Raman spectroscopy in the study of amyloid formation and phase separation.

Neurodegenerative diseases, such as Alzheimer's and Parkinson's, share a common pathological feature of amyloid structure accumulation. However, the structure-function relationship between these well-ordered, ß-sheet-rich, filamentous protein deposits and disease etiology remains to be defined. Recently, an emerging hypothesis has linked phase separation, a process involved in the formation of protein condensates, to amyloid formation, suggesting that liquid protein droplets serve as loci for amyloid initiation. To elucidate how these processes contribute to disease progression, tools that can directly report on protein secondary structural changes are needed. Here, we review recent studies that have demonstrated Raman spectroscopy as a powerful vibrational technique for interrogating amyloid structures; one that offers sensitivity from the global secondary structural level to specific residues. This probe-free technique is further enhanced via coupling to a microscope, which affords structural data with spatial resolution, known as Raman spectral imaging (RSI). In vitro and in cellulo applications of RSI are discussed, highlighting studies of protein droplet aging, cellular internalization of fibrils, and Raman imaging of intracellular water. Collectively, utilization of the myriad Raman spectroscopic methods will contribute to a deeper understanding of protein conformational dynamics in the complex cellular milieu and offer potential clinical diagnostic capabilities for protein misfolding and aggregation processes in disease states.

Entropy Tug-of-War Determines Solvent Effects in the Liquid-Liquid Phase Separation of a Globular Protein.

Liquid-liquid phase separation (LLPS) plays a key role in the compartmentalization of cells via the formation of biomolecular condensates. Here, we combined atomistic molecular dynamics (MD) simulations and terahertz (THz) spectroscopy to determine the solvent entropy contribution to the formation of condensates of the human eye lens protein γD-Crystallin. The MD simulations reveal an entropy tug-of-war between water molecules that are released from the protein droplets and those that are retained within the condensates, two categories of water molecules that were also assigned spectroscopically. A recently developed THz-calorimetry method enables quantitative comparison of the experimental and computational entropy changes of the released water molecules. The strong correlation mutually validates the two approaches and opens the way to a detailed atomic-level understanding of the different driving forces underlying the LLPS.

Hydration makes a difference! How to tune protein complexes between liquid-liquid and liquid-solid phase separation.

Understanding how protein rich condensates formed upon liquid-liquid phase separation (LLPS) evolve into solid aggregates is of fundamental importance for several medical applications, since these are suspected to be hot-spots for many neurotoxic diseases. This requires developing experimental approaches to observe in real-time both LLPS and liquid-solid phase separation (LSPS), and to unravel the delicate balance of protein and water interactions dictating the free energy differences between the two. We present a vibrational THz spectroscopy approach that allows doing so from the point of view of hydration water. We focus on a cellular prion protein of high medical relevance, which we can drive to undergo either LLPS or LSPS with few mutations. We find that it is a subtle balance of hydrophobic and hydrophilic solvation contributions that allows tuning between LLPS and LSPS. Hydrophobic hydration provides an entropic driving force to phase separation, through the release of hydration water into the bulk. Water hydrating hydrophilic groups provides an enthalpic driving force to keep the condensates in a liquid state. As a result, when we modify the protein by a few mutations to be less hydrophilic, we shift from LLPS to LSPS. This molecular understanding paves the way for a rational design of proteins.

Water bend-libration as a cellular Raman imaging probe of hydration.

Water is a ubiquitous and vital component of living systems. Hydration, which is the interaction between water and intracellular biomolecules, plays an important role in cellular processes. However, it is technically challenging to study water structure within cells directly. Here, we demonstrate the utility and power of the water bend-libration combination band as a unique Raman spectral imaging probe of cellular hydration. Hydration maps reveal distinct water environments within subcellular compartments (e.g., nucleolus and lipid droplet) due to the spectral sensitivity of this coupled vibrational band. Spectroscopic studies using the water bend-libration are broadly applicable, offering the potential to capture the chemical complexity of hydration in numerous systems.

Real-time measure of solvation free energy changes upon liquid-liquid phase separation of α-elastin.

