Biophysics and Physicobiology

We report here a technical advancement that enables time-resolved fluorescence spectroscopy in spatially resolved domains of a living cell at low temperatures. The technique is based on a combination of the self-developed cryo-confocal microscope system and the streak-camera technology. An instrumental response time of ca. 24 ps was achieved. This technique was applied to reveal the light-harvesting dynamics in local domains within single Chlamydomonas reinhardtii cells. Organisms performing oxygenic photosynthesis, like Chlamydomonas, have evolved a regulation mechanism called state transitions (ST), which maintains the excitation balance between PSI and PSII. ST relies on the shuttling of light-harvesting chlorophyll protein complex II (LHCII) between the two PSs. In the present experiment, cells were induced either to state1, where LHCII is bound to PSII, or state2, where LHCII moved and is bound to PSI. After the induction of ST, cells were immediately cooled to ca. 80 K, where PSI and PSII show clearly separated fluorescence emission bands, enabling the visualization of these components separately. Based on kinetic analyses of the time-resolved fluorescence spectra in both PSI-rich and PSII-rich local domains, we concluded that (1) the intracellular inhomogeneity in the PSII/PSI fluorescence ratio comes from that in the PSII/PSI stoichiometry, not from that in the antenna sizes of the PSs, and (2) the antenna size of PSI in state2 cells may larger in intact cells than that of the isolated PSI-LHCI-LHCII super-complex reported so far.

The collective vibration of many biomolecules such as the skeleton vibration, dipole rotation and conformational bending falls in the terahertz (THz) frequency domain. Terahertz time-domain spectroscopy (THZ-TDS), which is very sensitive to the conformational changes, can be used to characterize the collective vibration of biomolecules. In this study, we investigated the low-frequency THz absorption spectra of two rhodopsin mimics using transmission THz-TDS. Using the normal model analysis (NMA), we successfully modelled the experimental terahertz absorption curve and attributed a unique collective motion pattern to each distinctive terahertz absorption frequency. By comparing the terahertz absorption spectra between without and with retinal, we show that the retinal binding can significantly alters the terahertz absorption spectra as well as the vibration modes. Furthermore, by comparing the terahertz absorption spectra between the two mutants, we observed that the single mutation can significantly change the influence of retinal binding on the terahertz absorption spectrum.

The transmembrane-electrostatically localized protons/cations charges (TELC, also known as TELP) model may serve as a unified framework to explain a wide range of bioenergetic phenomenon. Transient TELC and transmembrane potential in a laser flash-energized bacteriorhodopsin (bR) purple membrane (PM) open flat sheet are now better analyzed. Under the Heberle et al. 1994 experimental conditions, the number of bR molecules is now calculated to be 8200 per PM open flat sheet with a diameter of 600 nm. With a single-turnover laser flash intensity of 3 mJ/cm2 to photoexcite 10% of the bR molecules, the number of laser flash-induced peak TELC density is calculated to be 2900 per {micro}m2 of PM, which translates to a peak transient transmembrane potential of 50 mV. The bR protonic outlet protrudes into the liquid phase outside the putative "potential well/barrier". The observation is in line with the TELP model; but does not support the "potential well/barrier" model. The author encourages research on more relevant protonic cell systems that have transmembrane potential with TELC comprising excess positive charges at one side and excess anions at the other side of the membrane.

Forster resonance energy transfer (FRET) is a physicochemical phenomenon involving non-radiative energy transfer between donor and acceptor fluorophores. While FRET efficiency primarily depends on the proximity between fluorophores, additional factors also substantially influence the efficiency in living cells. However, how non-distance factors modulate live-cell FRET efficiency remains poorly understood. Here, we report the significant role of N- and C-terminal topology in determining live-cell FRET efficiency, independent of fluorophore proximity, donor variants, and subcellular compartment. Using acceptor photobleaching and sensitized emission measurements in living cells, we found that FRET efficiencies of mCherry-EGFP or mCherry-EYFP (acceptor-donor) were significantly higher than those of EGFP-mCherry or EYFP-mCherry (donor-acceptor), respectively. These efficiencies were comparable between the nucleus and cytoplasm. An orientation index analysis showed that the acceptor-donor configuration is more favorable than the donor-acceptor configuration regardless of donor variants and subcellular localization. FRET efficiencies were also higher with EYFP than with EGFP as the donor. AlphaFold2-based structural modeling suggested similar proximity with structurally heterogeneous and loosely constrained geometry of donor and acceptor fluorophores. Collectively, these results demonstrate that topological arrangement, rather than simple distance considerations, plays a significant role in FRET efficiency in living cells, providing molecular implications for the design of intramolecular FRET-based biosensors.

