Cavity-Amplified Scattering Spectroscopy Reveals the Dynamics of Proteins and Nanoparticles in Quasi-transparent and Miniature Samples.

Dynamic light scattering techniques can give access to the motion spectrum of microscopic objects and are therefore routinely used for numerous industrial and research applications ranging from particle sizing to the characterization of the viscoelastic properties of materials. However, such measurements are impossible when samples do not scatter light enough, i.e., when light undergoes too few scattering events when passing through a sample, either due to excessively small scattering cross sections or due to low concentrations of scatterers. Here, we propose to amplify the light scattering efficiency by placing weakly scattering samples inside a Lambertian cavity with high-reflectance walls. When injected with laser light, the cavity produces a 3D isotropic and homogeneous light field, effectively elongates the photon scattering path length through the sample by 2-3 orders of magnitude, and leads to a dramatic increase in sensitivity. With a 104-fold increase in sensitivity compared to classical techniques, we potentially expand the applications of light scattering to miniaturized microfluidics samples and to weakly scattering samples in general. We show that we can access the short-time dynamics of low-turbidity samples and demonstrate our sensitivity gain by measuring the diffusion coefficient and, therefore, the size of particles ranging from 5 nm to 20 µm with volume fractions as low as 10-9 in volumes as low as 100 µL and in solvents with refractive index mismatches down to Δn ≈ 0.01. Beyond the realm of current applications of light scattering techniques, our cavity-amplified scattering spectroscopy method (CASS) and its high sensitivity represent a significant methodological step toward the study of short-time dynamics problems such as the ballistic limit of Brownian motion, the internal dynamics of proteins, or the dielectric dynamics of liquids.

Reaction Cycle of Operating Pump Protein Studied with Single-Molecule Spectroscopy.

Biological machinery relies on nonequilibrium dynamics to maintain stable directional fluxes through complex reaction cycles. For such reaction cycles, the presence of microscopically irreversible conformational transitions of the protein, and the accompanying entropy production, is of central interest. In this work, we use multidimensional single-molecule fluorescence lifetime correlation spectroscopy to measure the forward and reverse conformational transitions of bacteriorhodopsin during trans-membrane H+ pumping. We quantify the flux, affinity, enthalpy and entropy production through portions of the reaction cycle as a function of temperature. We find that affinity of irreversible conformational transitions decreases with increasing temperature, resulting in diminishing flux and entropy production. We show that the temperature dependence of the transition affinity is well fit by the Gibbs-Helmholtz relation, allowing the ΔHtrans to be experimentally extracted.

Protein-Bath Coupling of an Internal Reaction Coordinate at Intermediate Time Scales.

Thermally activated barrier-crossing processes are central to protein reaction kinetics. A determining factor for such kinetics is the extent to which the protein's motions are coupled to the surrounding bath. It is understood that slow large-scale conformational motions are strongly coupled to the environment, while fast librational motions are uncoupled. However, less is known about protein-bath coupling of reaction coordinates located on the interior of a protein and with dynamics on intermediate time scales. In this work, we use single molecule 2D fluorescence lifetime correlation spectroscopy to study the microsecond chemical reaction occurring in the chromophore pocket of eGFP. The equilibrium reaction involves a dihedral rotation of a glutamic acid residue and a rearrangement of the local hydrogen-bonding network surrounding the endogenous chromophore, with no accompanying large-scale conformational changes. We observe that the internal chemical reaction is coupled to the solvent viscosity, though the scaling deviates from Kramers' behavior. We attribute this deviation to the internal friction of the protein, which weakens the protein-solvent coupling at high viscosity and intermediate time scales.

Fast and robust two-dimensional inverse Laplace transformation of single-molecule fluorescence lifetime data.

