13) and extended the analysis to kinetics (14). Following their work, experiments from other laboratories have reported probe-dependent folding equilibria and kinetics in a number of small proteins (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). However, because the spectroscopic signals do not directly report on structure and are often subject to interferences from nonstructural effects that lead to ambiguities in their interpretations, the challenge lies in relating the observed experimental data to the underlying structural and energetic states of the protein.…”

Applications of two-dimensional infrared spectroscopy

“…10, 74–76, 81–83 For example, 2D IR spectroscopy has been extensively applied toward the study of amyloid fibril formation. 77, 78 Figure 7 shows 2D IR spectra from one recent study of human γD-crystallin protein that employed a combination of 2D IR spectroscopy and segmental 13 C labeling to elucidate the involvement of the N- and C-terminal domains in the formation of amyloid fibrils.…”

Direct observation of ground-state lactam–lactim tautomerization using temperature-jump transient 2D IR spectroscopy

“…Nevertheless, the time window for these experiments is limited to the vibrational lifetime of the systems, typically a few picoseconds. To circumvent this limitation, one can perform transient 2D IR (t-2D IR) experiments that follow reequilibration after an abrupt perturbation (25)(26)(27)(28)(29)(30). Our strategy to monitor ground-state reactivity is to perform t-2D IR triggered by a nanosecond T-jump and observe the tautomeric interconversion in real time during the reequilibration.…”