Protein dynamics at the sub-nanosecond timescale and the [A] length-scale has widely been studied using quasi-elastic neutron scattering (QENS). In almost all QENS studies on hydrogenated proteins in D2O buffers, analysis of the spectra after buffer subtraction is carried out under the assumption that the remaining spectra arise from incoherent scattering of proteins while the contribution of coherent scattering is negligible. On the contrary, a study using polarization analysis has shown that the coherent scattering accounts for more than 10% of the total scattering intensity of hydrogenated proteins (Gaspar et al., Biochim. Biophys. Acta 1804:76-82 (2010)). In addition, the effects of coherent scattering on the values of dynamical parameters of proteins obtained by analysis of QENS spectra remain unclear. Here, molecular dynamics (MD) simulation on hen egg white lysozyme was used to investigate this issue. QENS spectra containing only incoherent scattering and those containing both incoherent and coherent scattering were calculated from the MD trajectory. Dynamical parameters were then extracted from the two simulated QENS spectra. Comparison of the resultant dynamical parameters has shown that the error in the values of the dynamical parameters induced by coherent scattering is at most 6%. This error is unlikely to significantly affect the results of QENS studies that investigate the relative changes in protein dynamics caused by different physicochemical conditions such as temperature unless dynamical parameters need to be determined with high precision at the absolute scale.

Quasi-elastic neutron scattering (QENS) is a powerful technique to study protein dynamics. In general, QENS measurements are carried out in D2O solvent whereas functional studies of proteins are conducted in H2O solvent. Therefore, to link the QENS studies with the functional studies and then to understand the molecular basis of protein functions in detail, it is important to investigate the effects of solvent isotopic change on dynamical parameters obtained by QENS. For this purpose, in this study, MD simulations were carried out on hen egg white lysozyme, a well-folded and characterized protein, in H2O and in D2O. The dynamical parameters were extracted from the QENS spectra calculated from the MD trajectories. It was found that isotopic effects depend on energy resolutions and that at the energy resolutions that recent QENS studies often employ, the local dynamical behavior of proteins characterized in D2O more or less reflects that in H2O.

Opsins underlie diverse physiological responses to light in animals. In the dark, most opsins bind the chromophore 11-cis retinal, which isomerizes to all-trans form upon light absorption, representing the initial key step in signaling. Maintenance of opsin function therefore requires continuous regeneration of the inactive, 11-cis-retinal-bound state. Here, we report a novel type of opsin, AtAntho2c, from a reef-building coral, whose active form, bound to all-trans retinal, can thermally revert to the initial dark state bound to 11-cis retinal. A cysteine residue in extracellular loop 2 region plays a key role in the self-regeneration ability. Using time-resolved and low-temperature spectroscopies, we identify two spectrally distinct photointermediates prior to the all-trans to 11-cis isomerization in AtAntho2c, whose formation rates and yields are found to vary depending on temperature and pH conditions. The active form of AtAntho2c activates Gi/o G protein, resulting in a transient and repeatable decrease in cellular cAMP levels upon repeated light stimulations, even in the absence of exogenous retinal in cultured cells. Furthermore, we confirm that cells expressing AtAntho2c exhibit membrane hyperpolarization via GIRK channel activation light-dependently. These properties highlight the potential of AtAntho2c as a versatile optogenetic actuator capable of repeatedly modulate Gi/o signaling without retinal supplementation. Significance StatementLight-sensitive proteins, opsins, form active states upon light absorption, leading to intracellular G protein signaling and various cellular outputs. The active states require specific enzymatic machinery or another photon absorption to regenerate inactive opsins ready to respond to repeated light stimuli and maintain continuous responsiveness. In our study, we identify and analyze a coral opsin of which the active state rapidly and autonomously reverts to the inactive state in the dark through thermal isomerization of the retinal chromophore within the opsin. This regeneration mechanism allows the opsin to respond to repeated light stimuli at high temporal resolution and maintain large signal amplitude without the need for exogenous retinal making this opsin potentially useful for developing versatile optogenetic tools.