Fluorescence spectroscopy at the single-molecule scale has been indispensable for studying conformational dynamics and rare states of biological macromolecules. Single-molecule two-dimensional (2D) fluorescence lifetime correlation spectroscopy is an emerging technique that holds promise for the study of protein and nucleic acid dynamics, as the technique is 1) capable of resolving conformational dynamics using a single chromophore, 2) resolves forward and reverse transitions independently, and 3) has a dynamic window ranging from microseconds to seconds. However, the calculation of a 2D fluorescence relaxation spectrum requires an inverse Laplace transform (ILT), which is an ill-conditioned inversion that must be estimated numerically through a regularized minimization. Current methods for performing ILTs of fluorescence relaxation can be computationally inefficient, sensitive to noise corruption, and difficult to implement. Here, we adopt an approach developed for NMR spectroscopy (T1-T2 relaxometry) to perform one-dimensional (1D) and 2D-ILTs on single-molecule fluorescence spectroscopy data using singular-valued decomposition and Tikhonov regularization. This approach provides fast, robust, and easy to implement Laplace inversions of single-molecule fluorescence data. We compare this approach to the widely used maximal entropy method.

Phase separation of DNA: From past to present.

Phase separation of biological molecules, such as nucleic acids and proteins, has garnered widespread attention across many fields in recent years. For instance, liquid-liquid phase separation has been implicated not only in membraneless intracellular organization but also in many biochemical processes, including transcription, translation, and cellular signaling. Here, we present a historical background of biological phase separation and survey current work on nuclear organization and its connection to DNA phase separation from the perspective of DNA sequence, structure, and genomic context.

Liquid-Liquid Phase Separation of Histone Proteins in Cells: Role in Chromatin Organization.

Liquid-liquid phase separation (LLPS) of proteins and nucleic acids has emerged as an important phenomenon in membraneless intracellular organization. We demonstrate that the linker histone H1 condenses into liquid-like droplets in the nuclei of HeLa cells. The droplets, observed during the interphase of the cell cycle, are colocalized with DNA-dense regions indicative of heterochromatin. In vitro, H1 readily undergoes LLPS with both DNA and nucleosomes of varying lengths but does not phase separate in the absence of DNA. The nucleosome core particle maintains its structural integrity inside the droplets, as demonstrated by FRET. Unexpectedly, H2A also forms droplets in the presence of DNA and nucleosomes in vitro, whereas the other core histones precipitate. The phase diagram of H1 with nucleosomes is invariant to the nucleosome length at physiological salt concentration, indicating that H1 is capable of partitioning large segments of DNA into liquid-like droplets. Of the proteins tested (H1, core histones, and the heterochromatin protein HP1α), this property is unique to H1. In addition, free nucleotides promote droplet formation of H1 nucleosome in a nucleotide-dependent manner, with droplet formation being most favorable with ATP. Although LLPS of HP1α is known to contribute to the organization of heterochromatin, our results indicate that H1 also plays a role. Based on our study, we propose that H1 and DNA act as scaffolds for phase-separated heterochromatin domains.

Depletion layer dynamics of polyelectrolyte solutions under Poiseuille flow.

Complex liquids flow through channels faster than expected, an effect attributed to the formation of low-viscosity depletion layers at the boundaries. Characterization of depletion layer length scale, concentration, and dynamics has remained elusive due in large part to the lack of suitable real-space experimental techniques. The short length scales associated with depletion layers have traditionally prohibited direct imaging. By overcoming this limitation via adaptations of stimulated emission depletion (STED) microscopy, we directly measure the concentration profile of polymer solutions at a nonadsorbing wall under Poiseuille flow. Using this approach, we 1) confirm the theoretically predicted concentration profile governed by entropically driven depletion, 2) observe depletion layer narrowing at low to intermediate shear rates, and 3) report depletion layer composition that approaches pure solvent at unexpectedly low shear rates.

DNA Local-Flexibility-Dependent Assembly of Phase-Separated Liquid Droplets.

