A billion years of evolution manifest in nanosecond protein dynamics.

Protein dynamics form a critical bridge between protein structure and function, yet the impact of evolution on ultrafast processes inside proteins remains enigmatic. This study delves deep into nanosecond-scale protein dynamics of a structurally and functionally conserved protein across species separated by almost a billion years, investigating ten homologs in complex with their ligand. By inducing a photo-triggered destabilization of the ligand inside the binding pocket, we resolved distinct kinetic footprints for each homolog via transient infrared spectroscopy. Strikingly, we found a cascade of rearrangements within the protein complex which manifest in time points of increased dynamic activity conserved over hundreds of millions of years within a narrow window. Among these processes, one displays a subtle temporal shift correlating with evolutionary divergence, suggesting reduced selective pressure in the past. Our study not only uncovers the impact of evolution on molecular processes in a specific case, but has also the potential to initiate a field of scientific inquiry within molecular paleontology, where species are compared and classified based on the rapid pace of protein dynamic processes; a field which connects the shortest conceivable time scale in living matter (10[Formula: see text] s) with the largest ones (10[Formula: see text] s).

Transient 2D IR Spectroscopy and Multiscale Simulations Reveal Vibrational Couplings in the Cyanobacteriochrome Slr1393-g3.

Cyanobacteriochromes are bistable photoreceptor proteins with desirable photochemical properties for biotechnological applications, such as optogenetics or fluorescence microscopy. Here, we investigate Slr1393-g3, a cyanobacteriochrome that reversibly photoswitches between a red-absorbing (Pr) and green-absorbing (Pg) form. We applied advanced IR spectroscopic methods to track the sequence of intermediates during the photocycle over many orders of magnitude in time. In the conversion from Pg to Pr, we have revealed a new intermediate with distinct spectroscopic features in the IR, which precedes Pr formation using transient IR spectroscopy. In addition, stationary and transient 2D IR experiments measured the vibrational couplings between different groups of the chromophore and the protein in these intermediate states, as well as their structural disorder. Anharmonic QM/MM calculations predict spectra in good agreement with experimental 2D IR spectra of the initial and final states of the photocycle. They facilitate the assignment of the IR spectra that serve as a basis for the interpretation of the spectroscopic results and suggest structural changes of the intermediates along the photocycle.

MCL-1 promiscuity and the structural resilience of its binding partners.

The allosteric protein MCL-1 and its natural inhibitors, the BH3-only proteins PUMA, BIM, and NOXA regulate apoptosis by interacting promiscuously within an entangled binding network. Little is known about the transient processes and dynamic conformational fluctuations that are the basis for the formation and stability of the MCL-1/BH3-only complex. In this study, we designed photoswitchable versions of MCL-1/PUMA and MCL-1/NOXA, and investigated the protein response after an ultrafast photo-perturbation with transient infrared spectroscopy. We observed partial α-helical unfolding in all cases, albeit on strongly varying timescales (1.6 ns for PUMA, 9.7 ns for the previously studied BIM, and 85 ns for NOXA). These differences are interpreted as a BH3-only-specific "structural resilience" to defy the perturbation while remaining in MCL-1's binding pocket. Thus, the presented insights could help to better understand the differences between PUMA, BIM, and NOXA, the promiscuity of MCL-1, in general, and the role of the proteins in the apoptotic network.

The molecular mechanism of light-induced bond formation and breakage in the cyanobacteriochrome TePixJ.

Cyanobacteriochromes (CBCRs) are small and versatile photoreceptor proteins with high potential for biotechnological applications. Among them, the so-called DXCF-CBCRs exhibit an intricate secondary photochemistry: miliseconds after activation with light, a covalent linkage between a conserved cysteine residue and the light-absorbing tetrapyrrole chromophore is reversibly formed or broken. We employed time-resolved IR spectroscopy over ten orders of magnitude in time in conjunction with 2D-IR spectroscopy to investigate the molecular mechanism of this intriguing reaction in the DXCF-CBCR model system TePixJ from T. elongatus. The crosspeak pattern in the 2D-IR spectrum facilitated the assignment of the dominant signals to vibrational modes of the chromophore, which in turn enabled us to construct a mechanistic model for the photocycle reactions from the time-resolved IR spectra. Here, we assigned the time-resolved signals to several proton transfer steps and distinct geometric changes of the chromophore. We propose a model that describes how these events lead to the rearrangement of charges in the chromophore binding pocket, which serves as the trigger for the light-induced bond formation and breakage with the nearby cysteine.

Signal Propagation Within the MCL-1/BIM Protein Complex.

