Buried Residues Research Articles

The conformational landscape of fold-switcher KaiB is tuned to the circadian rhythm timescale

How can a single protein domain encode a conformational landscape with multiple stably folded states, and how do those states interconvert? Here, we use real-time and relaxation-dispersion NMR to characterize the conformational landscape of the circadian rhythm protein KaiB from Rhodobacter sphaeroides. Unique among known natural metamorphic proteins, this KaiB variant spontaneously interconverts between two monomeric states: the "Ground" and "Fold-switched" (FS) states. KaiB in its FS state interacts with multiple binding partners, including the central KaiC protein, to regulate circadian rhythms. We find that KaiB itself takes hours to interconvert between the Ground and FS state, underscoring the ability of a single-sequence to encode the slow process needed for function. We reveal the rate-limiting step between the Ground and FS state is the cis-trans isomerization of three prolines in the fold-switching region by demonstrating interconversion acceleration by the prolyl isomerase Cyclophilin A. The interconversion proceeds through a "partially disordered" (PD) state, where the C-terminal half becomes disordered while the N-terminal half remains stably folded. We found two additional properties of KaiB's landscape. First, the Ground state experiences cold denaturation: At 4 °C, the PD state becomes the majorly populated state. Second, the Ground state exchanges with a fourth state, the "Enigma" state, on the millisecond-timescale. We combine AlphaFold2-based predictions and NMR chemical shift predictions to predict this Enigma state is a beta-strand register shift that relieves buried charged residues, and support this structure experimentally. These results provide mechanistic insight into how evolution can design a single-sequence that achieves specific timing needed for its function.

Effect of pH-Shift Treatment on IgE-Binding Capacity and Conformational Structures of Peanut Protein.

Hypoallergenic processing is an area worthy of continued exploration. In the treatment of the peanut protein (PP), pH shift was applied by acidic (pH 1.0-4.0) and alkaline (pH 9.0-12.0) treatment, after which the pH was adjusted to 7.0. Following pH-shift treatment, PP showed a larger particle size than in neutral solutions. SDS-PAGE, CD analysis, intrinsic fluorescence, UV spectra, and surface hydrophobicity indicated the protein conformation was unfolded with the exposure of more buried hydrophobic residues. Additionally, the IgE-binding capacity of PP decreased after pH-shift treatment on both sides. Label-free LC-MS/MS results demonstrated that the pH-shift treatment induced the structural changes on allergens, which altered the abundance of peptides after tryptic digestion. Less linear IgE-binding epitopes were detected in PP with pH-shift treatment. Our results suggested the pH-shift treatment is a promising alternative approach in the peanut hypoallergenic processing. This study also provides a theoretical basis for the development of hypoallergenic food processing.

Dissecting diazirine photo-reaction mechanism for protein residue-specific cross-linking and distance mapping

While photo-cross-linking (PXL) with alkyl diazirines can provide stringent distance restraints and offer insights into protein structures, unambiguous identification of cross-linked residues hinders data interpretation to the same level that has been achieved with chemical cross-linking (CXL). We address this challenge by developing an in-line system with systematic modulation of light intensity and irradiation time, which allows for a quantitative evaluation of diazirine photolysis and photo-reaction mechanism. Our results reveal a two-step pathway with mainly sequential generation of diazo and carbene intermediates. Diazo intermediate preferentially targets buried polar residues, many of which are inaccessible with known CXL probes for their limited reactivity. Moreover, we demonstrate that tuning light intensity and duration enhances selectivity towards polar residues by biasing diazo-mediated cross-linking reactions over carbene ones. This mechanistic dissection unlocks the full potential of PXL, paving the way for accurate distance mapping against protein structures and ultimately, unveiling protein dynamic behaviors.

The conformational landscape of fold-switcher KaiB is tuned to the circadian rhythm timescale.