Phase separation of intracellular components has been recently realized as a mechanism by which cells achieve membraneless organization. Here, we study the associative liquid-liquid phase separation (LLPS) of DNA upon complexation with cationic polypeptides. Comparing the phase behavior of different single-stranded DNA as well as double-stranded DNA (dsDNA) sequences that differ in persistence lengths, we find that DNA local flexibility, not simply charge density, determines the LLPS. Furthermore, in a nucleotide- and DNA-dependent manner, free nucleotide triphosphates promote LLPS of polypeptide-dsDNA complexes that are otherwise prone to precipitation. Under these conditions, dsDNA undergoes a secondary phase separation forming liquid-crystalline subcompartments inside the droplets. These results point toward a role of local DNA flexibility, encoded in the sequence, in the regulation and selectivity of multicomponent LLPS in membraneless intracellular organization.

Non-Fickian Molecular Transport in Protein-DNA Droplets.

Bulk-level measurements of dynamics have suggested that phase-separated, protein-nucleic acid rich droplets can be viewed as simple liquids. In this report, we show that histone proteins spontaneously phase separate into liquid-like droplets in the presence of DNA. Using super-resolution fluorescence microscopy, we find that molecular transport in these droplets is non-Fickian (subdiffusive) at nanoscopic length scales. This observation cannot be explained by charge-charge interactions. Instead, our results strongly suggest that cation-π interactions drive the non-Fickian behavior. Given the ubiquity of cationic and aromatic moieties in protein-nucleic acid rich liquid-like phases observed in cells, we anticipate that non-Fickian diffusion is a general transport mechanism in such phases.

Operating organic light-emitting diodes imaged by super-resolution spectroscopy.

Super-resolution stimulated emission depletion (STED) microscopy is adapted here for materials characterization that would not otherwise be possible. With the example of organic light-emitting diodes (OLEDs), spectral imaging with pixel-by-pixel wavelength discrimination allows us to resolve local-chain environment encoded in the spectral response of the semiconducting polymer, and correlate chain packing with local electroluminescence by using externally applied current as the excitation source. We observe nanoscopic defects that would be unresolvable by traditional microscopy. They are revealed in electroluminescence maps in operating OLEDs with 50 nm spatial resolution. We find that brightest emission comes from regions with more densely packed chains. Conventional microscopy of an operating OLED would lack the resolution needed to discriminate these features, while traditional methods to resolve nanoscale features generally cannot be performed when the device is operating. This points the way towards real-time analysis of materials design principles in devices as they actually operate.

Histidine Orientation Modulates the Structure and Dynamics of a de Novo Metalloenzyme Active Site.

The ultrafast dynamics of a de novo metalloenzyme active site is monitored using two-dimensional infrared spectroscopy. The homotrimer of parallel, coiled coil α-helices contains a His3-Cu(I) metal site where CO is bound and serves as a vibrational probe of the hydrophobic interior of the self-assembled complex. The ultrafast spectral dynamics of Cu-CO reveals unprecedented ultrafast (2 ps) nonequilibrium structural rearrangements launched by vibrational excitation of CO. This initial rapid phase is followed by much slower ∼40 ps vibrational relaxation typical of metal-CO vibrations in natural proteins. To identify the hidden coupled coordinate, small molecule analogues and the full peptide were studied by QM and QM/MM calculations, respectively. The calculations show that variation of the histidines' dihedral angles in coordinating Cu controls the coupling between the CO stretch and the Cu-C-O bending coordinates. Analysis of different optimized structures with significantly different electrostatic field magnitudes at the CO ligand site indicates that the origin of the stretch-bend coupling is not directly due to through-space electrostatics. Instead, the large, ∼3.6 D dipole moments of the histidine side chains effectively transduce the electrostatic environment to the local metal coordination orientation. The sensitivity of the first coordination sphere to the protein electrostatics and its role in altering the potential energy surface of the bound ligands suggests that long-range electrostatics can be leveraged to fine-tune function through enzyme design.

Dynamics of rhenium photocatalysts revealed through ultrafast multidimensional spectroscopy.