Cotranslational folding is vital for proteins to form correct structures in vivo. However, it is still unclear how a nascent chain folds at atomic resolution during the translation process. Previously, we have built a model of ribosomal exit tunnel and investigated cotranslational folding of a three-helices protein by using all-atom molecular dynamics simulations. Here we shall study the cotranslational folding of three mainly-{beta} proteins using the same method and find that cotranslational folding can enhance helical population in most cases and reduce nonnative long-range contacts before emerging from the ribosomal exit tunnel. After exiting the tunnel, all proteins fall into local minimal states and structural ensembles in cotranslational folding are more helical than in free folding. Importantly, for GTT WW domain, one local minimal state in cotranslational folding is known as correct folding intermediate, which is not found in free folding. This result suggests that cotranslational folding may directly increase folding efficiency by accelerating sampling more than by avoiding the misfolded state, which is a mainstream viewpoint in present. In addition, our method can serve as a general scheme to study cotranslational folding process of proteins. Statement of SignificanceIn cell, the formations of correct three-dimensional structures of proteins, namely protein folding, are essential to human health. Misfolding can lead to serious diseases such as Alzheimers disease and mad cow disease. As the first step of in vivo folding, the effect of cotranslational folding on the correct folding of proteins has been the focus of scientific research in this century. Although some experiments have shown that cotranslational folding can improve the efficiency of folding, its microscopic mechanism is not yet clear. In this paper, we study the process of cotranslational folding of three proteins by using all-atom molecular dynamics simulations, and try to reveal some aspects of the mechanism of cotranslational folding from a microscopic perspective.

Spiroplasma swim by switching their helical body into right- and left-handed. Helicity formation and switching can be reconstituted in an immotile minimal synthetic bacterium, JCVI-syn3B by introducing the pair of bacterial actin, MreB4 and MreB5. Here, we analyzed MreB behaviors optically, to investigate this unknown mechanism. We tried MreB4 fluorescence labeling by protein fusion. The labeling was not achieved, because the fusion of fluorescent protein or peptide to 16 positions resulted in immotile constructs. These results may suggest that MreB4 has many interaction interfaces with other proteins. Induced expression of MreB4 to cells with constitutive MreB5 expression resulted in earlier and higher frequency of motile cells, distinct from the results of constitutive MreB4 and inducible MreB5. Next, the behavior of labeled MreB5 was analyzed. Photobleaching and photoactivation suggested static behavior of MreB5 during cell movements. Cell treatment by A22, a MreB polymerization inhibitor caused helix deformation, movement stall, and diffusion of MreB5 fluorescence, suggesting that A22 sensitive MreB5 interaction should be involved in helix formation and motility. SignificanceSpiroplasma MreB is a unique bacterial actin that causes motility with a different mechanism from other actins. Here, we investigated the helicity switching involved in the motility. Fluorescently traced MreB behaviors suggested that the motility mechanism is not coupled with its replacement in the internal structure.