How can a single protein domain encode a conformational landscape with multiple stably-folded states, and how do those states interconvert? Here, we use real-time and relaxation-dispersion NMR to characterize the conformational landscape of the circadian rhythm protein KaiB from Rhodobacter sphaeroides. Unique among known natural metamorphic proteins, this variant of KaiB spontaneously interconverts between two monomeric states: the "Ground" and "Fold-switched" (FS) state. KaiB in its FS state interacts with multiple binding partners, including the central KaiC protein, to regulate circadian rhythms. We find that KaiB itself takes hours to interconvert between the Ground and KaiC binding-competent FS state, underscoring the ability of a single protein sequence to encode the slow process needed for its biological function. We reveal that the rate-limiting step between the Ground and FS state is the cis-trans isomerization of three prolines in the C-terminal fold-switching region by demonstrating acceleration of interconversion by the prolyl cis/trans isomerase CypA. The interconversion proceeds through a "partially disordered" (PD) state, where the N-terminal half remains stably folded while the C-terminal half becomes disordered. We discovered two additional properties of KaiB's landscape: firstly, the Ground state experiences cold denaturation: at 4°C, the PD state becomes the majorly populated state. Secondly, the Ground state exchanges with a fourth state, the "Enigma" state, on the millisecond timescale. We combine AlphaFold2-based predictions and NMR chemical shift predictions to suggest that this "Enigma" state represents a beta-strand register shift that eases buried charged residues in the Ground state. These results provide mechanistic insight in how evolution can design a single sequence that achieves specific timing needed for its function.

A unified analysis of evolutionary and population constraint in protein domains highlights structural features and pathogenic sites

Protein evolution is constrained by structure and function, creating patterns in residue conservation that are routinely exploited to predict structure and other features. Similar constraints should affect variation across individuals, but it is only with the growth of human population sequencing that this has been tested at scale. Now, human population constraint has established applications in pathogenicity prediction, but it has not yet been explored for structural inference. Here, we map 2.4 million population variants to 5885 protein families and quantify residue-level constraint with a new Missense Enrichment Score (MES). Analysis of 61,214 structures from the PDB spanning 3661 families shows that missense depleted sites are enriched in buried residues or those involved in small-molecule or protein binding. MES is complementary to evolutionary conservation and a combined analysis allows a new classification of residues according to a conservation plane. This approach finds functional residues that are evolutionarily diverse, which can be related to specificity, as well as family-wide conserved sites that are critical for folding or function. We also find a possible contrast between lethal and non-lethal pathogenic sites, and a surprising clinical variant hot spot at a subset of missense enriched positions.

Elucidating the microscopic origin of observed anti-correlation between the protein-protein and protein-water interaction energies

Water as a solvent plays an important role in dictating the structure and dynamics of the large biomolecules like proteins. It has been proposed that the protein dynamics and energetics are closely coupled to the water molecules in the hydration shell. In particular, it has been shown earlier that the fluctuations in intra-protein (protein-protein) interaction energy is strongly anti-correlated with the protein-water interaction energy (solvation energy). Moreover, the dynamical signatures of water gets reflected into the protein dynamics in terms of a dominant power law behavior. In this work, we have carried out systematic molecular dynamics (MD) simulations of three different protein systems to understand the key physical factors that contribute to such anti-correlations. First, we compare the dynamics in globular folded proteins like Lysozyme with intrinsically disordered peptides (IDPs) like Amyloid beta to show that enhanced conformational fluctuations lead to significantly stronger anti-correlation. Trp-cage miniprotein shows a similar behavior distributed over two sub-ensembles of folded and unfolded states. We also investigate what type of interactions and molecular processes are responsible for the observed anti-correlation. In particular, we observe that the extent of correlation decreases significantly in the order of charged, polar and non-polar amino acid residues. Charged residues that form salt bridge interactions show a dominant anti-correlation. Surprisingly, a few non-polar (hydrophobic and buried) residues demonstrate significant anti-correlation as well. We attribute this to the structural fluctuations that lead to an equilibrium between buried and solvent exposed states of these residues, where the protein-protein and protein-water interactions compensate each other. Our studies indicate that the molecular origin of the observed anti-correlation can be vary depending on the type of the interaction. The overall coupling/correlation is dominated by the electrostatic interactions and charged residues due to a general dielectric screening effect, whereas a few buried hydrophobic residues may also show such coupling due to structural dynamics between buried and exposed states.

