DEAD-box RNA helicases are central regulators of RNA metabolism, employing ATP-dependent mechanisms to remodel RNA structure and RNA-protein interactions, yet how helicase catalysis is coordinated with multi-subunit interactions between RNA and protein remains unresolved. Translation initiation helicase, eukaryotic initiation factor 4A (eIF4A), which acts as an intrinsically non-processive enzyme, is essential for unwinding structured mRNAs, relies on cofactors to achieve physiological activity. Here we uncover an unexpected RNA-helicase state of eIF4A, demonstrating that eIF4A forms nanometer-scale RNA-protein clusters (RPCs) of ~2-5 MDa in presence of its physiological cofactors eIF4B and eIF4G, RNA and ATP under near-physiological concentrations. Using a single molecule approach, we directly resolve the formation of discrete clusters that recruit multiple copies of proteins with RNA upon ATP addition and show that RPC formation correlates with helicase activity in vitro. Further, we find eIF4B as a key determinant of this multi-subunit assembly. Its intrinsically disordered regions (IDRs) together with structured RNA-recognition motifs (RRMs) drive multivalent RNA-dependent clustering, critical for efficient helicase activity. Disrupting eIF4B-RNA interactions through a targeted point mutation (F139A) in the RRM reduces both the cluster size and the helicase activity, further establishing a functional link between cluster formation and catalytic activity. Consistent with these findings, in-cell diffusion measurements reveal markedly slower diffusion of wild-type eIF4B compared with the RNA-binding-deficient mutant, indicative of RPC formation within the cellular environment. Together, our results reveal regulated helicase clustering as a previously unrecognized characteristic of the translation initiation machinery, linking ATP-dependent DEAD-box helicase activity to nanometer-scale RNA-protein clusters and translation initiation regulation.

Ongoing improvements of genetically encoded fluorescent proteins have enhanced cellular localization studies and performance of biosensors, such as environmentally or mechanically sensitive fluorescence resonance energy transfer pairs, in cell biological and biophysical research. The brightest yellow fluorescent protein, widely used in these studies is YPet, derived from the jellyfish Aequorea victoria via the GFP derivative Venus. YPet dimerizes at concentrations used in cellular studies (KD1-2 = 3.4 μM) which impacts quantitative interpretation of emission intensity, rotational freedom, energy transfer, and lifetime. Although YPet is nearly 30% brighter than Venus, no atomic structures of YPet have been reported to ascertain the structural differences leading to the higher brightness, possibly due to the tendency to dimerize or oligomerize. Here, we report properties of a new YPet derivative, mCLIFY, a monomeric, bright, yellow, and long-lived fluorescent protein created by circular permutation of YPet and substitution of the amino acid residues thought to mediate dimerization. mCLIFY retains the advantageous photophysical properties of YPet but does not dimerize at least up to 40 μM concentration. We determined the atomic structure of mCLIFY at 1.57-Å resolution. Extensive characterization of the photophysical and structural properties of YPet and mCLIFY allowed us to elucidate the bases of their long lifetimes, enhanced brightness, and the difference in propensity to dimerize.

Mutations in the RNA helicase DDX3X, implicated in various cancers and neurodevelopmental disorders, often impair RNA unwinding and translation. However, the mechanisms underlying the impairment and the differential interactions of DDX3X mutants with wild-type (WT) X-linked DDX3X and Y-linked homolog DDX3Y remain elusive. This study reveals that specific DDX3X mutants more frequently found in disease form distinct hollow condensates in cells. Using a combined structural, biochemical, and single-molecule microscopy study, we show that reduced ATPase and RNA release activities contribute to condensate formation and these catalytic deficits result from inhibiting the catalytic cycle at multiple steps. Proteomic investigations further demonstrate that these hollow condensates sequester WT DDX3X/DDX3Y and other proteins crucial for diverse signaling pathways. WT DDX3X enhances the dynamics of heterogeneous mutant/WT hollow condensates more effectively than DDX3Y. These findings offer valuable insights into the catalytic defects of specific DDX3X mutants and their differential interactions with wild-type DDX3X and DDX3Y, potentially explaining sex biases in disease. Here the authors find that DDX3X mutants with catalytic impairments form hollow condensates in cells that have slower dynamics when co-condensed with DDX3Y than with WT DDX3X, revealing potential mechanisms of sex biases in DDX3X-related diseases.