Biological condensates are known to retain a large fraction of water to remain in a liquid and reversible state. Local solvation contributions from water hydrating hydrophilic and hydrophobic protein surfaces were proposed to play a prominent role for the formation of condensates through liquid-liquid phase separation (LLPS). However, although the total free energy is accessible by calorimetry, the partial solvent contributions to the free energy changes upon LLPS remained experimentally inaccessible so far. Here, we show that the recently developed THz calorimetry approach allows to quantify local hydration enthalpy and entropy changes upon LLPS of α-elastin in real time, directly from experimental THz spectroscopy data. We find that hydrophobic solvation dominates the entropic solvation term, whereas hydrophilic solvation mainly contributes to the enthalpy. Both terms are in the order of hundreds of kJ/mol, which is more than one order of magnitude larger than the total free energy changes at play during LLPS. However, since we show that entropy/enthalpy mostly compensates, a small entropy/enthalpy imbalance is sufficient to tune LLPS. Theoretically, a balance was proposed before. Here we present experimental evidence based on our spectroscopic approach. We finally show that LLPS can be steered by inducing small changes of solvation entropy/enthalpy compensation via concentration or temperature in α-elastin.

Liquid-Liquid Phase Separation? Ask the Water!

Water is more than an inert spectator during liquid-liquid phase separation (LLPS), the reversible compartmentalization of protein solutions into a protein-rich and a dilute phase. We show that LLPS is driven by changes in hydration entropy and enthalpy. Tuning LLPS by adjusting experimental parameters, e.g., addition of co-solutes, is a major goal for biological and medical applications. This requires a general model to quantify thermodynamic driving forces. Here, we develop such a model based on the measured amplitudes of characteristic THz-features of two hydration populations: "Cavity-wrap" water hydrating hydrophobic patches is released during LLPS leading to an increase in entropy. "Bound" water hydrating hydrophilic patches is retained since it is enthalpically favorable. We introduce a THz-phase diagram mapping these spectroscopic/thermodynamic changes. This provides not only a precise understanding of hydrophobic and hydrophilic hydration driving forces as a function of temperature and concentration but also a rational means to tune LLPS.

Observation of dissipating solvated protons upon hydrogel formation.

Aqueous hyaluronan solutions form an elastic hydrogel within a narrow pH range, around pH 2.4, making this a model system to study the conformational changes of the hydrogen bond network upon gelation. This pH-dependent behavior allows us to probe water surrounding a biologically relevant molecule in different environments (liquid versus elastic state) which change due to an environmental stimulus. Here, we use Terahertz (THz) reflection absorption spectroscopy in attenuated total reflection (ATR) geometry as a tool to study gelation. THz spectroscopy is sensitive to changes in the hydrogen-bonded water network, and here we show that we can correlate changes in macroscopic properties to changes in the solvation of hyaluronan. Above and below the gelation pH, solvated protons are present in the solutions, however, this spectral signature is completely absent between pH 2.4-2.8, which is the pH at which hyaluronan forms a hydrogel. We propose that solvated protons are forming ion pairs with hyaluronan in this pH range. Adding urea or glucose to hyaluronan solutions changes their elasticity, in which an increase or decrease in elasticity can be linked to the formation and destruction of these ion pairs, respectively.

Spectroscopic Fingerprints of Cavity Formation and Solute Insertion as a Measure of Hydration Entropic Loss and Enthalpic Gain.

Hydration free energies are dictated by a subtle balance of hydrophobic and hydrophilic interactions. We present here a spectroscopic approach, which gives direct access to the two main contributions: Using THz-spectroscopy to probe the frequency range of the intermolecular stretch (150-200 cm-1 ) and the hindered rotations (450-600 cm-1 ), the local contributions due to cavity formation and hydrophilic interactions can be traced back. We show that via THz calorimetry these fingerprints can be correlated 1 : 1 with the group specific solvation entropy and enthalpy. This allows to deduce separately the hydrophobic (i.e. cavity formation) and hydrophilic contributions to thermodynamics, as shown for hydrated alcohols as a case study. Accompanying molecular dynamics simulations quantitatively support our experimental results. In the future our approach will allow to dissect hydration contributions in inhomogeneous mixtures and under non-equilibrium conditions.

Active Site Hydrogen Bonding Induced in Cytochrome P450cam by Effector Putidaredoxin.

