The Journal of Physical Chemistry Letters

Chlorophylls (Chls) are known for fast, sub-picosecond internal conversion (IC) from ultraviolet/blue absorbing ("B" or "Soret" states) to the energetically lower, red light-absorbing Q states. Consequently, excitation energy transfer (EET) in photosynthetic pigment-protein complexes involving the B states has so far not been considered. We present, for the first time, a theoretical framework for the existence of B-B EET in tightly coupled Chl aggregates, such as photosynthetic pigment-protein complexes. We show that according to a simple Forster resonance energy transport (FRET) scheme, unmodulated B-B EET likely poses an existential threat, in particular the photochemical reaction centers (RCs). This insight leads to so-far undescribed roles for carotenoids (Crts, this article) and Chl b (next article in this series) of possibly primary importance. Here we show that B [->] Q IC is assisted by the symmetry-allowed Crt state (S2) by using the plant antenna complex CP29 as a model: The sequence is B [->] S2 (Crt, unrelaxed) [->]S2 (Crt, relaxed) [->] Q. This sequence has the advantage of preventing ~ 39% of Chl-Chl B-B EET, since the Crt S2 state is a highly efficient FRET acceptor. The likelihood of CP29 to forward potentially harmful B excitations towards the photosynthetic reaction center (RC) is thus reduced. In contrast to the B band of Chls, most Crt energy donation is energetically located near the Q band, which allows for 74/80% backdonation (from lutein/violaxanthin) to Chls. Neoxanthin, on the other hand, likely donates in the B band region of Chl b, with 76% efficiency. The latter is discussed in more detail in the next article in this series. Crts thus do not only act in their currently proposed photoprotective roles, but also as a crucial building block for any system that could otherwise deliver harmful "blue" excitations to the RCs.

Retinal is a chromophore covalently bound to various photoreceptors. Its photo-induced isomerization triggers a series of structural changes named photocycle, leading to diverse biological functions. Despite tremendous advances in structural biology and artificial intelligence-driven structure prediction, it remains challenging to analyze all photocyclic intermediates. Here, we present an optimized computational approach to calculate RSBH+ isomerization and its induced structural changes based on a classical molecular mechanics approach using quantum mechanically improved retinal force field parameters. Isomerization is induced by an excited state restraint which is subsequently relaxed to allow the return to the electronic ground state. We applied this approach to the key protein of optogenetics, Channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Besides the reformation of the alltrans/CN-anti ground state, we observed the production of a mixture of two isomeric states 13-cis/CN- anti and 13-cis/CN-syn. These findings agree with the previously found branched photocycle model based on experimental data. Our calculations show an asymmetric potential energy landscape of the excited state leading to a corresponding isomerization state distribution. Unlike earlier publications, our procedure describes the retinal photoisomerization on the natural timescale of 500 fs. As our newly derived retinal force field parameter set precisely relies on quantum biological knowledge, it assists to improve the refinement of experimental structure biological data. Our readily customizable strategy provides mechanistic insights at high spatio-temporal resolution, which permits accurate structural predictions of early photocycle intermediates. These insights will stimulate the rational design of optogenetic tools thus providing improved diagnostic and therapeutic approaches for neuronal and other diseases. HighlightsO_LIuniversal method to study molecular mechanism of optogenetic tools C_LIO_LIretinal photo-isomerization calculation in real time C_LIO_LIprediction of branched photo cycle agrees with experimental IR spectroscopic results C_LIO_LIdetected asymmetric excited state potential energy landscape C_LIO_LIassists to improve structural model refinement of retinal proteins C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=116 HEIGHT=200 SRC="FIGDIR/small/707937v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@71f7fdorg.highwire.dtl.DTLVardef@503482org.highwire.dtl.DTLVardef@1a77120org.highwire.dtl.DTLVardef@1f410a0_HPS_FORMAT_FIGEXP M_FIG C_FIG