DEAD-box helicases, crucial for many aspects of RNA metabolism, often contain intrinsically disordered regions (IDRs) whose functions remain unclear. Using multiparameter confocal microscopy, we reveal that sex chromosome-encoded homologous RNA helicases, DDX3X and DDX3Y, form nanometer-scale RNA-protein clusters (RPCs) that foster their catalytic activities in vitro and in cells. The IDRs are critical for the formation of these RPCs. A thorough analysis of the catalytic cycle of DDX3X and DDX3Y by ensemble biochemistry and single-molecule photon bursts in the confocal microscope showed that RNA release is a major step that differentiates the unwinding activities of DDX3X and DDX3Y. The N-terminal IDRs of DDX3X and DDX3Y are both the drivers of RPC formation and the major differentiators of their enzymatic activities. Our findings provide new insights that the nanoscale helicase RPCs may be the normal state of these helicases under non-stressed conditions that promote their RNA unwinding and might act as nucleation points for stress granule formation. This mechanism may apply broadly among other members of the DEAD-box helicase family.

mCLIFY is a novel circularly permuted variant of the fluorescent protein (FP) YPet, a Venus-derivative. Using a battery of methods, we have shown that mCLIFY is exclusively monomeric retaining the favorable spectral and photophysical properties of the dimerizing YPet. We determined its atomic structure to 1.57 Å resolution and found that it is nearly identical to Venus, confirming that circular permutation did not alter its canonical structure. mCLIFY, YPet, and Venus are highly similar, only differing by six amino acids.

DEAD-box helicases, which are crucial for many aspects of RNA metabolism, often contain intrinsically disordered regions (IDRs), whose functions remain unclear. Using multiparameter confocal microscopy, we reveal that sex chromosome-encoded homologous RNA helicases, DDX3X and DDX3Y, form nano-sized RNA-protein clusters (RPCs) that foster their catalytic activities in vitro and in cells. The IDRs are critical for the formation of these RPCs. A thorough analysis of the catalytic cycle of DDX3X and DDX3Y by ensemble biochemistry and single molecule photon bursts in the confocal microscope showed that RNA release is a major step that differentiates the unwinding activities of DDX3X and DDX3Y. Our findings provide new insights that the nano-sized helicase RPCs may be the normal state of these helicases under non-stressed conditions that promote their RNA unwinding and act as nucleation points for liquid-liquid phase separation under stress. This mechanism may apply broadly among other members of the DEAD-box helicase family.

Fluorescent proteins (FP) have become widely used biophysical and cell biological tools that report on a variety of processes within cells. The popular yellow FP, YPet, serves as either a donor or acceptor for many FRET-based biosensors, yet it has been difficult to fully characterize. In studying our circularly permuted and equally bright variant, mCLIFY, features common to YPet were revealed, including β-barrel and chromophore sequence homology. We determined the atomic structure of mCLIFY to 1.6 Å resolution, which reveals a remarkably well-aligned π-interaction between the chromophore with Tyr37, as well as a water-mediated network of hydrogen bonds which supports the chromophore. Both features are likely promoting the robust photophysical properties of mCLIFY. To better understand the self-association properties of YPet, known to be a weak dimer, sedimentation velocity analytical ultracentrifugation (SV-AUC) determined a monomer-dimer dissociation constant KD of 3.4 μM, whereas mCLIFY did not dimerize. Size-exclusion chromatography in-line with synchrotron small-angle X-ray scattering and multi-angle light scattering (SEC-SAXS-MALS) confirmed these findings. SAXS studies show that mCLIFY correlates well to the solution monomer, whereas YPET exists as an antiparallel dimer in solution. Their fluorescence intensities and anisotropies were compared via FLIM (Fluorescence Lifetime Imaging Microscopy) of FPs expressed in E. coli. Protein concentrations increased above 5 μM for both FPs within 45 minutes of IPTG induction. Unexpectedly, average anisotropy of YPet significantly decreased from 0.322 to 0.183 with increasing protein concentration presumably due to HOMO-FRET and/or trivial reabsorption of emitted photons. Anisotropy for mCLIFY (0.316) decreased at much higher concentrations. The data presented here suggest that potential quantitative errors from overexpression of non-monomeric FPs may be avoided with newly available, more fully studied monomeric variants like mCLIFY.