Rhenium catalysts have shown promise to promote carbon neutrality by reducing a prominent greenhouse gas, CO2, to CO and other starting materials. Much research has focused on identifying intermediates in the photocatalysis mechanism as well as time scales of relevant ultrafast processes. Recent studies have implemented multidimensional spectroscopies to characterize the catalyst's ultrafast dynamics as it undergoes the many steps of its photocycle. Two-dimensional infrared (2D-IR) spectroscopy is a powerful method to obtain molecular structure information while extracting time scales of dynamical processes with ultrafast resolution. Many observables result from 2D-IR experiments including vibrational lifetimes, intramolecular redistribution time scales, and, unique to 2D-IR, spectral diffusion, which is highly sensitive to solute-solvent interactions and motional dynamics. Spectral diffusion, a measure of how long a vibrational mode takes to sample its frequency space due to multiple solvent configurations, has various contributing factors. Properties of the solvent, the solute's structural flexibility, and electronic properties, as well as interactions between the solvent and solute, complicate identifying the origin of the spectral diffusion. With carefully chosen experiments, however, the source of the spectral diffusion can be unveiled. Within the context of a considerable body of previous work, here we discuss the spectral diffusion of several rhenium catalysts at multiple stages in the catalysis. These studies were performed in multiple polar liquids to aid in discovering the contributions of the solvent. We also performed electronic ground state 2D-IR and electronic excited state transient-2D-IR experiments to observe how spectral diffusion changes upon electronic excitation. Our results indicate that with the original Lehn catalyst in THF, relative to the ground state, the spectral diffusion slows by a factor of 3 in the equilibrated triplet metal-to-ligand charge transfer state. We attribute this slowdown to a decrease in dielectric friction as well as an increase in molecular flexibility. It is possible to partially simulate the charge transfer by altering the electron density moderately by adding electron donating or withdrawing substituents symmetrically to the bipyridine ligand. We find that unlike the significant electronic structure change induced by MLCT, such small substituent effects do not influence the spectral diffusion. A solvent study in THF, DMSO, and CH3CN found there to be an explicit solvent dependence that we can correlate to the solvent donicity, which is a measure of its nucleophilicity. Future studies focused on the solvent effects on spectral diffusion in the crucial photoinitiated state can illuminate the role the solvent plays in the catalysis.

Equilibrium excited state dynamics of a photoactivated catalyst measured with ultrafast transient 2DIR.

A detailed understanding of photocatalyzed reaction dynamics requires a sensitive means of investigating the transient catalytically active species. Ideally, the method should be able to compare the electronically excited photocatalyst directly to the ground state species. We use equilibrium and transient two-dimensional infrared (2DIR and t-2DIR) spectroscopy to study the ground and excited state spectral dynamics of [Re(CO)3(bpy)Cl] in tetrahydrofuran (THF). We leverage the long-lived triplet excited state of the molecule to re-establish an equilibrated state relative to intersystem crossing dynamics and external solvent fluctuations, allowing access to the dynamics experienced by the excited state photocatalyst. The decay of frequency correlations within the excited triplet state species differs significantly from the ground state (slower by a factor of 3), indicating that the electronic excitation and subsequent metal-to-ligand charge transfer and associated structural changes are sufficient to perturb the spectral dynamics as sensed by the carbonyl ligands. In addition, we observe a 2-fold slowdown in ground state spectral dynamics around the in-phase symmetric vibrational mode compared to the two lower frequency, out-of-phase symmetric and asymmetric modes. Following electronic absorption and metal-to-ligand charge transfer the symmetry of the vibrational modes are disrupted, and all vibrational modes experience inhomogeneous broadening and spectral diffusion. The qualitative change in broadening mechanisms arises from the charge redistribution, indicating that direct comparisons of vibrational spectral dynamics on different electronic states-reported here for the first time-can be highly sensitive indicators of changes in electronic structure and in the concomitant solvation dynamics that underlie the microscopic details of charge transfer reactions.

Super-resolution study of polymer mobility fluctuations near c*.