Triplet state spectroscopy reveals involvement of the buried tryptophan residue 310 in Glyceraldehyde-3-phosphate dehydrogenase (GAPD) in the interaction with acrylamide

Using conventional steady state and time resolved fluorescence study of the interaction between a multi-tryptophan protein and a quencher, it is difficult, if not impossible to identify the particular tryptophan residue/residues involved in the interaction. In this work we have exemplified the above contention using a multi-tryptophan protein, Glyceraldehyde-3-phosphate dehydrogenase (GAPD) from rabbit muscle having three tryptophan (Trp) residues at positions 84, 193 and 310 and a neutral quencher acrylamide in Tris buffer of pH 7.5. From the steady state and time resolved fluorescence quenching (at 298 K) with acrylamide Ksv, K and kq for the system have been calculated. Low temperature phosphorescence (LTP) spectra at 77 K of GAPD in suitable cryosolvent is known to exhibit two (0,0) bands corresponding to two tryptophan residues 193 and 310. Using the LTP study of free GAPD and GAPD – acrylamide it is possible to identify that the buried Trp 310 residue is specifically involved in the interaction with acrylamide. This is possible without doing any site-directed mutagenesis of GAPD which contains Trp residues at 84, 193 and 310. Tyrosine 320 is also specifically quenched. The results have been corroborated using the molecular docking studies. Molecular Dynamics simulation supports our contention of the involvement of Trp 310 and also shows that the other nearest residues of acrylamide are Val175 and Val232.

Surface exposed and charged residues drive thermostability in fungi.

Fungi, though mesophilic, include thermophilic and thermostable species, as well. The thermostability of proteins observed in these fungi is most likely to be attributed to several molecular factors, such as the presence of salt bridges and hydrogen bond interactions between side chains. These factors cannot be generalized for all fungi. Factors impacting thermostability can guide how fungal thermophilic proteins gain thermostability. We curated a dataset of proteins for 14 thermophilic fungi and their evolutionarily closer mesophiles. Additionally, the proteome of Chaetomium thermophilum and its evolutionarily related mesophile Chaetomium globosum was analyzed. Using eggNOG, we categorized the proteomes into clusters of orthologous groups (COGs). While the individual count of proteins is over-represented in mesophiles (for COGs S, G, L, and Q), there are certain features that are significantly enriched in thermophiles (such as charged residues, exposed residues, polar residues, etc.). Since fungi are known to be cellulolytic and chitinolytic by nature, we selected 37 existing carbohydrate-active enzymes (CAZyme) families in Eurotiales, Mucorales, and Sordariales. We looked at closely similar sequences and their modeled structures for further comparison. Comparing solvent accessibilities of thermophilic and mesophilic proteins, exposed and intermediate residues are observed higher in thermophiles whereas buried residues are observed higher in mesophiles. For specific five CAZYme families (GH7, GH11, GH18, GH45, and CBM1) we looked at position-specific substitutions between thermophiles and mesophiles. We also found that there are relatively more intramolecular interactions in thermophiles compared to mesophiles. Thus, we found factors such as surface exposed residues and charged residues that are highly likely to impart thermostability in fungi, and this study sets the stage for further studies in the area of fungal thermostability.

Effect of ultrasonic assisted grafting on the structural and functional properties of mung bean protein isolate conjugated with maltodextrin through maillard reaction

Four different methods of maillard reaction including ultrasound (150 W, 10 min) assisted, classical wet heating (80 °C, 60min), moderate water bath heating (60°C, 12 to 30 h) and dry state method (60 °C, 79 % relative humidity and 48 h) were used to Mung bean protein isolate – Maltodexrtin conjugates (MPI-MD) preparation. The samples prepared under ultrasound and wet heating were chosen for further analysis according to degree of graft and UV-absorbance at 420 nm. Higher glycosylation at short time and lower browning were obtained under ultrasound treatment. Covalent attachment in conjugates confirmed by SDS-polyacrylamide gel electrophoresis. The structural analysis revealed prominent unfolding effect of ultrasound waves on the protein's molecules. The decrease of α-helix content was related to the exposure of buried amino group residues during reaction. Glycation of MPI under ultrasound caused changes in tertiary structure of protein and leads to decrease in the fluorescence intensity compared with native and wet heating treatments. FTIR spectra confirmed the conjugation of the MPI and MD and suggested that protein structure was changed and ultrasound promoted the graft reaction more than wet heating treatment. Conjugated MPI showed higher emulsification and solubility index than MPI, moreover the effect of ultrasonic waves on ameliorated functional properties was impressive than those for wet heating treatment. Overall, this study showed use of ultrasonication in maillard reaction was a suitable method for producing MPI- MD conjugates and improved the efficiency of graft reaction and functional properties of grafts.

