Driving forces of the complex formation between highly charged disordered proteins.

Highly disordered complexes between oppositely charged intrinsically disordered proteins present a new paradigm of biomolecular interactions. Here, we investigate the driving forces of such interactions for the example of the highly positively charged linker histone H1 and its highly negatively charged chaperone, prothymosin α (ProTα). Temperature-dependent single-molecule Förster resonance energy transfer (FRET) experiments and isothermal titration calorimetry reveal ProTα-H1 binding to be enthalpically unfavorable, and salt-dependent affinity measurements suggest counterion release entropy to be an important thermodynamic driving force. Using single-molecule FRET, we also identify ternary complexes between ProTα and H1 in addition to the heterodimer at equilibrium and show how they contribute to the thermodynamics observed in ensemble experiments. Finally, we explain the observed thermodynamics quantitatively with a mean-field polyelectrolyte theory that treats counterion release explicitly. ProTα-H1 complex formation resembles the interactions between synthetic polyelectrolytes, and the underlying principles are likely to be of broad relevance for interactions between charged biomolecules in general.

Extreme dynamics in a biomolecular condensate.

Proteins and nucleic acids can phase-separate in the cell to form concentrated biomolecular condensates1-4. The functions of condensates span many length scales: they modulate interactions and chemical reactions at the molecular scale5, organize biochemical processes at the mesoscale6 and compartmentalize cells4. Understanding the underlying mechanisms of these processes will require detailed knowledge of the rich dynamics across these scales7. The mesoscopic dynamics of biomolecular condensates have been extensively characterized8, but their behaviour at the molecular scale has remained more elusive. Here, as an example of biomolecular phase separation, we study complex coacervates of two highly and oppositely charged disordered human proteins9. Their dense phase is 1,000 times more concentrated than the dilute phase, and the resulting percolated interaction network10 leads to a bulk viscosity 300 times greater than that of water. However, single-molecule spectroscopy optimized for measurements within individual droplets reveals that at the molecular scale, the disordered proteins remain exceedingly dynamic, with their chain configurations interconverting on submicrosecond timescales. Massive all-atom molecular dynamics simulations reproduce the experimental observations and explain this apparent discrepancy: the underlying interactions between individual charged side chains are short-lived and exchange on a pico- to nanosecond timescale. Our results indicate that, despite the high macroscopic viscosity of phase-separated systems, local biomolecular rearrangements required for efficient reactions at the molecular scale can remain rapid.

Interaction Dynamics of Intrinsically Disordered Proteins from Single-Molecule Spectroscopy.

Many proteins contain large structurally disordered regions or are entirely disordered under physiological conditions. The functions of these intrinsically disordered proteins (IDPs) often involve interactions with other biomolecules. An important emerging effort has thus been to identify the molecular mechanisms of IDP interactions and how they differ from the textbook notions of biomolecular binding for folded proteins. In this review, we summarize how the versatile tool kit of single-molecule fluorescence spectroscopy can aid the investigation of these conformationally heterogeneous and highly dynamic molecular systems. We discuss the experimental observables that can be employed and how they enable IDP complexes to be probed on timescales from nanoseconds to hours. Key insights include the diverse structural and dynamic properties of bound IDPs and the kinetic mechanisms facilitated by disorder, such as fly-casting; disorder-mediated encounter complexes; and competitive substitution via ternary complexes, which enables rapid dissociation even for high-affinity complexes. We also discuss emerging links to aggregation, liquid-liquid phase separation, and cellular processes, as well as current technical advances to further expand the scope of single-molecule spectroscopy.

Polyelectrolyte interactions enable rapid association and dissociation in high-affinity disordered protein complexes.