Sex differences are pervasive in human health and disease with the most striking differences lying in the sex chromosomes, which encode a group of sex-specific protein homologs. DDX3X and DDX3Y are a pair of sexually dimorphic non-processive ATP-dependent RNA helicases. We previously showed that DDX3Y forms larger, less dynamic phase separated condensates compared to DDX3X, possibly due to the lower ATPase activity of DDX3Y and different dynamic interactions with RNA. We used single-molecule FRET of immobilized, fluorescently labeled RNA duplexes and reported that DDX3X and DDX3Y show different binding dynamics and partial unwinding activities (Shen and Yanas et al., Molecular Cell 2022). To further investigate the enzymatic activities of DDX3, we used multiparameter confocal spectroscopy to obtain unwinding, time-resolved anisotropy, diffusion coefficients, and structural changes by FRET of freely diffusing RNA duplexes upon addition of DDX3, with and without ATP. Duplex RNA with a 5’ overhang was labeled with a Cy3 and Alexa647 FRET pair at the chain termini. The proportion of duplex RNA was quantified and used as a readout for helicase activity, and DDX3X shows higher helicase activity than DDX3Y. With ns time-resolved fluorescence anisotropy, we found that the shorter, non-overhanging strand RNA is released with helicase activity, whereas protein remains bound to the longer RNA strand. Diffusion constants from fluorescence correlation spectroscopy revealed formation of enzymatically active protein clusters at low DDX concentrations. Removal of N- and C-terminal intrinsically disordered regions suppressed formation of clusters and reduced enzymatic activity. We hypothesize that these clusters act as “helicase hubs,” possibly tiny nuclei of liquid granules, which may be translational centers in the cell, leading to efficient translation initiation.

Mutations in the RNA helicase DDX3X, implicated in various cancers and neurodevelopmental disorders, often impair RNA unwinding and translation. However, the mechanisms underlying this impairment and the differential interactions of DDX3X mutants with wild-type (WT) X-linked DDX3X and Y-linked homolog DDX3Y remain elusive. This study reveals that specific DDX3X mutants more frequently found in disease form distinct hollow condensates in cells. Using a combined structural, biochemical, and single-molecule microscopy study, we show that reduced ATPase and RNA release activities contribute to condensate formation and the catalytic deficits result from inhibiting the catalytic cycle at multiple steps. Proteomic investigations further demonstrate that these hollow condensates sequester WT DDX3X/DDX3Y and other proteins crucial for diverse signaling pathways. WT DDX3X enhances the dynamics of heterogeneous mutant/WT hollow condensates more effectively than DDX3Y. These findings offer valuable insights into the catalytic defects of specific DDX3X mutants and their differential interactions with wild-type DDX3X and DDX3Y, potentially explaining sex biases in disease.

Understanding molecular interactions and dynamics of proteins and DNA in a cell-like crowded environment is crucial for predicting their functions within the cell. Noncanonical G-quadruplex DNA (GqDNA) structures adopt various topologies that were shown to be strongly affected by molecular crowding. However, it is unknown how such crowding affects the solvation dynamics in GqDNA. Here, we study the effect of cosolvent (acetonitrile) crowding on ligand (DAPI) solvation dynamics within human telomeric antiparallel GqDNA through direct comparison of time-resolved fluorescence Stokes shift (TRFSS) experiments and molecular dynamics (MD) simulations results. We show that ligand binding affinity to GqDNA is drastically affected by acetonitrile (ACN). Solvation dynamics probed by DAPI in GqDNA groove show dispersed dynamics from ∼100 fs to 10 ns in the absence and presence of 20% and 40% (v/v) ACN. The nature of dynamics remain similar in buffer and 20% ACN, although in 40% ACN, distinct dynamics is observed in <100 ps. MD simulations performed on GqDNA/DAPI complex reveal preferential solvation of ligand by ACN, particularly in 40% ACN. Simulated solvation time-correlation functions calculated from MD trajectories compare very well to the overall solvation dynamics of DAPI in GqDNA, observed in experiments. Linear response decomposition of simulated solvation correlation functions unfolds the origin of dispersed dynamics, showing that the slower dynamics is dominated by DNA-motion in the presence of ACN (and also by the ACN dynamics at higher concentration). However, water-DNA coupled motion controls the slow dynamics in the absence of ACN. Our data, thus, unravel a detailed molecular picture showing that though ACN crowding affect ligand binding affinity to GqDNA significantly, the overall dispersed solvation dynamics in GqDNA remain similar in the absence and the presence of 20% ACN, albeit with a small effect on the dynamics in the presence of 40% ACN due to preferential solvation of ligand by ACN.