Influence of linoleic acid on the immunodetection of shrimp (Litopenaeus vannamei) tropomyosin and the mechanism investigation via multi-spectroscopic and molecular modeling techniques

The effect of linoleic acid (LA) on the IgG/IgE recognition, in vitro digestibility and immunodetection of shrimp tropomyosin (TM) was investigated. Subsequently, the simultaneous binding of LA-TM was explored using multi-spectroscopic and molecular modeling techniques. Our findings reveled that the addition of LA significantly reduced TM’s IgG/IgE immunoreactivity, digestibility, and immunodetection. Further analysis using multi-spectroscopic and molecular modeling techniques indicated that while TM’s secondary structure remained largely unchanged, its 3-D structure showed significant alterations such as increased particle size and hydrophobic surface area, and a higher number of buried hydrophobic residues exposed due to the binding of LA to TM. These structural changes rendered it difficult for target antibodies and digestive enzymes to interact with related epitopes and cleavage sites buried inside the molecule. The results obtained in this study provide valuable insights into the molecular mechanism of poor immunodetection caused by food matrix interference.

Two sequence- and two structure-based ML models have learned different aspects of protein biochemistry

Deep learning models are seeing increased use as methods to predict mutational effects or allowed mutations in proteins. The models commonly used for these purposes include large language models (LLMs) and 3D Convolutional Neural Networks (CNNs). These two model types have very different architectures and are commonly trained on different representations of proteins. LLMs make use of the transformer architecture and are trained purely on protein sequences whereas 3D CNNs are trained on voxelized representations of local protein structure. While comparable overall prediction accuracies have been reported for both types of models, it is not known to what extent these models make comparable specific predictions and/or generalize protein biochemistry in similar ways. Here, we perform a systematic comparison of two LLMs and two structure-based models (CNNs) and show that the different model types have distinct strengths and weaknesses. The overall prediction accuracies are largely uncorrelated between the sequence- and structure-based models. Overall, the two structure-based models are better at predicting buried aliphatic and hydrophobic residues whereas the two LLMs are better at predicting solvent-exposed polar and charged amino acids. Finally, we find that a combined model that takes the individual model predictions as input can leverage these individual model strengths and results in significantly improved overall prediction accuracy.

Proteome-wide structural analysis identifies warhead- and coverage-specific biases in cysteine-focused chemoproteomics

Covalent drug discovery has undergone a resurgence over the past two decades and reactive cysteine profiling has emerged in parallel as a platform for ligand discovery through on- and off-target profiling; however, the scope of this approach has not been fully explored at the whole-proteome level. We combined AlphaFold2-predicted side-chain accessibilities for >95% of the human proteome with a meta-analysis of eighteen public cysteine profiling datasets, totaling 44,187 unique cysteine residues, revealing accessibility biases in sampled cysteines primarily dictated by warhead chemistry. Analysis of >3.5 million cysteine-fragment interactions further showed that hit elaboration and optimization drives increased bias against buried cysteine residues. Based on these data, we suggest that current profiling approaches cover a small proportion of potential ligandable cysteine residues and propose future directions for increasing coverage, focusing on high-priority residues and depth. All analysis and produced resources are freely available and extendable to other reactive amino acids.

Electrostatic Modulation of Intramolecular and Intermolecular Interactions during the Formation of an Amyloid-like Assembly.

The mechanism of protein aggregation can be broadly viewed as a shift from the native-state stabilizing intramolecular to the aggregated-phase sustaining intermolecular interactions. Understanding the role of electrostatic forces on the extent of modulation of this switch has recently evolved as a topic of monumental significance as protein aggregation has lately been connected to charge modifications of an aging proteome. To decipher the distinctive role of electrostatic forces on the extremely complicated phase separation landscape, we opted for a combined in vitro-in silico approach to ascertain the structure-dynamics-stability-aggregability relationship of the functional tandem RRM domains of the ALS-related protein TDP-43 (TDP-43tRRM), under a bivariate solution condition in terms of pH and salt concentration. Under acidic pH conditions, the native TDP-43tRRM protein creates an aggregation-prone entropically favorable partially unfolded conformational landscape due to enthalpic destabilization caused by the protonation of the buried ionizable residues and consequent overwhelming fluctuations of selective segments of the sequence leading to anti-correlated movements of the two domains of the protein. The evolved fluffy ensemble with a comparatively exposed backbone then easily interacts with incoming protein molecules in the presence of salt via typical amyloid-aggregate-like intermolecular backbone hydrogen bonds with a considerable contribution originating from the dispersion forces. Subsequent exposure to excess salt at low pH conditions expedites the aggregation process via an electrostatic screening mechanism where salt shows preferential binding to the positively charged side chain. The applied target observable-specific approach complementarity unveils the hidden information landscape of an otherwise complex process with unquestionable conviction.