The protein MCL-1 is a crucial factor in regulating apoptosis, the programmed cell death, and thus plays a major role in numerous cancer types. The allosteric protein MCL-1 is naturally moderated by the BH3-only peptide BIM, which binds at its canonical binding groove. In its isolated form, BIM is disordered but assumes an α-helical shape when bound by MCL-1. The underlying binding mechanism (i.e., induced fit vs conformational selection), as well as the time scales of the signal cascade subsequent to binding, are not understood. Here, an artificially photoswitchable variant of the MCL-1/BIM complex was designed and investigated by transient infrared spectroscopy. By destabilizing the α-helix of BIM with a covalently linked azobenzene photoswitch, the dynamical response of the whole complex upon an ultrafast photo-perturbation was characterized. While the destabilized and partially unfolded BIM still binds to MCL-1, a step-like cascade of structural rearrangements of both, MCL-1 and BIM was detected, spanning a wide range of time scales from pico- to microseconds. The results indicate that BIM binds according to an induced fit mechanism, while the structural adaptations of MCL-1 may constitute an allosteric signal.

Sequence of Events during Peptide Unbinding from RNase S: A Complete Experimental Description.

The phototriggered unbinding of the intrinsically disordered S-peptide from the RNase S complex is studied with the help of transient IR spectroscopy, covering a wide range of time scales from 100 ps to 10 ms. To that end, an azobenzene moiety has been linked to the S-peptide in a way that its helicity is disrupted by light, thereby initiating its complete unbinding. The full sequence of events is observed, starting from unfolding of the helical structure of the S-peptide on a 20 ns time scale while still being in the binding pocket of the S-protein, S-peptide unbinding after 300 µs, and the structural response of the S-protein after 3 ms. With regard to the S-peptide dynamics, the binding mechanism can be classified as an induced fit, while the structural response of the S-protein is better described as conformational selection.

Needles in a haystack: H-bonding in an optogenetic protein observed with isotope labeling and 2D-IR spectroscopy.

Recently, re-purposing of cyanobacterial photoreceptors as optogentic actuators enabled light-regulated protein expression in different host systems. These new bi-stable optogenetic tools enable interesting new applications, but their light-driven working mechanism remains largely elusive on a molecular level. Here, we study the optogenetic cyanobacteriochrome Am1-c0023g2 with isotope labeling and two dimensional infrared (2D-IR) spectroscopy. Isotope labeling allows us to isolate two site-specific carbonyl marker modes from the overwhelming mid-IR signal of the peptide backbone vibrations. Unlike conventional difference-FTIR spectroscopy, 2D-IR is sensitive to homogeneous and inhomogeneous broadening mechanisms of these two vibrational probes in the different photostates of the protein. We analyse the 2D-IR line shapes in the context of available structural models and find that they reflect the hydrogen-bonding environment of these two marker groups.

The Speed of Allosteric Signaling Within a Single-Domain Protein.

While much is known about different allosteric regulation mechanisms, the nature of the allosteric signal and the time scale on which it propagates remains elusive. The PDZ3 domain from postsynaptic density-95 protein is a small protein domain with a terminal third α-helix, i.e., the α3-helix, which is known to be allosterically active. By cross-linking the allosteric helix with an azobenzene moiety, we obtained a photocontrollable PDZ3 variant. Photoswitching triggers its allosteric transition, resulting in a change in binding affinity of a peptide to the remote binding pocket. Using time-resolved infrared and UV/vis spectroscopy, we follow the allosteric signal transduction and reconstruct the timeline in which the allosteric signal propagates through the protein within 200 ns.

A stop-flow sample delivery system for transient spectroscopy.

A stop-flow sample delivery system for transient spectroscopy is presented, which is, in particular, suited for laser-based instruments (quantum-cascade lasers or amplified femtosecond lasers) with excitation pulse repetition rates in the range 10-100 Hz. Two pulsing micro-valves are mounted onto a flow cuvette designed for transient IR spectroscopy, which is integrated into a flow cycle driven by a peristaltic pump. The performance of the system is demonstrated with transient IR experiments of the trans-to-cis photoisomerization of a water-soluble azobenzene derivative. The sample stands still when the micro-valves are closed and is pushed out from the probe beam focus on a 1 ms timescale when opening the micro-valves. The setup is extremely sample efficient. It needs only small sample volumes, and at the same time, it enables excitation of a large fraction of molecules in solution.

Vibrational Couplings in Hydridocarbonyl Complexes: A 2D-IR Perspective.

Hydridocarbonyl complexes, a class of industrially relevant catalysts, contain both the M-H and M-CO moieties. Here, using two-dimensional infrared spectroscopy, we examine the coupling of the typically weak M-H stretching mode and the intense M(C≡O) mode. By studying a series of Ir(I)- and Ir(III)-based hydridocarbonyl complexes, we show that the arrangement of the H and CO ligands in a trans configuration leads to strong vibrational coupling and mode delocalization. In contrast, a cis arrangement leads to no coupling, with the localized M-H mode having a much larger anharmonicity. These results highlight a promising strategy for enhancing the M-H vibration by intensity borrowing from the strong C≡O modes in a trans configuration, allowing for direct determination by infrared spectroscopy of both the oxidation (by frequency shifts) and the protonation state (via vibrational coupling) of the complex, in mechanistic studies of proton-coupled electron transfer reactions.