Base stacking is fundamentally important to the stability of double-stranded DNA. However, few experiments can directly probe the local conformations and conformational fluctuations of the DNA bases. Here we report a new spectroscopic approach to study the local conformations of DNA bases using the UV-absorbing fluorescent guanine analogue, 6-methyl isoxanthopterin (6-MI), which can be used as a site-specific probe to label DNA. In these experiments, we apply a two-photon excitation (2PE) approach to two-dimensional fluorescence spectroscopy (2DFS), which is a fluorescence-detected nonlinear Fourier transform spectroscopy. In 2DFS, a repeating sequence of four collinear laser pulses (with center wavelength [~] 675 nm and relative phases swept at radio frequencies) is used to excite the lowest energy electronic-vibrational (vibronic) transitions of 6-MI (with center wavelength [~] 340 nm). The ensuing low flux fluorescence is phase-synchronously detected at the level of individual photons and as a function of inter-pulse delay. The 2PE transition pathways that give rise to electronically excited state populations include optical coherences between electronic ground and excited states and non-resonant (one-photon-excited) virtual states. Our results indicate that 2PE-2DFS experiments can provide information about the electronic-vibrational spectrum of the 6-MI monomer, in addition to the conformation-dependent exciton coupling between adjacent 6-MI monomers within a (6-MI)2 dimer. In principle, this approach can be used to determine the local base-stacking conformations of (6-MI)2 dimer-substituted DNA constructs.

Most temperate plants can tolerate both chilling and freezing temperatures. Plants have developed self-defense mechanisms to perceive cold signal, trigger the cold tolerance for the cold response ICE1-CBF-COR pathway by packing a cascade of kinase phosphorylation/dephosphorylation events into a functional module. Linked to the central ICE1-CBF-COR model, two sub-models were adopted for the second messenSger Ca2+ -- a one-compartment model for Ca2+ transient and a two-compartment model for a train of Ca2+ spikes. Numerical simulation verified the expression patterns of the cold-induced genes as observed in Arabidopsis thaliana, Brassica napa and Solanum lycopersicum, including wild types and mutants. Moreover, the desensitization and resensitization of CBF3 and COR15A were displayed as well as the dynamics in the gradually decreasing temperature response to cold stress. The duration of cold tolerance was predicted for approximate 10 days. Interpreting and predicting the dynamical behaviors of the cold signaling pathway are valuable and time-saving for understanding mechanisms of cold acclimation. Author summaryIn higher organisms, CBF3 transcriptional level are output in a pulse and COR15A transcriptional level are maintained at a high homeostatic level to respond cold stress, which is determined by a complex set of regulatory mechanisms. Feedback loops from Ca2+ perception onto CaM, kinases, cold-acclimated genes in sequence, which have been experimentally reported, are thought to induce COR15A transcript accumulation under cold temperature exposure. The cold response pathway has been modeled based on a computational model. However, protein kinase cascades made the model relatively complicated and the cold-response gene COR15A was absent. Here, we develop a compact model with cold tolerance target gene COR15A. We show that this model could reproduce the temporal dynamics and characteristics of the core gene variables in different plants with wild type and mutant. Reduction of CaM proportion and augment of negative feedback control by ZAT12 in the model results in the desensitization of CBF3 and COR15A. Less than 24-hr warm treatment loses the ability of resensitization of CBF3 and COR15A after 14-day cold exposure. We further predict that the duration of cold tolerance is maintained at about 10 days in our model.

Forster resonance energy transfer (FRET) between fluorescent proteins (FPs) is widely used in the design of genetically encoded fluorescent biosensors, which are powerful tools for monitoring the dynamics of biochemical activities in live cells. FRET ratio, defined as the ratio between acceptor and donor signals, is often used as a proxy for the actual FRET efficiency, which must be corrected for signal crosstalk using donor-only and acceptor-only samples. However, the FRET ratio is highly sensitive to imaging conditions, making direct comparisons across different experiments and over time challenging. Inspired by a method for multiplexed biosensor imaging using barcoded cells, we reasoned that calibration standards with fixed FRET efficiency can be introduced into a subset of cells for normalization of biosensor signals. Our theoretical analysis indicated that the FRET ratio of high-FRET species relative to non-FRET species slightly decreases at high excitation intensity, suggesting the need for calibration using both high and low FRET standards. To test these predictions, we created FRET donor-acceptor pairs locked in "FRET-ON" and "FRET-OFF" conformations and introduced them into a subset of barcoded cells. Our results confirmed the theoretical predictions and showed that the calibrated FRET ratio is independent of imaging settings. We also provided a strategy for calculating the FRET efficiency. Together, our study presents a simple strategy for calibrated and highly multiplexed imaging of FRET biosensors, facilitating reliable comparisons across experiments and supporting long-term imaging applications.