Phosphorylation-Competent Metastable State of Escherichia coli Toxin HipA.

Phosphorylation is a key post-translational modification that alters the functional state of many proteins. The Escherichia coli toxin HipA, which phosphorylates glutamyl-tRNA synthetase and triggers bacterial persistence under stress, becomes inactivated upon autophosphorylation of Ser150. Interestingly, Ser150 is phosphorylation-incompetent in the crystal structure of HipA since it is deeply buried ("in-state"), although in the phosphorylated state it is solvent exposed ("out-state"). To be phosphorylated, a minor population of HipA must exist in the phosphorylation-competent "out-state" (solvent-exposed Ser150), not detected in the crystal structure of unphosphorylated HipA. Here we report a molten-globule-like intermediate of HipA at low urea (∼4 kcal/mol unstable than natively folded HipA). The intermediate is aggregation-prone, consistent with a solvent exposed Ser150 and its two flanking hydrophobic neighbors (Val/Ile) in the "out-state". Molecular dynamics simulations showed the HipA "in-out" pathway to contain multiple free energy minima with an increasing degree of Ser150 solvent exposure with the free energy difference between the "in-state" and the metastable exposed state(s) to be ∼2-2.5 kcal/mol, with unique sets of hydrogen bonds and salt bridges associated with the metastable loop conformations. Together, the data clearly identify the existence of a phosphorylation-competent metastable state of HipA. Our results not only suggest a mechanism of HipA autophosphorylation but also add to a number of recent reports on unrelated protein systems where the common proposed mechanism for phosphorylation of buried residues is their transient exposure even without phosphorylation.

Solution NMR studies reveal novel CLIC4 chaperone activity with respect to profilin-1.

Despite its name, chloride intracellular channel 4 (CLIC4) is a soluble enzyme that can catalyze glutathione-dependent thiol transfers. It is unclear how this enzymatic activity relates to its biological role in remodeling the actin cytoskeleton. Previous studies have shown that CLIC4 interacts with profilin-1, a critical regulator of actin polymerization. We confirmed a direct interaction, 170 ± 20 μM by microscale thermophoresis. However, using solution nuclear magnetic resonance (NMR) spectroscopy, we demonstrate that CLIC4 has a higher affinity for profilin when it is unfolded. Amide hydrogen exchange studies demonstrate that the N- and C-terminal helices of profilin readily unfold, whereas the central β-beta sheet core remains relatively intact. Interaction with CLIC4 increases amide exchange rates throughout profilin, indicating preferential stabilization of partially unfolded intermediates. We note that profilin is glutathionylated in proteomic studies of cells exposed to oxidative stress. Profilin is unfolded by glutathionylation of buried cysteine residues in its N- or C-terminal helices, and this disrupts its ability to bind actin, as measured by pyrene-actin polymerization assay. We are currently attempting to demonstrate that CLIC4 can reverse glutathionylation of profilin-1 and promote its refolding in cells and in vitro.

Constant pH molecular dynamics simulations of membrane proteins.

Environment-driven dynamic protonation of titratable residues in a protein plays a key role in membrane transport and pH response in various biological processes. The dominant protonation state of a titratable amino-acid residue depends on environmental conditions like pH, dielectric environment (ionic strength, proximity to water-membrane interfaces), other amino-acid residues in its vicinity, etc. Constant pH Molecular Dynamics (CpHMD) techniques enable the study of dynamic protonation events in a molecular simulation and can be regarded as an in-silico titration process. In CpHMD techniques, the protonation state of a titratable residue is allowed to propagate between the different ionization states. As the protonation states of different residues change, the charge of the total system fluctuates. In hybrid solvent-based schemes, where the changes in the protonation states are handled using an implicit solvent representation, these charge fluctuations do not pose a problem. However, in case of an explicit-solvent representation, where the electrostatics are handled using a particle mesh ewald (PME) scheme, the varying charge poses a problem in reliable evaluation of energetics and thereby the pKa of the titratable residues. A large aqueous fraction in the simulation box is known to mitigate the charge artifacts. However, compared to soluble proteins, membrane protein simulation systems have a large non-aqueous volume fraction, owing to the lipid membrane. In this study, we compare different CpHMD methodologies for estimation of pKa in membrane proteins. These methods include both an explicit solvent-based CpHMD as well as hybrid solvent-based CpHMD. We present the pKa values of the acidic residues (aspartic acid, glutamic acid) from these simulations. In general, the hybrid solvent-based scheme showed a faster convergence compared to the explicit solvent-based scheme. However, in case of buried residues, the hybrid solvent-based scheme took a longer time to converge.