The structural dynamics of a molecule are determined by the underlying potential energy landscape. Conical intersections are funnels connecting otherwise separate energy surfaces. Posited almost a century ago 1, conical intersections remain the subject of intense scientific investigation 2-4. In biology, they play a pivotal role in vision, photosynthesis, and DNA stability 5,6. In ultrafast radiationless de-excitation 1,7, they are vital to ameliorating photon-induced damage. In chemistry, they tightly couple the normally separable nuclear and electronic degrees of freedom, precluding the Born-Oppenheimer approximation 8. In physics, they manifest a Berry phase, giving rise to destructive interference between clockwise and anti-clockwise trajectories around the conical intersection 9. Accurate theoretical methods for examining conical intersections are at present limited to small molecules. Experimental investigations are challenged by the required time resolution and sensitivity. Current structure-dynamical understanding of conical intersections is thus limited to simple molecules with around 10 atoms, on timescales of about 100 fs or longer 10. Spectroscopy can achieve better time resolution, but provides only indirect structural information. Here, we present single-femtosecond, atomic-resolution movies of a 2,000-atom protein passing through a conical intersection. These movies, extracted from experimental data by geometric machine learning, reveal the dynamical trajectories of de-excitation via a conical intersection, yield the key parameters of the conical intersection controlling the de-excitation process, and elucidate the topography of the electronic potential energy surfaces involved.

Yersinia pestis, the causative agent of plague, is capable to evade human immune system response by recruiting the plasma circulating vitronectin proteins, which acts as a shield and avoids its lysis. Vitronectin recruitment is mediated by its interaction with the bacterial transmembrane protein Ail, protruding from Y. pestis outer membrane. By using all atom long-scale molecular dynamic simulations of Ail embedded in a realistic model of the bacterial membrane, we have shown that vitronectin forms a stable complex, mediated by interactions between the disordered moieties of the two proteins. The main amino acids driving the complexation have also been evidenced, thus favoring the possible rational design of specific peptides which, by inhibiting vitronectin recruitment, could act as original antibacterial agents. TOC ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/574511v1_ufig1.gif" ALT="Figure 1"> View larger version (140K): org.highwire.dtl.DTLVardef@1f43519org.highwire.dtl.DTLVardef@1ca1b0forg.highwire.dtl.DTLVardef@1b68510org.highwire.dtl.DTLVardef@1d9fe1d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Chlorophylls (Chls) are known for fast, sub-picosecond internal conversion (IC) from ultraviolet/blue absorbing ("B" or "Soret" states) to the energetically lower, red light-absorbing Q states. Consequently, excitation energy transfer (EET) in photosynthetic pigment-protein complexes involving the B states has so far not been considered. We present, for the first time, a theoretical framework for the existence of B-B EET in tightly coupled Chl aggregates, such as photosynthetic pigment-protein complexes. We show that according to a simple Forster resonance energy transport (FRET) scheme, unmodulated B-B EET likely poses an existential threat, in particular the photochemical reaction centers (RCs). This insight leads to so-far undescribed roles for carotenoids (Crts, cf. previous article in this series) and Chl b (this article) of possibly primary importance. It is demonstrated how pigments in a photosynthetic antenna pigment-protein complex (CP29) undergo FRET. Here, the focus is on the role of Chl b for EET in the Q and B bands. Further, the initial excited pigment distribution in the B band is computed for relevant solar irradiation and wavelength-centered laser pulses. It is found that both accessory pigment classes compete efficiently with Chl a absorption in the B band, leaving only 40% of B band excitations for Chl a. B state population is preferentially relocated to Chl b after excitation of any Chls, due to a near-perfect match of Chl b B band absorption with Chl a B state emission spectra. This results in an efficient depletion of the Chl a population (0.66 per IC/EET step, as compared to 0.21 in a Chl a-only system). Since Chl b only occurs in the peripheral antenna complexes, and RCs contain only Chl a, this would automatically trap potentially dangerous B state population distantly from the RCs.