Sex differences are pervasive in human health and disease. The most striking differences lie in the sex chromosomes, which encode a group of sex-specific protein homologs. Although the functions of the X chromosome proteins are well appreciated, how they compare to their Y chromosome homologs remains elusive. DDX3X and DDX3Y are one such pair of sexually dimorphic non-processive ATP-dependent RNA helicases. Herein, we demonstrate that DDX3Y forms larger RNA-dependent, phase separated liquid condensates compared to DDX3X, which is possibly due to differences in kinetics and dynamic interactions with RNA. By a malachite green phosphate assay and MESG continuous phosphate assay, we find that DDX3X has higher ATPase activity than DDX3Y. We used single-molecule FRET of immobilized Cy3- and Alexa647-labeled RNA duplexes, and report that DDX3X and DDX3Y show different binding and possibly different unwinding activities. The addition of ATP to either DDX3X or DDX3Y shifted FRET efficiency from E = 0.8 to E = 0. The low FRET population was larger for DDX3X than for DDX3Y. Data at three protein concentrations suggest cooperative interaction of the proteins with RNA. DDX3X showed a larger proportion of dynamic FRET recordings upon addition of ATP compared to DDX3Y. Freely diffusing complexes were studied by multiparameter confocal spectroscopy to obtain unwinding, anisotropy, diffusion coefficient, and FRET of the RNA duplex upon addition of the helicases in the presence and absence of ATP. Collectively, the kinetics and smFRET data support that DDX3Y has weaker ATPase activity, leading to the less dynamic RNA-DDX3Y complexes. Decreased dynamics, in turn, may contribute to the weaker disassembly of DDX3Y condensates upon addition of ATP compared to DDX3X condensates. Comparison of sex chromosome-encoded protein homologs may provide insights into sexual differences in RNA metabolism and human diseases.

The important biological processes that help communicate between intra- and extracellular environments take place at cell membrane/water interface. However, molecular interactions at these interfaces are strongly affected by the depth-dependent hydration and local environmental polarity across the lipid/water interface and also the lipid ordering. A new family of membrane probes based on 4-aminophthalimide (4AP-Cn) have been synthesized, which are of particular interest here because of their extreme sensitivity towards sensing depth-dependent polarity, hydration and energy transfer dynamics at model lipid/water interfaces in sub-nanometre length scale. We envisage that these new probes will be extremely useful for characterizing the static and dynamic properties of naturally occurring membrane/water interfaces as well. This chapter presents the protocol for 4AP-Cn synthesis, as well as detailed experimental and MD simulation methods for measuring depth-dependent polarity, hydration and multi-molecular energy transfer dynamics at lipid/water interfaces of gel and fluid phases of lipid bilayer.

Although, currently available fluorescent proteins (FPs) provide an exceptional variety of high-performance options, new variations with improved properties are emerging. YPet is considered to be the brightest of the YFP variants, but it has the tendency to form dimers or oligomers under physiological conditions, compromising quantification of biophysical signals such as anisotropy and FRET. Here, we introduce mCLFY, a new monomeric bright yellow FP based on circular permutation of the amino acid sequence of YPet, separating the N- and C-termini of mCLFY from the protective barrel caps to potentially improve chromophore stability. The new N- and C-termini of mCLFY are located on the opposite end of the β barrel at YPet residues 175 in the loop arising from β-strand 8 and β-strand 9 residue 176. Fluorescence correlation spectroscopy (FCS) yielded a diffusion constant for mCLFY of D = 111 ± 1 μm2/s (s.e.m., n = 14), whereas for YPet, D = 57 ± 2 μm2/s (n = 14). Native gel electrophoresis supports these observations showing faster migration of mCLFY compared to YPet. Quantum yield (QY) of mCLFY is 0.76, using the reported 0.77 QY of YPet as a standard. Molar extinction coefficients were determined at λ = 517 nm, ε = 121 ± 7 x 103 and 124 x 103 M−1·cm−1 for mCLFY and YPet, respectively. mCLFY shows reduced pH sensitivity of brightness relative to YPet. Structural details that lead to the improved stability of mCLFY require further investigation. Using time correlated single photon counting, we determined fluorescence lifetimes of mCLFY and YPet to be 3.29 ± 0.04 ns and 3.27 ± 0.09 ns (n = 8 ea.), respectively. mCLFY is an improved FP similar to YPet, but with less tendency to dimerize and less pH sensitivity.