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.

Single-molecule fluorescence imaging has extensively revealed the dynamic and structural characteristics of biomolecules. However, its application is limited by the upper concentration of fluorophore-tagged biomolecules, which is in the sub-ten nanomolar range. We found that the signal-to-noise ratio (SNR) in single-molecule fluorescence imaging is strongly influenced by the size of fluorophore-labeled molecules in solution. Our computational simulations suggest that the faster diffusion of background fluorophores can enhance the SNR of target molecules. Moreover, we identified that the molecular motion through fluid flow can improve SNR. This study provides a novel perspective by emphasizing the importance of molecular motion in SNR and propose a rapid barrier-free method to increase the upper concentration limit in single-molecule imaging.

Microbial rhodopsin, an important photoreceptor protein, has been widely used in several fields, such as optogenetics, biotechnology, and biodevices etc. However, current microbial rhodopsins are all transmembrane proteins, which both complicates the investigation on the photoreaction mechanism and limits their further applications. Therefore, a suitable mimic for microbial rhodopsin can not only provide a better model for understanding the mechanism, but also can extend the applications. The human protein CRABPII turns out to be a good template for design mimics on rhodopsin, due to the convenience in synthesis and the stability after mutations. Recently, Geiger et al. designed a new CRABPII-based mimic M1-L121E on microbial rhodopsin with the correct 13-cis (13C) isomerization after irritation. However, it still remains a question how similar it is compared with the natural microbial rhodopsin, in particular in the aspect of the photoreaction dynamics. In this article, we investigated the excited-state dynamics of this mimic by measuring its transient absorption spectra. Our results reveal that there are two components in the solution of mimic M1-L121E at PH=8, known as protonated Schiff base (PSB) and unprotonated Schiff base (USB) states. In both states, the photoreaction process from 13-cis (13C) to all-trans (AT) is faster than that from the inverse direction. In addition, the photoreaction process in PSB state is faster than that in the USB state. In the end, we compared the isomerization time of the PSB state with the properties of the microbial rhodopsin, and confirmed that the mimic M1-L121E indeed captures the main feature of the rhodopsin and is a good model of microbial rhodopsin in the photoreaction dynamics. However, our results also reveal significant differences in the excited-state dynamics of the mimic relative to the natural microbial rhodopsin, including the slower PSB isomerization rates in both 13C-AT and AT-13C directions, as well as the unusual USB photoreaction dynamics at PH=8. Such unique properties have not been observed in the natural rhodopsin, which could further deepen the understanding in photoreaction mechanism of the photosensitive proteins.

The question of whether physical phenomena at a quantum level significantly impact aspects of macroscopic life has long remained unanswered. Histone modification by acetylation regulates the transcriptional activity of genes, and thereby broadly impacts cellular metabolism. In chemical reactions, the quantum tunneling effect is a phenomenon in which a small quantum particle of the reactant can pass through the potential energy barrier, even if it does not have sufficient energy to overcome the barrier. Here, we demonstrated that quantum effects are involved in the enzymatic reaction of histone deacetylation, by monitoring kinetic isotope effects due to hydrogen isotopes of water molecules and their temperature dependence as indicators. Due to the kinetic isotope effects associated with the quantum effects, the reaction rate balance between histone acetylation and deacetylation in cells was altered with heavy water, which changed epigenetic transcription regulation in the cells. Thus, microscopic quantum mechanisms exist in histone deacetylation, thereby broadly impacting macroscopic life phenomena through epigenetic regulation. TeaserQuantum effects in enzymatic reaction of histone deacetylation latently influence life phenomena through epigenetic regulation.