Intermolecular spin relaxation by translational motion of spin pairs have been widely used to study properties of the biomolecules in liquids. Notably, solvent paramagnetic relaxation enhancement (sPRE) arising from paramagnetic cosolutes has gained attentions for various applications, including the structural refinement of intrinsically disordered proteins, cosolute-induced protein denaturation, and the characterization of residue-specific effective near-surface electrostatic potentials (ENS). Among these applications, the transverse sPRE rate known as {Gamma} 2 has been predominantly been interpreted empirically as being proportional to <r-6>norm. In this study, we present a rigorous theoretical interpretation of {Gamma} 2 that it is instead proportional to <r-4>norm and provide explicit formula for calculating <r-4>norm without any adjustable parameters. This interpretation is independent of the type or strength of interactions and can be broadly applied, including to the precise interpretation of ENS.

Despite the vital role of nicotinamide adenine dinucleotide (NAD) as a cofactor in all living organisms, the diversity of its functions is poorly understood. Particularly in interaction with ultraviolet (UV) light, a variety of photorelaxation channels can be accessed, which current models lack to explain. In this work, for the first time, we used picosecond UV pump, mid-infrared (mIR) probe spectroscopy and accurate quantum-chemical calculations to elucidate the ultrafast photodynamics of NAD+ and NADH to unify contradictory mechanisms from the past decades in the big picture. We found direct evidence for a long-lived ([~]900 ps) charge-separated state in NADH, which has been unobserved previously and results in the parallel population of a fluorescent state. The photochemical pathways demonstrated here open up functions of NAD in chemistry and molecular biology, such as an electron donor, as a FRET agent or as a redox pair switch, which have not been considered previously. TOC GRAPHICS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/563164v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@740620org.highwire.dtl.DTLVardef@13e2c71org.highwire.dtl.DTLVardef@d7cb0corg.highwire.dtl.DTLVardef@153ab51_HPS_FORMAT_FIGEXP M_FIG C_FIG

Micron-scale electron transfer through polymeric cytochrome nanowires powers prokaryotic life from hydrothermal vents to terrestrial soils in ways not fully understood. How much structural diversity optimizes electrical conductivity for survival in these different habitats is challenging to assess experimentally. Herein, physiologically relevant redox conduction is computationally assessed in cytochrome filaments from Geobacter sulfurreducens (OmcE, OmcS, and OmcZ), Pyrobaculum calidifontis (A3MW92), and Archaeoglobus veneficus (F2KMU8). A newly implemented Python program, BioDC, is used and validated against redox currents predicted from considerably more expensive molecular dynamics and quantum mechanical/molecular mechanical calculations. BioDC uses the heme solvent accessibility, stacking geometry, and redox-linked change in electrostatic energy to estimate electron transfer energetics. Leveraging this efficiency, structurally diverse cytochrome nanowires from different organisms are shown to have similar redox conductivities. A functionally robust heme chain packaged in habitat-customized proteins is proposed to be a general evolutionary design principle for cytochrome nanowires widely distributed among prokaryotes. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/544705v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@ac61d9org.highwire.dtl.DTLVardef@14c2e87org.highwire.dtl.DTLVardef@82d60corg.highwire.dtl.DTLVardef@1b2cbd6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Guanine-quadruplexes (G4s) are non-canonical DNA structures that play important protective and regulatory roles within cells, influencing, for instance, gene expression. Although the secondary structure of many human G4s is well characterized, in several gene-promoter regions multiple G4s are located in close proximity and may form three-dimensional structures which could ultimately influence their biological roles. In this contribution, we analyze the interplay between the three neighboring G4s present in the c-KIT proto-oncogene promoter, namely WK1, WSP and WK2. In particular, we highlight how these three G4s are structurally linked and how their crosstalk favors the formation of a parallel structure for WSP, differently from what observed for this isolated G4 in solution. Relying on all-atom molecular dynamic simulations exceeding the s time-scale and using enhanced sampling methods, we provide the first computationally-resolved structure of a well-organized G4 cluster in the promoter of a crucial gene involved in cancer development. Our results indicate that neighboring G4s influence their mutual three-dimensional arrangement and provide a powerful tool to predict and interpret complex DNA structures that ultimately can be used as starting point for drug discovery purposes.