Highly charged intrinsically disordered proteins can form complexes with very high affinity in which both binding partners fully retain their disorder and dynamics, exemplified by the positively charged linker histone H1.0 and its chaperone, the negatively charged prothymosin α. Their interaction exhibits another surprising feature: The association/dissociation kinetics switch from slow two-state-like exchange at low protein concentrations to fast exchange at higher, physiologically relevant concentrations. Here we show that this change in mechanism can be explained by the formation of transient ternary complexes favored at high protein concentrations that accelerate the exchange between bound and unbound populations by orders of magnitude. Molecular simulations show how the extreme disorder in such polyelectrolyte complexes facilitates (i) diffusion-limited binding, (ii) transient ternary complex formation, and (iii) fast exchange of monomers by competitive substitution, which together enable rapid kinetics. Biological polyelectrolytes thus have the potential to keep regulatory networks highly responsive even for interactions with extremely high affinities.

Phase Separation Comes of Age: From Phenomenology to Single Molecules.

In this issue of Molecular Cell, Pitchiaya et al. (2019) use high-resolution single-molecule microscopy to dissect the localization of different types of RNAs with processing bodies (PBs) in cells, revealing novel insights about their dynamic recruitment to PBs.

Mechanism-Dependent Modulation of Ultrafast Interfacial Water Dynamics in Intrinsically Disordered Protein Complexes.

The recognition of intrinsically disordered proteins (IDPs) is highly dependent on dynamics owing to the lack of structure. Here we studied the interplay between dynamics and molecular recognition in IDPs with a combination of time-resolving tools on timescales ranging from femtoseconds to nanoseconds. We interrogated conformational dynamics and surface water dynamics and its attenuation upon partner binding using two IDPs, IBB and Nup153FG, both of central relevance to the nucleocytoplasmic transport machinery. These proteins bind the same nuclear transport receptor (Importinß) with drastically different binding mechanisms, coupled folding-binding and fuzzy complex formation, respectively. Solvent fluctuations in the dynamic interface of the Nup153FG-Importinß fuzzy complex were largely unperturbed and slightly accelerated relative to the unbound state. In the IBB-Importinß complex, on the other hand, substantial relative slowdown of water dynamics was seen in a more rigid interface. These results show a correlation between interfacial water dynamics and the plasticity of IDP complexes, implicating functional relevance for such differential modulation in cellular processes, including nuclear transport.

Comment on "Innovative scattering analysis shows that hydrophobic disordered proteins are expanded in water".

Editors at Science requested our input on the above discussion (comment by Best et al and response by Riback et al) because both sets of authors use our data from Fuertes et al (2017) to support their arguments. The topic of discussion pertains to the discrepant inferences drawn from SAXS versus FRET measurements regarding the dimensions of intrinsically disordered proteins (IDPs) in aqueous solvents. Using SAXS measurements on labeled and unlabeled proteins, we ruled out the labels used for FRET measurements as the cause of discrepant inferences between the two methods. Instead, we propose that FRET and SAXS provide complementary readouts because of a decoupling of size and shape fluctuations that is intrinsic to finite-sized, heteropolymeric IDPs. Accounting for this decoupling resolves the discrepant inferences between the two methods, thus making a case for the utility of both methods.

Reversible inactivation of yeast mitochondrial phenylalanyl-tRNA synthetase under oxidative stress.

BACKGROUND: Under oxidative stress cytoplasmic aminoacyl-tRNA synthetase (aaRSs) substrate specificity can be compromised, leading to tRNA mischarging and mistranslation of the proteome. Whether similar processes occur in mitochondria, which are major cellular sources of reactive oxygen species (ROS), is unknown. However, relaxed substrate specificity in yeast mitochondrial phenylalanyl-tRNA synthetase (ScmitPheRS) has been reported to increase tRNA mischarging and blocks mitochondrial biogenesis. METHODS: Non-reducing denaturing PAGE, cysteine reactivity studies, MALDI-TOF mass spectrometry, enzyme assay, western blot, growth assay, circular dichroism, dynamic light scattering and fluorescence spectroscopy were used to study the effect of oxidative stress on ScmitPheRS activity. RESULTS: ScmitPheRS is reversibly inactivated under oxidative stress. The targets for oxidative inactivation are two conserved cysteine residues resulting in reversible intra-molecular disulfide bridge formation. Replacement of either conserved cysteine residue increased viability during growth under oxidative stress. CONCLUSION: Formation of intra-molecular disulfide bridge under oxidative stress hinders the tRNAPhe binding of the enzyme, thus inactivating ScmitPheRS reversibly. GENERAL SIGNIFICANCE: The ScmitPheRS activity is compromised under oxidative stress due to formation of intra-molecular disulfide bridge. The sensitivity of ScmitPheRS to oxidation may provide a protective mechanism against error-prone translation under oxidative stress.

