Direct Monitoring of β‑Sheet Formation in the Outer Membrane Protein TtoA Assisted by TtOmp85

Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was applied to investigate the folding of an outer membrane protein, TtoA, assisted by TtOmp85, both from the thermophilic eubacterium <i>Thermus thermophilus</i>. To directly monitor the formation of β-sheet structure in TtoA and to analyze the function of TtOmp85, we immobilized unfolded TtoA on an ATR crystal. Interaction with TtOmp85 initiated TtoA folding as shown by time-dependent spectra recorded during the folding process. Our ATR-FTIR experiments prove that TtOmp85 possesses specific functionality to assist β-sheet formation of TtoA. We demonstrate the potential of this spectroscopic approach to study the interaction of outer membrane proteins in vitro and in a time-resolved manner

Devising Self-Assembled-Monolayers for Surface-Enhanced Infrared Spectroscopy of pH-Driven Poly‑l‑lysine Conformational Changes

Surface-enhanced infrared absorption spectroscopy (SEIRA) is applied to study protein conformational changes. In general, the appropriate functionalization of metal surfaces with biomolecules remains a challenge if the conformation and activity of the biomolecule shall be preserved. Here we present a SEIRA study to monitor pH-induced conformational changes of poly-l-lysine (PLL) covalently bound to a thin gold layer via self-assembled monolayers (SAMs). We demonstrate that the composition of the SAM is crucial. A SAM of 11-mercaptoundecanonic acid (MUA) can link PLL to the gold layer, but pH-driven conformational transitions were hindered compared to poly-l-lysine in solution. To address this problem, we devised a variety of SAMs, i.e., mixed SAMs of MUA with either octanethiol (OT) or 11-mercapto-1-undecanol (MUoL) and furthermore SAMs of MT­(PEG)₄ and NHS-PEG_10k-SH. These mixed SAMs modify the surface properties by changing the polarity and the morphology of the surface present to nearby PLL molecules. Our experiments reveal that mixed SAMs of MUA-MUoL and SAMs of NHS-PEG_10k-SH-MT­(PEG)₄ are suitable to monitor pH-driven conformational changes of immobilized PLL. These SAMs might be applicable for chemoselective protein immobilization in general

Effect of Hydrophobic Interactions on the Folding Mechanism of β‑Hairpins

Hydrophobic interactions are essential in stabilizing protein structures. How they affect the folding pathway and kinetics, however, is less clear. We used time-resolved infrared spectroscopy to study the dynamics of hydrophobic interactions of β-hairpin variants of the sequence Trpzip2 (SWTW­ENGKW­TWK-NH2) that is stabilized by two cross-strand Trp–Trp pairs. The hydrophobicity strength was varied by substituting the tryptophans pairwise by either tyrosines or valines. Relaxation dynamics were induced by a laser-excited temperature jump, which separately probed for the loss of the cross-strand β-hairpin interaction and the rise of the disordered structure. All substitutions tested result in reduced thermal stability, lower transition temperatures, and faster dynamics compared to Trpzip2. However, the changes in folding dynamics depend on the amino acid substituted for Trp. The aromatic substitution of Tyr for Trp results in the same kinetics for the unfolding of sheet and growth of disorder, with similar activation energies, independent of the substitution position. Substitution of Trp with a solely hydrophobic Val results in even faster kinetics than substitution with Tyr but is additionally site-dependent. If the hairpin has a Val pair close to its termini, the rate constants for loss of sheet and gain of disorder are the same, but if the pair is close to the turn, the sheet and disorder components show different relaxation kinetics. The Trp → Val substitutions reveal that hydrophobic interactions alone weakly stabilize the hairpin structure, but adding edge-to-face aromatic interaction strengthens it, and both modify the complex folding process

Isotopically Site-Selected Dynamics of a Three-Stranded -Sheet Peptide Detected with Temperature-Jump IR-Spectroscopy