Motional properties of individual amino acids in proteins are strongly modulated by their specific surrounding. The dynamics of tightly interacting residues can form intramolecular dynamic networks, which influence various features of protein function and serve as an access point for their modulation within signaling cascades. However, the possible formation of intermolecular networks shared between natural or constructed interaction partners has escaped thorough experimental assessment. Here, using fast-MAS solid-state NMR spectroscopy, we contrast the absence of a cross-talk between different residues in an apo protein with a recoupling of s timescale dynamics effective via a mediating crystal-crystal contact. The data show that dynamic allostery is not necessarily restricted to motionally coupled elements within a single protein but can traverse molecular boundaries. Interrogation of intermolecular dynamic networks by the strategies proposed here may shed light on the mechanisms underlying allosteric modulation of protein function in biological, pharmacological, and biotechnological studies.

A substantial number of Orange Carotenoid Protein (OCP) studies have aimed to describe the evolution of singlet excited states leading to the formation of photo-activated form, OCPR. The most recent one suggests that three picosecond-lived excited states are formed after the sub-100 fs decay of the initial S2 state. The S* state which has the longest reported lifetime of a few to tens of picoseconds is considered to be the precursor of the first red photoproduct P1. Here, we report the ultrafast photo-dynamics of the OCP from Synechocystis PCC 6803, carried out using Visible-NIR femtosecond time-resolved absorption spectroscopy as a function of the excitation pulse power and wavelength. We found that a carotenoid radical cation can form even at relatively low excitation power, obscuring the determination of photo-activation yields for P1. Moreover, the comparison of green (540 nm) and blue (470 nm) excitations revealed the existence of an hitherto uncharacterized excited state, denoted as S[~], living a few tens of picoseconds and formed only upon 470 nm excitation. Since neither the P1 quantum yield nor the photo-activation speed over hundreds of seconds vary under green and blue continuous irradiation, this S[~] species is unlikely to be involved in the photo-activation mechanism leading to OCPR. We also addressed the effect of His-tagging at the N- or C-termini on excited state photo-physical properties. Differences in spectral signatures and lifetimes of the different excited states were observed, at variance with the usual assumption that His-tagging hardly influences protein dynamics and function. Altogether our results advocate for careful consideration of the excitation power and His-tag position when comparing the photo-activation of different OCP variants, and beg to revisit the notion that S* is the precursor of photoactivated OCPR. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/474187v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1064473org.highwire.dtl.DTLVardef@77e3c7org.highwire.dtl.DTLVardef@10b1e0eorg.highwire.dtl.DTLVardef@e24977_HPS_FORMAT_FIGEXP M_FIG C_FIG

Photosystem II (PSII) is powered by the light-capturing properties of chlorophyll a pigments that define the spectral range of oxygenic photosynthesis. Some photosynthetic cyanobacteria can acclimate to growth in longer wavelength light by replacing five chlorophylls for long wavelength pigments in specific locations, including one in the reaction center (RC). However, the exact location and the nature of this long wavelength pigment still remain uncertain. Here we have addressed the color-tuning mechanism of the farred light PSII (FRL-PSII) by excited state calculations at both the ab initio correlated (ADC2) and linear-response time-dependent density functional theory (LR-TDDFT) levels in combination with large-scale hybrid quantum/classical (QM/MM) simulations and atomistic molecular dynamics. We show that substitution of a single chlorophyll pigment (ChlD1) at the RC by chlorophyll d leads to a spectral shift beyond the far-red light limit, as a result of the protein electrostatic, polarization and electronic coupling effects that reproduce key structural and spectroscopic observations. Pigment substitution at the ChlD1 site further results in a low site energy within the RC that could function as a sink for the excitation energy and initiate the primary charge separation reaction, driving the water oxidation. Our findings provide a basis for understanding color-tuning mechanisms and bioenergetic principles of oxygenic photosynthesis at the far-red light limit.

