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.

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.

Using azobenzene photocontrol to set proteins in motion.

Controlling the activity of proteins with azobenzene photoswitches is a potent tool for manipulating their biological function. With the help of light, it is possible to change binding affinities, control allostery or manipulate complex biological processes, for example. Additionally, owing to their intrinsically fast photoisomerization, azobenzene photoswitches can serve as triggers that initiate out-of-equilibrium processes. Such switching of the activity initiates a cascade of conformational events that can be accessed with time-resolved methods. In this Review, we show how the potency of azobenzene photoswitching can be combined with transient spectroscopic techniques to disclose the order of events and experimentally observe biomolecular interactions in real time. This strategy will further our understanding of how a protein can accommodate, adapt and readjust its structure to answer an incoming signal, revealing more of the dynamical character of proteins.

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.

Intrinsic Dynamics of Protein-Peptide Unbinding.

The dynamics of peptide-protein binding and unbinding of a variant of the RNase S system has been investigated. To initiate the process, a photoswitchable azobenzene moiety has been covalently linked to the S-peptide, thereby switching its binding affinity to the S-protein. Transient fluorescence quenching was measured with the help of a time-resolved fluorometer, which has been specifically designed for these experiments and is based on inexpensive light-emitting diodes and laser diodes only. One mutant shows on-off behavior with no specific binding detectable in one of the states of the photoswitch. Unbinding is faster by at least 2 orders of magnitude, compared to that of other variants of the RNase S system. We conclude that unbinding is essentially barrier-less in that case, revealing the intrinsic dynamics of the unbinding event, which occurs on a time scale of a few hundred microseconds in a strongly stretched-exponential manner.

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.

Sensing the allosteric force.

Allosteric regulation is an innate control in most metabolic and signalling cascades that enables living organisms to adapt to the changing environment by tuning the affinity and regulating the activity of target proteins. For a microscopic understanding of this process, a protein system has been designed in such a way that allosteric communication between the binding and allosteric site can be observed in both directions. To that end, an azobenzene-derived photoswitch has been linked to the α3-helix of the PDZ3 domain, arguably the smallest allosteric protein with a clearly identifiable binding and allosteric site. Photo-induced trans-to-cis isomerisation of the photoswitch increases the binding affinity of a small peptide ligand to the protein up to 120-fold, depending on temperature. At the same time, ligand binding speeds up the thermal cis-to-trans back-isomerisation rate of the photoswitch. Based on the energetics of the four states of the system (cis vs trans and ligand-bound vs free), the concept of an allosteric force is introduced, which can be used to drive chemical reactions.

Photocontrolling Protein-Peptide Interactions: From Minimal Perturbation to Complete Unbinding.

An azobenzene-derived photoswitch has been covalently cross-linked to two sites of the S-peptide in the RNase S complex in a manner that the α-helical content of the S-peptide reduces upon cis-to-trans isomerization of the photoswitch. Three complementary experimental techniques have been employed, isothermal titration calorimetry, circular dichroism spectroscopy and intrinsic tyrosine fluorescence quenching, to determine the binding affinity of the S-peptide to the S-protein in the two states of the photoswitch. Five mutants with the photoswitch attached to different sites of the S-peptide have been explored, with the goal to maximize the change in binding affinity upon photoswitching, and to identify the mechanisms that determine the binding affinity. With regard to the first goal, one mutant has been identified, which binds with reasonable affinity in the one state of the photoswitch, while specific binding is completely switched off in the other state. With regard to the second goal, accompanying molecular dynamics simulations combined with a quantitative structure activity relationship revealed that the α-helicity of the S-peptide in the binding pocket correlates surprisingly well with measured dissociation constants. Moreover, the simulations show that both configurations of all S-peptides exhibit quite well-defined structures, even in apparently disordered states.

Comparative stability of ficin and papain in acidic conditions and the presence of ethanol.

Proteolytic enzymes are used for proteolysis and peptide synthesis which can be run in various conditions including low pH value and the presence of ethanol. The most common cysteine protease applied in acidic-alcoholic conditions is well-characterized papain. Ficin, which is closely related to papain in terms of proteolytic activity and substrate specificity, could potentially be applied in the alcoholic beverage industry and peptide synthesis. The aim of this study was to compare papain and ficin stability in process conditions. Comparative stability study showed that ficin as a mixture of different isoforms has a broader range of stability in respect of pH and cold storage stability, in comparison to papain. It retains about 70% of initial activity after 3-week cold storage at low pH and in the presence of ethanol. Unlike ficin, papain loses about 70% of initial activity in the same incubation period as it is more prone to non-native aggregation that was confirmed by FTIR analysis. The presence of multiple isoforms of ficin stabilizes the protease against cold denaturation and aggregation, making it more suitable for biotechnological and laboratory usage than single papain isoform. It is more cold-stable in alcoholic-acidic and acidic conditions suggesting possible replacement of papain with even lower enzyme concentration.