Cytochrome P450s are diverse and powerful catalysts that can activate molecular oxygen to oxidize a wide variety of substrates. Catalysis relies on effective uptake of two electrons and two protons. For cytochrome P450cam, an archetypal member of the superfamily, the second electron must be supplied by the redox partner putidaredoxin (Pdx). Pdx also plays an effector role beyond electron transfer, but after decades the mechanism remains under investigation. We applied infrared spectroscopy to heme-ligated CN- to examine the influence of Pdx binding. The results indicate that Pdx induces the population of a conformation wherein the CN- ligand forms a strong hydrogen bond to a solvent water molecule, experimentally corroborating the formation of a proposed proton delivery network. Further, characterization of T252A P450cam implicates the side chain of Thr252 in regulating the population equilibrium of hydrogen-bonded states within the P450cam/Pdx complex, which could underlie its role in directing activated oxygen toward product formation and preventing reaction uncoupling through peroxide release.

Dynamics underlying hydroxylation selectivity of cytochrome P450cam.

Structural heterogeneity and the dynamics of the complexes of enzymes with substrates can determine the selectivity of catalysis; however, fully characterizing how remains challenging as heterogeneity and dynamics can vary at the spatial level of an amino acid residue and involve rapid timescales. We demonstrate the nascent approach of site-specific two-dimensional infrared (IR) spectroscopy to investigate the archetypical cytochrome P450, P450cam, to better delineate the mechanism of the lower regioselectivity of hydroxylation of the substrate norcamphor in comparison to the native substrate camphor. Specific locations are targeted throughout the enzyme by selectively introducing cyano groups that have frequencies in a spectrally isolated region of the protein IR spectrum as local vibrational probes. Linear and two-dimensional IR spectroscopy were applied to measure the heterogeneity and dynamics at each probe and investigate how they differentiate camphor and norcamphor recognition. The IR data indicate that the norcamphor complex does not fully induce a large-scale conformational change to a closed state of the enzyme adopted in the camphor complex. Additionally, a probe directed at the bound substrate experiences rapidly interconverting states in the norcamphor complex that explain the hydroxylation product distribution. Altogether, the study reveals large- and small-scale structural heterogeneity and dynamics that could contribute to selectivity of a cytochrome P450 and illustrates the approach of site-selective IR spectroscopy to elucidate protein dynamics.

Site-Specific 1D and 2D IR Spectroscopy to Characterize the Conformations and Dynamics of Protein Molecular Recognition.

Proteins exist as ensembles of interconverting states on a complex energy landscape. A complete, molecular-level understanding of their function requires knowledge of the populated states and thus the experimental tools to characterize them. Infrared (IR) spectroscopy has an inherently fast time scale that can capture all states and their dynamics with, in principle, bond-specific spatial resolution, and 2D IR methods that provide richer information are becoming more routine. Although application of IR spectroscopy for investigation of proteins is challenged by spectral congestion, the issue can be overcome by site-specific introduction of amino acid side chains that have IR probe groups with frequency-resolved absorptions, which furthermore enables selective characterization of different locations in proteins. Here, we briefly introduce the biophysical methods and summarize the current progress toward the study of proteins. We then describe our efforts to apply site-specific 1D and 2D IR spectroscopy toward elucidation of protein conformations and dynamics to investigate their involvement in protein molecular recognition, in particular mediated by dynamic complexes: plastocyanin and its binding partner cytochrome f, cytochrome P450s and substrates or redox partners, and Src homology 3 domains and proline-rich peptide motifs. We highlight the advantages of frequency-resolved probes to characterize specific, local sites in proteins and uncover variation among different locations, as well as the advantage of the fast time scale of IR spectroscopy to detect rapidly interconverting states. In addition, we illustrate the greater insight provided by 2D methods and discuss potential routes for further advancement of the field of biomolecular 2D IR spectroscopy.

Heterogeneous and Highly Dynamic Interface in Plastocyanin-Cytochrome f Complex Revealed by Site-Specific 2D-IR Spectroscopy.

Transient protein complexes are crucial for sustaining dynamic cellular processes. The complexes of electron-transfer proteins are a notable example, such as those formed by plastocyanin (Pc) and cytochrome f (cyt f) in the photosynthetic apparatus. The dynamic and heterogeneous nature of these complexes, however, makes their study challenging. To better elucidate the complex of Nostoc Pc and cyt f, 2D-IR spectroscopy coupled to site-specific labeling with cyanophenylalanine infrared (IR) probes was employed to characterize how the local environments at sites along the surface of Pc were impacted by cyt f binding. The results indicate that Pc most substantially engages with cyt f via the hydrophobic patch around the copper redox site. Complexation with cyt f led to an increase in inhomogeneous broadening of the probe absorptions, reflective of increased heterogeneity of interactions with their environment. Notably, most of the underlying states interconverted very rapidly (1 to 2 ps), suggesting a complex with a highly mobile interface. The data support a model of the complex consisting of a large population of an encounter complex. Additionally, the study demonstrates the application of 2D-IR spectroscopy with site-specifically introduced probes to reveal new quantitative insight about dynamic biochemical systems.