The clock protein of cyanobacteria KaiC forms a homohexamer with two ring-shaped domains, C1 and C2. These domains undergo several domain-specific conformational transitions and allosterically communicate to generate a circadian rhythm. Interestingly, experiments show a possibility that C2 is independent of C1. However, detailed interplay among them remains elusive. Here we propose a mathematical model, which explicitly considers the interplay. The allostery in KaiC is here modeled to be unidirectional from C2 to C1. We demonstrate that the unidirectional allostery is sufficient for the circadian rhythm by showing the quantitative reproducibility of various experimental data, including temperature dependence of both phosphorylation oscillation and ATPase activity. Based on the present model, we further discuss possible functional roles of the unidirectional allostery particularly in the period robustness against both protein concentration and temperature.

1The spike protein of SARS-CoV-2 is a highly flexible membrane receptor that triggers the translocation of the virus into cells by attaching to the human receptors. Like other type I membrane receptors, this protein has several extracellular domains connected by flexible hinges. The presence of these hinges results in high flexibility, which consequently results in challenges in defining the conformation of the protein. Here, We developed a new method to define the conformational space based on a few variables inspired by the robotic fields methods to determine a robotic arms forward kinematics. Using newly performed atomistic molecular dynamics (MD) simulations and publicly available data, we found that the Denavit-Hartenberg (DH) parameters can reliably show the changes in the local conformation. Furthermore, the rotational and translational components of the homogenous transformation matrix constructed based on the DH parameters can identify the changes in the global conformation of the spike and also differentiate between the conformation with a similar position of the spike head, which other types of parameters, such as spherical coordinates, fail to distinguish between such conformations. Finally, the new method will be beneficial for looking at the conformational heterogeneity in all other type I membrane receptors.

Time-resolved resonance Raman spectroscopy with continuous-wave excitation is a fundamental technique that has contributed substantially to the understanding of structure and dynamics of bacteriorhodopsin and related retinal proteins. However, the underlying principles were developed about fifty years ago for instrumentation that is hardly in use any more. Thus, the adaptation of the technique to current state-of-the art equipment is needed to satisfy the increasing demand for the spectroscopic characterization of microbial retinal proteins. In this work, we focus on pump-probe time-resolved resonance Raman experiments with a confocal spectrometer using a rotating cell. We discuss the boundary conditions that fulfill the fresh sample conditions and the photochemical innocence of the probe beam as a prerequisite for studying parent or intermediate states of retinal proteins that undergo a cyclic photoinduced reaction sequence. For the measurements of intermediate states and reaction kinetics, pump-probe experiments are required in which the two laser beams hit the flowing sample with a defined but variable delay time. An appropriate set-up for such two-beam experiments with a confocal spectrometer is proposed and tested in time-resolved experiments of bacteriorhodopsin. The comparison with the results obtained with previous classical slit spectrometers with 90-degree-scattering illustrates the advantages and disadvantages of the confocal arrangement. It is shown that modern confocal spectrometers substantially decrease the spectra acquisition time but require a more demanding optical set-up. Furthermore, the extent of photoconversion by the pump beam is lower than for the 90-degree-scattering arrangement which lowers the accuracy of kinetic measurements.

To advance the industrialization of cultured meat and regenerative medicine, scalable and efficient cell culture techniques are essential. Among these, the suspension culture method using microcarriers has emerged as a promising approach for the large-scale cell culture technique. However, monitoring cell growth on the microcarriers remains challenging, particularly in developing cell counting techniques that can be seamlessly integrated into bioprocess workflows without cell detachment, fluorescence labeling and any parameter tuning in the analysis algorithm. In this study, we proposed a versatile image analysis-based cell counting method by using cellular autofluorescence without any parameter tuning. The proposed method estimates the number of cells by applying spatiotemporal averaging to the autofluorescence signals in the microscopic images. Using numerical and cell culture experiments, we demonstrated that the proposed method can estimates the number of cells accurately. This technique, which harnesses the ubiquitous autofluorescence inherent in living cells, offers a cost-effective and practical solution applicable to a broad range of fields requiring high-throughput cell quantification.