The measurement and understanding of collective solvation dynamics in DNA have vital biological implications, as protein and ligand binding to DNA can be directly controlled by complex electrostatic interactions of anionic DNA and surrounding dipolar water, and ions. Time-resolved fluorescence Stokes shift (TRFSS) experiments revealed anomalously slow solvation dynamics in DNA much beyond 100 ps that follow either power-law or slow multiexponential decay over several nanoseconds. The origin of such dispersed dynamics remains difficult to understand. Here we compare results of TRFSS experiments to molecular dynamics (MD) simulations of well-known 4',6-diamidino-2-phenylindole (DAPI)/Dickerson-Drew DNA complex over five decades of time from 100 fs to 10 ns to understand the origin of such dispersed dynamics. We show that the solvation time-correlation function (TCF) calculated from 200 ns simulation trajectory (total 800 ns) captures most features of slow dynamics as measured in TRFSS experiments. Decomposition of TCF into individual components unravels that slow dynamics originating from dynamically coupled DNA-water motion, although contribution from coupled water-Na+ motion is non-negligible. The analysis of residence time of water molecules around the probe (DAPI) reveals broad distribution from ∼6 ps to ∼3.5 ns: Several (49 nos.) water molecules show residences time greater than 500 ps, of which at least 14 water molecules show residence times of more than 1 ns in the first solvation shell of DAPI. Most of these slow water molecules are found to occupy two hydration sites in the minor groove near DAPI binding site. The residence time of Na+, however, is found to vary within ∼17-120 ps. Remarkably, we find that freezing the DNA fluctuations in simulation eliminates slower dynamics beyond ∼100 ps, where water and Na+ dynamics become faster, although strong anticorrelation exists between them. These results indicate that primary origin of slow dynamics lies within the slow fluctuations of DNA parts that couple with nearby slow water and ions to control the dispersed collective solvation dynamics in DNA minor groove.

The fact that ubiquitous water and ions are important for biological functions of biomolecules is well established. However, understanding the dynamics of water and ions near biomolecules such as DNA, protein and phospholipid is difficult, but essential, for comprehending biomolecular functions. While significant progress has been made to apprehend the hydration structure and dynamics around proteins and phospholipids, understanding dynamics of water and ions near DNA remains challenging. Time-resolved fluorescence Stokes shift (TRFSS) experiments and molecular dynamics (MD) simulation have unraveled several new insights about perturbed water and counterion dynamics near poly-anionic DNA from femtoseconds to nanoseconds time-scales, although with debated explanations of the dispersed DNA dynamics. Here we review the advances of TRFSS experiments and MD simulation studies that unfolded several fascinating, but complex, dynamical features of perturbed water and counterions near different DNA structures in solution.

Water around biomolecules is special for behaving strangely – both in terms of structure and dynamics, while ions are found to control various interactions in biomolecules such as DNA, proteins and lipids. The questions that how water and ions around these biomolecules behave in terms of their structure and dynamics, and how they affect the biomolecular functions have triggered tremendous research activities worldwide. Such activities not only unfolded important static and dynamic properties of water and ions around these biomolecules, but also provoked heated debate regarding their explanation and role in biological functions. DNA, being negatively charged, interacts strongly with the surrounding dipolar water and positively charged counterions, leading to complex electrostatic coupling of water and ions with the DNA. Recent time-resolved fluorescence Stokes shift experiments and related computer simulation studies from our and other laboratories have unfolded some unique dynamic characteristics of water and ions near different structures of DNA. These results are discussed here to showcase the specialty of water and ion dynamics around DNA.

Recognition of DNA base mismatches and their subsequent repair by enzymes is vital for genomic stability. However, it is difficult to comprehend such a process in which enzymes sense and repair different types of mismatches with different ability. It has been suggested that the differential structural changes of mismatched bases act as cues to the repair enzymes, although the effect of such DNA structural changes on surrounding water and ion dynamics is inevitable due to strong electrostatic coupling among them. Thus, collective dynamics of DNA, water, and ions near the mismatch site is believed to be important for mismatch recognition and repair mechanism. Here we show that introduction of a T·T mismatch in the minor groove of DNA induces dispersed (collective) power-law solvation dynamics (of exponent ∼0.24), measured by monitoring the time-resolved fluorescence Stokes shifts (TRFSS) of two popular minor groove binders (Hoechst 33258 and DAPI) over five decades of time from 100 fs to 10 ns. The same ligands however sense different dynamics (power-law of exponent ∼0.15 or power-law multiplied with biexponential relaxation) in the minor groove of normal-DNA. The similar fluorescence anisotropy decays of ligands measured in normal- and T·T-DNA suggest that Stokes shift dynamics and their changes in T·T-DNA purely originate from the solvation process, and not from any internal rotational motion of probe-ligands. The dispersed power-law solvation dynamics seen in T·T-DNA indicate that the ligands do not sense any particular (exponential) relaxation specific to T·T wobbling and/or other conformational changes. This could be the reason why T·T mismatch is recognized by enzymes with lower efficiency compared to purine-pyrimidine and purine-purine mismatches.