Site-specific 2D IR spectroscopy: a general approach for the characterization of protein dynamics with high spatial and temporal resolution.

The conformational heterogeneity and dynamics of protein side chains contribute to function, but investigating exactly how is hindered by experimental challenges arising from the fast timescales involved and the spatial heterogeneity of protein structures. The potential of two-dimensional infrared (2D IR) spectroscopy for measuring conformational heterogeneity and dynamics with unprecedented spatial and temporal resolution has motivated extensive effort to develop amino acids with functional groups that have frequency-resolved absorptions to serve as probes of their protein microenvironments. We demonstrate the full advantage of the approach by selective incorporation of the probe p-cyanophenylalanine at six distinct sites in a Src homology 3 domain and the application of 2D IR spectroscopy to site-specifically characterize heterogeneity and dynamics and their contribution to cognate ligand binding. The approach revealed a wide range of microenvironments and distinct responses to ligand binding, including at the three adjacent, conserved aromatic residues that form the recognition surface of the protein. Molecular dynamics simulations performed for all the labeled proteins provide insight into the underlying heterogeneity and dynamics. Similar application of 2D IR spectroscopy and site-selective probe incorporation will allow for the characterization of heterogeneity and dynamics of other proteins, how heterogeneity and dynamics are affected by solvation and local structure, and how they might contribute to biological function.

Conformational Change Induced by Putidaredoxin Binding to Ferrous CO-ligated Cytochrome P450cam Characterized by 2D IR Spectroscopy.

The importance of conformational dynamics to protein function is now well-appreciated. An outstanding question is whether they are involved in the effector role played by putidaredoxin (Pdx) in its reduction of the O2 complex of cytochrome P450cam (P450cam), an archetypical member of the cytochrome P450 superfamily. Recent studies have reported that binding of Pdx induces a conformational change from a closed to an open state of ferric P450cam, but a similar conformational change does not appear to occur for the ferrous, CO-ligated enzyme. To better understand the effector role of Pdx when binding the ferrous, CO-ligated P450cam, we applied 2D IR spectroscopy to compare the conformations and dynamics of the wild-type (wt) enzyme in the absence and presence of Pdx, as well as of L358P P450cam (L358P), which has served as a putative model for the Pdx complex. The CO vibrations of the Pdx complex and L358P report population of two conformational states in which the CO experiences distinct environments. The dynamics among the CO frequencies indicate that the energy landscape of substates within one conformation are reflective of the closed state of P450cam, and for the other conformation, differ from the free wt enzyme, but are equivalent between the Pdx complex and L358P. The two states co-populated by the Pdx complex are postulated to reflect a loosely bound encounter complex and a more tightly bound state, as is commonly observed for the dynamic complexes of redox partners. Significantly, this study shows that the binding of Pdx to ferrous, CO-ligated P450cam does perturb the conformational ensemble in a way that might underlie the effector role of Pdx.

Evaluation of p-(13C,15N-Cyano)phenylalanine as an Extended Time Scale 2D IR Probe of Proteins.

Two-dimensional infrared (2D IR) spectroscopy provides a powerful approach for the direct study of molecular dynamics with high spatial and temporal resolution. Its application for investigating specific locations in proteins requires the incorporation of IR probe groups with spectrally isolated absorptions to avoid the congestion inherent to protein spectra. This has motivated extensive efforts toward the development of new IR probes, but there remains a need for those that can extend the experimental time range, which is limited by their vibrational lifetimes. Toward this goal, isotopically labeled p-(13C15N-cyano)phenylalanine was synthesized, site-selectively incorporated into the protein plastocyanin, and evaluated for its potential as a 2D IR probe. The isotopic labeling increases the vibrational lifetime about 2-fold, which results in larger signals at longer time scales. However, isotopic labeling simultaneously shifts the absorption to a spectral region with greater water absorbance, which results in greater heating-induced signals in the background that overlap those of the nitrile probe. The study demonstrates the use of a new 2D IR probe to measure the side chain dynamics in a protein and also illustrates the multiple factors to consider in development of 2D IR probes for studying proteins.