Photoactivated intermolecular electron transfer (ET) in cryptochromes proceeds along chains of aromatic residues and creates a spatially separated pair of radical electrons. Ultrafast time-dependent spectroscopy can provide experimental insight into this process and theoretical estimates of charge transfer rates are commonly obtained via Marcus theory. Here, we present a new perspective on the ET in European robin cryptochrome 4a (ErCry4a) that synthesizes insights from real-time ET calculations, ultrafast spectroscopic measurements and analytical derivations. The simulations exemplify that molecular vibrations play an essential role in enabling the ET dynamics, which was further rationalized through analytical derivations. Ultrafast pump-probe spectroscopy provided experimental access to the first 1.5 ns of the ET cascade, where multiple radical pair recombination rates arise due to the dynamic equilibrium along the ET chain. We show that the motions of the protein environment and the ET dynamics are inseparably coupled, violating the timescale separation required for Marcus theory. The presented results highlight that non-equilibrium coupling between electronic and nuclear motion dominates ET kinetics in ErCry4a during the first nanosecond after photo-excitation. The findings exemplify the limits of Marcus theory and refine the interpretation of ultrafast spectroscopic signatures in cryptochromes.

The appearance of the novel betacoronavirus SARS-CoV-2 represents a major threat to human health, and its diffusion around the world is causing dramatic consequences. The knowledge of the 3D structures of SARS-CoV-2 proteins can facilitate the development of therapeutic and diagnostic molecules. Specifically, comparative analyses of the structures of SARS-CoV-2 proteins and homologous proteins from previously characterized viruses, such as SARS-CoV, can reveal the common and/or distinctive traits that underlie the mechanisms of recognition of cell receptors and of molecules of the immune system. Herein, we apply our recently developed energy-based methods for the prediction of antibody-binding epitopes and protein-protein interaction regions to the Receptor Binding Domain (RBD) of the Spike proteins from SARS-CoV-2 and SARS-CoV. Our analysis focusses only on the study of the structure of RBDs in isolation, without making use of any previous knowledge of binding properties. Importantly, our results highlight structural and sequence differences among the regions that are predicted to be immunoreactive and bind/elicit antibodies. These results provide a rational basis to the observation that several SARS-CoV RDB-specific monoclonal antibodies fail to appreciably bind the SARS-CoV-2 counterpart. Furthermore, we correctly identify the region of SARS-CoV-2 RBD that is engaged by the cell receptor ACE2 during viral entry into host cells. The data, sequences and structures we present here can be useful for the development of novel therapeutic and diagnostic interventions.

We examine how cellular interactions in an all-atom model of the Homo sapiens cytoplasm influence the early folding events of Protein B (PB), a three-helix bundle protein. While PB is known to fold during in vitro simulations in three microseconds, all three initially unfolded PB copies in our cytoplasm model never completely reached their native topology across our 31 microsecond simulation. We were able to capture initial formation of all three helices and a compact topology similar to the native state. Sticking interactions between PB and surrounding macromolecules, as well as other unfolded PBs, became competitive with PB folding. Interaction between PB copies seeded beta-strand formation, modeling initial events of protein aggregation. Finally, the fold-switching potential of PB related GA domains has been explored in previous studies, and the sticking and crowding in our model thus initiates sampling of helix/sheet structural plasticity of PB.