Nanoscale dynamic heterogeneities in synthetic polymer solutions are detected using super-resolution optical microscopy. To this end, we map concentration fluctuations in polystyrene-toluene solutions with spatial resolution below the diffraction limit, focusing on critical fluctuations near the polymer overlap concentration, c*. Two-photon super-resolution microscopy was adapted to be applicable in an organic solvent, and a home-built STED-FCS system with stimulated emission depletion (STED) was used to perform fluorescence correlation spectroscopy (FCS). The polystyrene serving as the tracer probe (670 kg mol(-1), radius of gyration RG ≈ 35 nm, end-labeled with a bodipy derivative chromophore) was dissolved in toluene at room temperature (good solvent) and mixed with matrix polystyrene (3,840 kg mol(-1), RG ≈ 97 nm, Mw/Mn = 1.04) whose concentration was varied from dilute to more than 10c*. Whereas for dilute solutions the intensity-intensity correlation function follows a single diffusion process, it splits starting at c* to imply an additional relaxation process provided that the experimental focal area does not greatly exceed the polymer blob size. We identify the slower mode as self-diffusion and the increasingly rapid mode as correlated segment fluctuations that reflect the cooperative diffusion coefficient, Dcoop. These real-space measurements find quantitative agreement between correlation lengths inferred from dynamic measurements and those from determining the limit below which diffusion coefficients are independent of spot size. This study is considered to illustrate the potential of importing into polymer science the techniques of super-resolution imaging.

Heterogeneous preferential solvation of water and trifluoroethanol in homologous lysozymes.

Cytoplasmic osmolytes can significantly alter the thermodynamic and kinetic properties of proteins relative to those under dilute solution conditions. Spectroscopic experiments of lysozymes in cosolvents indicate that such changes may arise from the heterogeneous, site-specific hydrophobic interactions between protein surface residues and individual solvent molecules. In pursuit of an accurate and predictive model for explaining biomolecular interactions, we study the averaged structural characteristics of mixed solvents with homologous lysozyme solutes using all-atom molecular dynamics. By observing the time-averaged densities of different aqueous solutions of trifluoroethanol, we deduce trends in the heterogeneous solvent interactions over each protein's surface, and investigate how the homology of protein structure does not necessarily translate to similarities in solvent structure and composition-even when observing identical side chains.

Crowding induced collective hydration of biological macromolecules over extended distances.

Ultrafast two-dimensional infrared (2D-IR) spectroscopy reveals picosecond protein and hydration dynamics of crowded hen egg white lysozyme (HEWL) labeled with a metal-carbonyl vibrational probe covalently attached to a solvent accessible His residue. HEWL is systematically crowded alternatively with polyethylene glycol (PEG) or excess lysozyme in order to distinguish the chemically inert polymer from the complex electrostatic profile of the protein crowder. The results are threefold: (1) A sharp dynamical jamming-like transition is observed in the picosecond protein and hydration dynamics that is attributed to an independent-to-collective hydration transition induced by macromolecular crowding that slows the hydration dynamics up to an order of magnitude relative to bulk water. (2) The interprotein distance at which the transition occurs suggests collective hydration of proteins over distances of 30-40 Å. (3) Comparing the crowding effects of PEG400 to our previously reported experiments using glycerol exposes fundamental differences between small and macromolecular crowding agents.

Ultrafast 2DIR probe of a host-guest inclusion complex: structural and dynamical constraints of nanoconfinement.

