Matthew J. Tucker

Tetrazine Phototriggers: Probes for Peptide Dynamics†

Ultrafast photochemical triggers hold the promise of providing information on the dynamics of peptide and protein folding.[1] Prior to photolysis, bonding to the trigger constrains the peptide to have a narrow structure distribution. Photochemical triggering releases the constraints, permitting the molecule to evolve to a different equilibrium distribution. The structure evolution, even when ultrafast, can be followed by infrared probe or two-dimensional infrared spectroscopy. Fast phototriggering can thus reveal early kinetic events in protein dynamics by providing a means to explore the free energy landscape of folding and misfolding. Several phototriggers have been developed for this purpose,[1,2] but there remain significant challenges. For example, disulfide bonds in peptides can be used as a phototriggers. Deep UV light severs the disulfide bond and releases the structural constraints; such experiments have been carried out in short helical peptides,[1a,b] cyclic peptides,[1d] and β hairpins.[1c] Although disulfide photolysis offers the capability of initiating ultrafast structure equilibration, limitations preclude broad generality of this technique. Homolytic S–S bond scission reveals two reactive radicals that can undergo geminate recombination, as well as reactions with protein sidechains. Moreover, the UV excitation required to dissociate the disulfide bond also excites the peptide backbone. Another example is azobenzene which undergoes fast, reversible photoisomerization. When designed into a peptide a light pulse can be used to cause the system to reversibly shift between significantly different equilibrium configurations.[3]

Using Two Fluorescent Probes to Dissect the Binding, Insertion, and Dimerization Kinetics of a Model Membrane Peptide

Helix-helix association within a membrane environment represents one of the fundamental processes in membrane protein folding. However, studying the kinetics of such processes has been difficult because most membrane proteins are insoluble in aqueous solution. Here we present a stopped-flow fluorescence study of the membrane-interaction kinetics of a designed, water-soluble transmembrane (TM) peptide, anti-alpha(IIb), which is known to dimerize in phospholipid bilayers. We show that by using two fluorescent amino acids, tryptophan and p-cyanophenylalanine, we are able to kinetically dissect distinct phases in the peptide-membrane interaction, representing membrane binding, membrane insertion, and TM helix-helix association. Our results further show that the last process occurs on a time scale of seconds, indicating that the association of two TM helices is an intrinsically slow event.

Nonequilibrium dynamics of helix reorganization observed by transient 2D IR spectroscopy

Significance Exploration of the helix–coil transition, a fundamental aspect in protein conformational dynamics, remains controversial, with published timescales differing by more than two orders of magnitude. We report measurements of the temporal evolution of local structural parameters within a helix, exploring the free energy landscape near but not initially at the equilibrium conformational distribution. The model helical peptide was constrained with a photosensitive cross-link that can be released in tens of picoseconds upon laser irradiation. Transient 2D IR spectroscopy was used to record structural snapshots of bond distance and bond angle during formation of a single helical turn in an already-nucleated helix upon photorelease. This nonequilibrium approach permits access to transient structures that are averaged out in standard methods. The relaxation of helical structures very close to equilibrium is observed via transient 2D IR spectroscopy. An initial distribution of synthetically distorted helices having an unnatural bridge linking the 10th and 12th residues of an alanine-rich α-helix is released to evolve into the equilibrium distribution of α-helix conformations. The bridge constrains the structure to be slightly displaced from the full α-helix equilibrium near these residues, yet the peptide is not unfolded completely. The release is accomplished by a subpicosecond pulse of UV irradiation. The resulting 2D IR signals are used to obtain snapshots of the ∼100-ps helical conformational reorganization of the distorted dihedral angle and distance between amide units at chemical bond length-scale resolution. The decay rates of the angle between the dipoles, dihedral angles, and distance autocorrelations obtained from molecular dynamics simulations support the experiments, providing evidence that the final helix collapse conforms to linear response theory.

Direct Assessment of the α-Helix Nucleation Time

The nucleation event in α-helix formation is a fundamental process in protein folding. However, determining how quickly it takes place based on measurements of the relaxation dynamics of helical peptides is difficult because such relaxations invariably contain contributions from various structural transitions such as from helical to nonhelical states and helical to partial-helical conformations. Herein, we measure the temperature-jump (T-jump) relaxation kinetics of three model peptides that fold into a single-turn α-helix, using time-resolved infrared spectroscopy, aiming to provide a direct assessment of the helix nucleation rate. The α-helical structure of these peptides is stabilized by a covalent cross-linker formed between the side chains of two residues at the i and i + 4 positions. If we assume that this cross-linker mimics the structural constraint arising from a strong side chain-side chain interaction (e.g., a salt bridge) in proteins, these peptides would represent good models for studying the nucleation process of an α-helix in a protein environment. Indeed, we find that the T-jump induced relaxation rate of these peptides is approximately (0.6 μs)(-1) at room temperature, which is slower than that of commonly studied alanine-based helical peptides but faster than that of a naturally occurring α-helix whose folded state is stabilized by a series of side chain-side chain interactions. Taken together, our results put an upper limit of about 1 μs for the helix nucleation time at 20 °C and suggest that the subsequent propagation steps occur with a time constant of about 240 ns.