The causative pathogen of Coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an enveloped virus assembled by a lipid envelope and multiple structural proteins. In this study, by integrating experimental data, structural modeling, and coarse-grained molecular dynamics simulations, we constructed multiscale models of SARS-CoV-2. Our 500-ns coarse-grained simulation of the intact virion allowed us to investigate the dynamic behavior of the membrane-embedded proteins and the surrounding lipid molecules in situ. Our results indicated that the membrane-embedded proteins are highly dynamic, and certain types of lipids exhibit various binding preferences to specific sites of the membrane-embedded proteins. The equilibrated virion model was transformed into atomic resolution, which provided a 3D structure for scientific demonstration and can serve as a framework for future exascale all-atom MD simulations.

Galectin-1 (Gal-1) is a galactose-binding protein involved in various cellular functions. Gal-1s activity has been suggested to be connected to two molecular concepts, which are however lacking experimental proof: a) enhanced binding affinity of Gal-1 towards membranes containing monosialotetrahexosylganglioside (GM1) over disialoganglioside GD1a and b) cross-linking of GM1s by homodimers of Gal-1. We provide evidence about the specificity and the nature of Gal-1 interaction with model membranes containing GM1 or GD1a, employing a broad panel of fluorescence-based and label-free experimental techniques, complemented by atomistic biomolecular simulations. Our study demonstrates that Gal-1 binds indeed specifically to GM1, and not to GD1a, when embedded in membranes over a wide range of concentrations (i.e., 30 nM to 10 M). The apparent binding constant is about tens of micromoles. On the other hand, no evidence of Gal-1/GM1 cross-linking was observed. Our findings suggest that cross-linking does not result from sole interactions between GM1 and Gal-1, indicating that in a physiological context, additional triggers are needed, which shift the GM1/Gal-1 equilibria towards the membrane-bound homodimeric Gal-1. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/614102v2_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@c4ff01org.highwire.dtl.DTLVardef@141c82eorg.highwire.dtl.DTLVardef@1bd7c0borg.highwire.dtl.DTLVardef@11af49e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Intrinsically disordered, low-complexity regions frequently cooperate with folded domains to mediate protein-protein interactions, yet accurately describing these mixed folded-disordered systems remains challenging. To visualize these mixed folded proteins, experimentally guided coarse-grained (CG) molecular dynamics simulations are often employed to extend the timescales required to capture the complex dynamics in play. However, the minimalistic nature of these approaches often compromises structural accuracy and can lead to inaccurate inter-domain interactions. Here we introduce backbone dihedral terms directly derived from NMR chemical shift data in CG-simulations to characterize the open state of a mixed-folded construct of the anti-aggregation chaperone DNAJB6 that contains a folded J-domain and a disordered GF linker. By tuning residue-specific backbone dihedral parameters to match NMR-derived secondary-structure propensities of the linker in CG-simulations, we generate conformational ensembles that yield accurate interdomain contact maps. In agreement with analysis of NMR relaxation data, the resulting ensembles show that even in the nominally open state the linker experiences motions that resemble those of the closed state driven by hydrophobic residues in GF. More generally we show that by expanding CG-simulations to allow them to capture both local and global structural properties, physically relevant interdomain contacts can be retrieved.

Multiparameter single-molecule fluorescence measurement is a promising technique for detecting and quantifying heterogeneity in freely diffusing molecules, albeit with challenges in data analysis due to limited photon numbers. Here, we present a universal analytical framework that leverages independent component analysis for unmixing multiparameter fluorescence signals. Applications to static and dynamic mixture systems demonstrate its potential allowing model-free separation of subpopulations with microsecond time resolution in nanomolar concentration regime.

While AlphaFold2 is rapidly being adopted as a new standard in protein structure predictions, it is limited to single structure prediction. This can be insufficient for the inherently dynamic world of biomolecules. Even with recent modifications towards conformational diversity, AlphaFold2 is devoid of providing thermodynamically ranked conformations. AlphaFold2-RAVE is an efficient protocol using the structural outputs from AlphaFold2 as initializations for AI augmented molecular dynamics. These simulations result in Boltzmann ranked ensembles, which we demonstrate on different proteins.