Two Differential Binding Mechanisms of FG-Nucleoporins and Nuclear Transport Receptors.

Phenylalanine-glycine-rich nucleoporins (FG-Nups) are intrinsically disordered proteins, constituting the selective barrier of the nuclear pore complex (NPC). Previous studies showed that nuclear transport receptors (NTRs) were found to interact with FG-Nups by forming an "archetypal-fuzzy" complex through the rapid formation and breakage of interactions with many individual FG motifs. Here, we use single-molecule studies combined with atomistic simulations to show that, in sharp contrast, FG-Nup214 undergoes a coupled reconfiguration-binding mechanism when interacting with the export receptor CRM1. Association and dissociation rate constants are more than an order of magnitude lower than in the archetypal-fuzzy complex between FG-Nup153 and NTRs. Unexpectedly, this behavior appears not to be encoded selectively into CRM1 but rather into the FG-Nup214 sequence. The same distinct binding mechanisms are unperturbed in O-linked ß-N-acetylglucosamine-modified FG-Nups. Our results have implications for differential roles of distinctly spatially distributed FG-Nup⋅NTR interactions in the cell.

p-Benzoquinone-induced aggregation and perturbation of structure and chaperone function of α-crystallin is a causative factor of cigarette smoke-related cataractogenesis.

Cigarette smoking is a significant risk factor for cataract. However, the mechanism by which cigarette smoke (CS) causes cataract remains poorly understood. We had earlier shown that in CS-exposed guinea pig, p-benzoquinone (p-BQ) derived from CS in the lungs is carried by the circulatory system to distant organs and induces various smoke-related pathogeneses. Here, we observed that CS exposure caused accumulation of the p-BQ-protein adduct in the eye lens of guinea pigs. We also observed accumulation of the p-BQ-protein adduct in resected lens from human smokers with cataract. No such accumulation was observed in the lens of never smokers. p-BQ is a strong arylating agent that forms Michael adducts with serum albumin and haemoglobin resulting in alterations of structure and function. A major protein in the mammalian eye lens is αA-crystallin, which is a potent molecular chaperone. αA-crystallin plays a key role in maintaining the integrity and transparency of the lens. SDS-PAGE indicated that p-BQ induced aggregation of αA-crystallin. Various biophysical techniques including UV-vis spectroscopy, fluorescence spectroscopy, FT-IR, bis-ANS titration suggested a perturbation of structure and chaperone function of αA-crystallin upon p-BQ modification. Our results indicate that p-BQ is a causative agent involved in the modification of αA-crystallin and pathogenesis of CS-induced cataract. Our findings would educate public about the impacts of smoking on eye health and help to discourage them from smoking. The study might also help scientists to develop new drugs for the intervention of CS-induced cataract at an early stage.

Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements.