Two-dimensional infrared (2DIR) spectroscopy is used to study the influence of nanoconfinement on the spectral diffusion dynamics of cyclopentadienyl manganese tricarbonyl (CpMn(CO)3, CMT) free in solution and confined in the cavity of ß-cyclodextrin. Contrary to the reorientation correlation function of the solvent molecules, determined through molecular dynamics simulations, measurements in three different solvents indicate that CMT confined in ß-cyclodextrin undergoes spectral diffusion that is faster than free CMT. To account for this discrepancy, we propose that spectral diffusion time scales contain a dynamical contribution that is dependent on the effective size of the conformational space presented by the solvation environment. This solvation state space size is related to the number of participating solvent molecules, which in turn is proportional to the solvent accessible surface area (SASA). We test the role of the number of participating solvent molecules using a simple Gaussian-Markov simulation and find that an increase in the number of participating solvent molecules indeed slows the time required to search the available conformational space. Finally, we test this dependence by comparing the spectral diffusion of a previously studied manganese carbonyl, dimanganese decacarbonyl (Mn2(CO)10, DMDC), to CMT and find that DMDC, which has a larger SASA, exhibits slower spectral diffusion. The experimental observations and the supporting simplistic solvation model suggest that vibrational probe molecules, such as CMT, might be able to function as sensors of conformational entropy.

Site-specific coupling of hydration water and protein flexibility studied in solution with ultrafast 2D-IR spectroscopy.

There is considerable evidence for the slaving of biomolecular dynamics to the motions of the surrounding solvent environment, but to date there have been few direct experimental measurements capable of site-selectively probing both the dynamics of the water and the protein with ultrafast time resolution. Here, two-dimensional infrared spectroscopy (2D-IR) is used to study the ultrafast hydration and protein dynamics sensed by a metal carbonyl vibrational probe covalently attached to the surface of hen egg white lysozyme dissolved in D(2)O/glycerol solutions. Surface labeling provides direct access to the dynamics at the protein-water interface, where both the hydration and the protein dynamics can be observed simultaneously through the vibrational probe's frequency-frequency correlation function. In pure D(2)O, the correlation function shows a fast initial 3 ps decay corresponding to fluctuations of the hydration water, followed by a significant static offset attributed to fluctuations of the protein that are not sampled within the <20 ps experimental window. Adding glycerol increases the bulk solvent viscosity while leaving the protein structurally intact and hydrated. The hydration dynamics exhibit a greater than 3-fold slowdown between 0 and 80% glycerol (v/v), and the contribution from the protein's dynamics is found to slow in a nearly identical fashion. In addition, the magnitude of the dynamic slowdown associated with hydrophobic hydration is directly measured and shows quantitative agreement with predictions from molecular dynamics simulations.

Ultrafast α-like relaxation of a fragile glass-forming liquid measured using two-dimensional infrared spectroscopy.

Ultrafast two-dimensional infrared (2D-IR) spectroscopy is used to study the picosecond dynamics of a vibrational probe molecule dissolved in a fragile glass former. The spectral dynamics are observed as the system is cooled to within a few degrees of the glass transition temperature (T(g)). We observe nonexponential relaxation of the frequency-frequency correlation function, similar to what has been reported for other dynamical correlation functions. In addition, we see evidence for α-like relaxation, typically associated with long-time, cooperative molecular motion, on the ultrafast time scale. The data suggests that the spectral dynamics are sensitive to cooperative motion occurring on time scales that are necessarily longer than the observation time.

Site-specific hydration dynamics of globular proteins and the role of constrained water in solvent exchange with amphiphilic cosolvents.

The thermodynamic driving forces for protein folding, association, and function are often determined by protein-water interactions. With a novel covalently bound labeling approach, we have used sensitive vibrational probes, site-selectively conjugated to two lysozyme variants-in conjunction with ultrafast two-dimensional infrared (2D-IR) spectroscopy-to investigate directly the protein-water interface. By probing alternatively a topologically flat, rigid domain and a flexible domain, we find direct experimental evidence for spatially heterogeneous hydration dynamics. The hydration environment around globular proteins can vary from exhibiting bulk-like hydration dynamics to dynamically constrained water, which results from stifled hydrogen bond switching dynamics near extended hydrophobic surfaces. Furthermore, we leverage preferential solvation exchange to demonstrate that the liberation of dynamically constrained water is a sufficient driving force for protein-surface association reactions. These results provide an intuitive picture of the dynamic aspects of hydrophobic hydration of proteins, illustrating an essential function of water in biological processes.