Analysis of 363 Genetic Variants in F5 via an Interactive Web Database Reveals New Insights into FV Deficiency and FV Leiden.

The inherited bleeding disorder Factor V (FV) deficiency and clotting risk factor FV Leiden are associated with genetic variants in the F5 gene. FV deficiency occurs with mild, moderate, severe, or asymptomatic phenotypes, and either dysfunctional or reduced amounts of plasma FV protein. Here we present an interactive web database containing 363 unique F5 variants derived from 801 patient records, with 199 FV deficiency-associated variants from 245 patient records. Their occurrence is rationalized based on the 2,224 residue sequence and new FV protein structures. The 199 FV deficiency variants correspond to 26 (13%) mild, 22 (11%) moderate, 49 (25%) severe, 35 (18%) asymptomatic, and 67 (34%) unreported phenotypes. Their variant distributions in the FV domains A1, A2, A3, B, C1 and C2 were 28 (14%), 32 (16%), 34 (17%), 42 (21%), 16 (8%), and 19 variants (10%), respectively, showing that these six regions contain similar proportions of variants. Variants associated with FV deficiency do not cluster near known protein-partner binding sites, thus the molecular mechanism leading to the phenotypes cannot be explained. However, the widespread distribution of FV variants in combination with a high proportion of buried variant residues indicated that FV is susceptible to disruption by small perturbations in its globular structure. Variants located in the disordered B domain also appear to disrupt the FV structure. We discuss how the interactive database provides an online resource that clarifies the clinical understanding of FV deficiency.

Fluorescence probing and molecular docking analysis of the binding interaction of bovine serum albumin (BSA) with the polarity probe AICCN.

In this work, the fluorescent probe 2-amino-4-(1H-indol-3-yl)-4H-chromene-3-carbonitrile (AICCN) has been used to evaluate its potential as a prospective polarity probe. From detailed fluorescence studies of the probe, it could be shown that AICCN can indeed function as an effective polarity probe. The calculated dipole moments of AICCN in both the ground state and excited state in various solvents lend support to the steady state fluorescence results. It was also shown that AICCN can be used to probe the micropolarity of micelles and can be used successfully for the determination of CMC of the surfactants. The binding process of the probe AICCN to BSA has been followed by plotting the binding isotherms and Scatchard Plots. The time-resolved fluorescence data indicate that the preferred binding site of AICCN in BSA lies close to the buried Trp residue Trp-213 in Domain II. This contention is further supported by the molecular docking studies. The interaction study of the probe AICCN with proteins is relevant for future use of AICCN as a hydrophobic drug. Information was also obtained about the effect of probe binding on the serum albumin structure, which may be correlated to its physiological activity. Thus, the probe AICCN can serve not only as a good reporter of polarity of the microenvironment in biological systems but also as an efficient fluorophore to monitor conformational changes in proteins in future.

Abstract 2748: Protein folding stabilities are a major determinant of oxidation rates for buried methionine residues

Abstract 2748: Protein folding stabilities are a major determinant of oxidation rates for buried methionine residues

Fused in sarcoma undergoes cold denaturation: Implications for phase separation.

The mediation of liquid-liquid phase separation (LLPS) for fused in sarcoma (FUS) protein is generally attributed to the low-complexity, disordered domains and is enhanced at low temperature. The role of FUS folded domains on the LLPS process remains relatively unknown since most studies are mainly based on fragmented FUS domains. Here, we investigate the effect of metabolites on full-length (FL) FUS LLPS using turbidity assays and differential interference contrast (DIC) microscopy, and explore the behavior of the folded domains by nuclear magnetic resonance (NMR) spectroscopy. FL FUS LLPS is maximal at low concentrations of glucose and glutamate, moderate concentrations of NaCl, Zn2+ , and Ca2+ and at the isoelectric pH. The FUS RNA recognition motif (RRM) and zinc-finger (ZnF) domains are found to undergo cold denaturation above 0°C at a temperature that is determined by the conformational stability of the ZnF domain. Cold unfolding exposes buried nonpolar residues that can participate in LLPS-promoting hydrophobic interactions. Therefore, these findings constitute the first evidence that FUS globular domains may have an active role in LLPS under cold stress conditions and in the assembly of stress granules, providing further insight into the environmental regulation of LLPS.