Unfolded states of proteins and native states of intrinsically disordered proteins (IDPs) populate heterogeneous conformational ensembles in solution. The average sizes of these heterogeneous systems, quantified by the radius of gyration (RG ), can be measured by small-angle X-ray scattering (SAXS). Another parameter, the mean dye-to-dye distance (RE ) for proteins with fluorescently labeled termini, can be estimated using single-molecule Förster resonance energy transfer (smFRET). A number of studies have reported inconsistencies in inferences drawn from the two sets of measurements for the dimensions of unfolded proteins and IDPs in the absence of chemical denaturants. These differences are typically attributed to the influence of fluorescent labels used in smFRET and to the impact of high concentrations and averaging features of SAXS. By measuring the dimensions of a collection of labeled and unlabeled polypeptides using smFRET and SAXS, we directly assessed the contributions of dyes to the experimental values RG and RE For chemically denatured proteins we obtain mutual consistency in our inferences based on RG and RE , whereas for IDPs under native conditions, we find substantial deviations. Using computations, we show that discrepant inferences are neither due to methodological shortcomings of specific measurements nor due to artifacts of dyes. Instead, our analysis suggests that chemical heterogeneity in heteropolymeric systems leads to a decoupling between RE and RG that is amplified in the absence of denaturants. Therefore, joint assessments of RG and RE combined with measurements of polymer shapes should provide a consistent and complete picture of the underlying ensembles.

Spectroscopic studies of the unfolding of a multimeric protein α-crystallin.

α-Crystallin is a multimeric eye lens protein having molecular chaperone-like function which is crucial for lens transparency. The stability and unfolding-refolding properties of α-crystallin plays important roles for its function. We undertook a multi probe based fluorescence spectroscopic approach to explore the changes in the various levels of organization of this protein at different urea concentration. Steady state fluorescence studies reveal that at 0.6M urea a compact structural intermediate is formed which has a native-like secondary structure with enhanced surface exposure of hydrophobic groups. At 2.8M urea the tertiary interactions are largely collapsed with partial collapse of secondary and quaternary structure. The surface solvation probed by picosecond time resolved fluorescence of acrylodan labeled α-crystallin revealed dry native-like core of α-crystallin at 0.6M urea compared to enhanced water penetration at 2.8M urea and extensive solvation at 6M urea. Activation energy for the subunit exchange decreased by 22 kJ mol(-1) on changing urea concentration from 0 to 0.6M compared with over 75 kJ mol(-1) on changing urea concentration from 0 to 2.8M. Light scattering and analytical ultracentrifugation techniques were used to determine size and oligomerization of the unfolding intermediates. The data indicated swelling but no oligomer breakdown at 0.6M urea. At 2.8M urea the oligomeric size is considerably reduced and a monomer is produced at 6M urea. The data clearly reveals that structural breakdown of α-crystallin does not follow hierarchical sequence as tertiary structure dissolution takes place before complete oligomeric dissociation.

Deoxycholate induced tetramer of αA-crystallin and sites of phosphorylation: fluorescence correlation spectroscopy and femtosecond solvation dynamics.

Structure and dynamics of acrylodan labeled αA-crystallin tetramer formed in the presence of a bile salt (sodium deoxycholate, NaDC) has been studied using fluorescence correlation spectroscopy (FCS) and femtosecond up-conversion techniques. Using FCS it is shown that, the diffusion constant (D(t)) of the αA-crystallin oligomer (mass ~800 kDa) increases from ~35 µm(2) s(-1) to ~68 µm(2) s(-1). This corresponds to a decrease in hydrodynamic radius (r(h)) from ~6.9 nm to ~3.3 nm. This corresponds to about 10-fold decrease in molecular mass to ~80 kDa and suggests formation of a tetramer (since mass of αA-crystallin monomer is ~20 kDa). The steady state emission maximum and average solvation time (<τ(s)>) of acrylodan labeled at cysteine 131 position of αA-crystallin is markedly affected on addition of NaDC, while the tryptophan (trp-9) becomes more exposed. This suggests that NaDC binds near the cys-131 and makes the terminal region of αA-crystallin exposed. This may explain the enhanced auto-phosphorylation activity of αA-crystallin near the terminus of the 173 amino acid protein (e.g., at the threonine 13, serine 45, or serine 169 and 172) and suggests that phosphorylation at ser-122 (close to cys-131) is relatively less important.