Ester Carbonyl Vibration as a Sensitive Probe of Protein Local Electric Field

The ability to quantify the local electrostatic environment of proteins and protein/peptide assemblies is key to gaining a microscopic understanding of many biological interactions and processes. Herein, we show that the ester carbonyl stretching vibration of two non-natural amino acids, L-aspartic acid 4-methyl ester and L-glutamic acid 5-methyl ester, is a convenient and sensitive probe in this regard, since its frequency correlates linearly with the local electrostatic field for both hydrogen-bonding and non-hydrogen-bonding environments. We expect that the resultant frequency-electric-field map will find use in various applications. Furthermore, we show that, when situated in a non-hydrogen-bonding environment, this probe can also be used to measure the local dielectric constant (ε). For example, its application to amyloid fibrils formed by Aβ(16-22) revealed that the interior of such β-sheet assemblies has an ε value of approximately 5.6.

Identification of Arginine Residues in Peptides by 2D-IR Echo Spectroscopy

The CN stretching vibrations of the guanidyl group in the arginine dipeptide side chain are examined by two-dimensional infrared spectroscopy. In D(2)O, the spectra display two distinct diagonal peaks. These nearly degenerate modes undergo ultrafast energy transfer. The energy-transfer rate was determined directly from the 2D-IR spectra to be 1/2.1 ps(-1). The cross peaks in 2D-IR arising from the energy transfer provide a definitive identification of arginine in larger proteins. An example of arginine in the transmembrane protein M2, found in influenza viruses, is given.

2D IR spectroscopy of histidine: probing side-chain structure and dynamics via backbone amide vibrations.

It is well known that histidine is involved in many biological functions due to the structural versatility of its side chain. However, probing the conformational transitions of histidine in proteins, especially those occurring on an ultrafast time scale, is difficult. Herein we show, using a histidine dipeptide as a model, that it is possible to probe the tautomer and protonation status of a histidine residue by measuring the two-dimensional infrared (2D IR) spectrum of its amide I vibrational transition. Specifically, for the histidine dipeptide studied, the amide unit of the histidine gives rise to three spectrally resolvable amide I features at approximately 1630, 1644, and 1656 cm–1, respectively, which, based on measurements at different pH values and frequency calculations, are assigned to a τ tautomer (1630 cm–1 component) and a π tautomer with a hydrated (1644 cm–1 component) or dehydrated (1656 cm–1 component) amide. Because of the intrinsic ultrafast time resolution of 2D IR spectroscopy, we believe that the current approach, when combined with the isotope editing techniques, will be useful in revealing the structural dynamics of key histidine residues in proteins that are important for function.

Design, synthesis, and photochemical validation of peptide linchpins containing the S,S-tetrazine phototrigger.

The design, solid-phase synthesis, and photochemical validation of diverse peptide linchpins, containing the S,S-tetrazine phototrigger, have been achieved. Steady state irradiation or femtosecond laser pulses confirm their rapid photofragmentation. Attachment of peptides to the C- and N-termini will provide access to diverse constrained peptide constructs that hold the promise of providing information about early peptide/protein conformational dynamics upon photochemical release.

The Design and Synthesis of Alanine-Rich α-Helical Peptides Constrained by an S,S-Tetrazine Photochemical Trigger: A Fragment Union Approach

The design and synthesis of alanine-rich α-helical peptides constrained in a partially unfolded state by incorporation of the S,S-tetrazine phototrigger has been achieved, permitting, upon photochemical release, observation by 2D-IR spectroscopy of the subnanosecond conformational dynamics that govern the early steps associated with α-helix formation. Solid-phase peptide synthesis was employed to elaborate the requisite fragments, with full peptide construction via solution-phase fragment condensation. The fragment union tactic was also employed to construct (13)C═(18)O isotopically edited amides to permit direct observation of conformational motion at or near specific peptide bonds.

Tyrosine as a Non-perturbing Site-Specific Vibrational Reporter for Protein Dynamics

The ability to detect changes in the local environment of proteins is pivotal to determining their dynamic nature during many biological processes. For this purpose, the utility of the tyrosine ring breathing vibration as a sensitive infrared reporter for measuring the local electric field in protein is investigated. Variations in the bandwidth of this vibrational transition in a variety of solvents indicate differences in microenvironment affect the inhomogeneous broadening and thus the frequency distribution. The ring mode is influenced by direct and indirect interactions associated with the charge distribution of the surrounding solvent molecules. Molecular dynamics simulations were implemented to obtain a correlation between the electric field induced by the solvent on the mode and the observed vibrational bandwidth. Moreover, the Trp-cage was synthesized as a model peptide system to access the efficacy of the correlation to predict the electric field strength within the hydrophobic core of the native and denatured states of the protein. The 2D IR spectra of tyrosine in dimethyl sulfoxide (DMSO) and water (D2O) show a two-fold difference in the time constant of the vibrational dynamics alluding to the dephasing mechanisms of the vibration and supporting the model put forth about the solvachromatic nature of the transition.