Infrared detected temperature jump (T-jump) spectroscopy and site-specific isotopic labeling were applied to study a model three-stranded beta-sheet peptide with the goal of individually probing the dynamics of strand and turn structural elements. This peptide had two DPro-Gly (pG) turn sequences to stabilize the two component hairpins, which were labeled with 13C=O on each of the Gly residues to resolve them spectroscopically. Labeling the second turn on the amide preceding the DPro (XxxDPro amide) provided an alternate turn label as a control. Placing 13C=O labels on specific in-strand residues gave shifted modes that overlap the XxxDPro amide I’ modes. Their impact could be separated from the turn dynamics by a novel difference-transient analysis approach. FTIR spectra were modeled with DFTcomputations which showed the local, isotope-selected vibrations were effectively uncoupled from the other amide I modes. Our T-jump dynamics results, combined with NMR structures and equilibrium spectral measurements, showed the first turn to be most stable and best formed with the slowest dynamics, while the second turn and first strand (N-terminus) had similar dynamics, and the third strand (C-terminus) had the fastest dynamics and was the least structured. The relative dynamics of the strands, XxxDPro amides and 13C-labeled Gly residues on the turns also qualitatively corresponded to molecular dynamics (MD) simulations of turn and strand fluctuations. MD trajectories indicated the turns to be bistable, with the first turn being Type I’ and the second turn flipping from I’ to II’. The differences in relaxation times for each turn and the separate strands revealed that the folding process of this turnstabilized beta-sheet structure proceeds in a multi-step process

Role of Aromatic Cross-Links in Structure and Dynamics of Model Three-Stranded β‑Sheet Peptides

A series of closely related peptide sequences that form triple-strand structures was designed with a variation of cross-strand aromatic interactions and spectroscopically studied as models for β-sheet formation and stabilities. Structures of the three-strand models were determined with NMR methods and temperature-dependent equilibrium studies performed using circular dichroism and Fourier transform infrared spectroscopies. Our equilibrium data show that the presence of a direct cross-strand aromatic contact in an otherwise folded peptide does not automatically result in an increased thermal stability and can even distort the structure. The effect on the conformational dynamics was studied with infrared-detected temperature-jump relaxation methods and revealed a high sensitivity to the presence and the location of the aromatic cross-links. Aromatic contacts in the three-stranded peptides slow down the dynamics in a site-specific manner, and the impact seems to be related to the distance from the turn. With a Xxx-^DPro linkage as a probe with some sensitivity for the turn, small differences were revealed in the relative relaxation of the sheet strands and turn regions. In addition, we analyzed the component hairpins, which showed less uniform dynamics as compared to the parent three-stranded β-sheet peptides

pH-Jump Induced Leucine Zipper Folding beyond the Diffusion Limit

The folding of a pH-sensitive leucine zipper, that is, a GCN4 mutant containing eight glutamic acid residues, has been investigated. A pH-jump induced by a caged proton (<i>o</i>-nitrobenzaldehyde, oNBA) is employed to initiate the process, and time-resolved IR spectroscopy of the amide I band is used to probe it. The experiment has been carefully designed to minimize the buffer capacity of the sample solution so that a large pH jump can be achieved, leading to a transition from a completely unfolded to a completely folded state with a single laser shot. In order to eliminate the otherwise rate-limiting diffusion-controlled step of the association of two peptides, they have been covalently linked. The results for the folding kinetics of the cross-linked peptide are compared with those of an unlinked peptide, which reveals a detailed picture of the folding mechanism. That is, folding occurs in two steps, one on an ∼1–2 μs time scale leading to a partially folded α-helix even in the monomeric case and a second one leading to the final coiled-coil structure on distinctively different time scales of ∼30 μs for the cross-linked peptide and ∼200 μs for the unlinked peptide. By varying the initial pH, it is found that the folding mechanism is consistent with a thermodynamic two-state model, despite the fact that a transient intermediate is observed in the kinetic experiment