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The potential barriers governing the motions of &alpha, synuclein (&alpha, S) variants&rsquo, hydration water, especially energetics of them, is in the focus of the work. The thermodynamical approach yielded essential information about distributions and heights of the potential barriers. The proteins&rsquo, structural disorder was measured by ratios of heterogeneous water-binding interfaces. They showed the &alpha, S monomers, oligomers and amyloids to possess secondary structural elements, although monomers are intrinsically disordered. Despite their disordered nature, monomers have 33% secondary structure, and therefore they are more compact than a random coil. At the lowest potential barriers with mobile hydration water, monomers are already functional, a monolayer of mobile hydration water is surrounding them. Monomers realize all possible hydrogen bonds with the solvent water. &alpha, S oligomers and amyloids have half of the mobile hydration water amount than monomers because aggregation involves less mobile hydration. The solvent-accessible surface of the oligomers is ordered or homogenous in its interactions with water to 66%. As a contrast, &alpha, S amyloids are disordered or heterogeneous to 75% of their solvent accessible surface and both wild type and A53T amyloids show identical, low-level hydration. Mobile water molecules in the first hydration shell of amyloids are the weakest bound compared to other forms.

Hydration properties of folded and unfolded/disordered miniproteins were monitored in frozen solutions by wide-line 1H-NMR. The amount of mobile water as function of T (−80 °C T 1H-NMR and molecular dynamics simulations we found that both the amount of mobile water surrounding proteins in ice, as well as their thaw profiles differs significantly as function of the compactness and conformational heterogeneity of their structure. We found that (i) at around −50 °C ~50 H2Os/protein melt (ii) if the protein is well-folded then this amount of mobile water remains quasi-constant up to −20 °C, (iii) if disordered then the quantity of the lubricating mobile water increases with T in a constant manner up to ~200 H2Os/protein by reaching −20 °C. Especially in the −55 °C ↔ −15 °C temperature range, wide-line 1H-NMR detects the heterogeneity of protein fold, providing the size of the hydration shell surrounding the accessible conformers at a given temperature. Results indicate that freezing of protein solutions proceeds by the gradual selection of the enthalpically most favored states that also minimize the number of bridging waters.

Wide-line (1) H NMR measurements were extended and all results were reinterpreted in a new thermodynamics-based approach to study aqueous solutions of thymosin-beta4 (Tbeta4 ), stabilin C-terminal domain (CTD) and their 1:1 complex. The energy distributions of the potential barriers, which control motion of protein-bound water molecules, were determined. Heterogeneous and homogeneous regions were found at the protein-water interface. The measure of heterogeneity gives a quantitative value for the portion of disordered parts in the protein. Ordered structural elements were found extending up to 20 % of the whole proteins. About 40 % of the binding sites of free Tbeta4 become involved in bonds holding the complex together. The complex has the most heterogeneous solvent accessible surface (SAS) in terms of protein-water interactions. The complex is more disordered than Tbeta4 or stabilin CTD. The greater SAS area of the complex is interpreted as a clear sign of its open structure.

Here we present a novel method for the characterization of the hydration of protein solutions based on measuring and evaluating two-component wide-line 1H NMR signals. We also provide a description of key elements of the procedure conceived for the thermodynamic interpretation of such results. These interdependent experimental and theoretical treatments provide direct experimental insight into the potential energy surface of proteins. The utility of our approach is demonstrated through the examples of two proteins of distinct structural classes: the globular, structured ubiquitin, and the intrinsically disordered ERD10 (early response to dehydration 10). We provide a detailed analysis and interpretation of data recorded earlier by cooling and slowly warming the protein solutions through thermal equilibrium states. We introduce and use order parameters that can be thus derived to characterize the distribution of potential energy barriers inhibiting the movement of water molecules bound to the surface of the protein. Our results enable a quantitative description of the ratio of ordered and disordered parts of proteins, and of the energy relations of protein–water bonds in aqueous solutions of the proteins.

Wide-line 1H NMR intensity and differential scanning calorimetry measurements were carried out on the intrinsically disordered 73-residue full transactivation domain (TAD) of the p53 tumor suppressor protein and two peptides: one a wild type p53 TAD peptide with a helix pre-structuring property, and a mutant peptide with a disabled helix-forming propensity. Measurements were carried out in order to characterize their water and ion binding characteristics. By quantifying the number of hydrate water molecules, we provide a microscopic description for the interactions of water with a wild-type p53 TAD and two p53 TAD peptides. The results provide direct evidence that intrinsically disordered proteins (IDPs) and a less structured peptide not only have a higher hydration capacity than globular proteins, but are also able to bind a larger amount of charged solute ions. [BMB Reports 2016; 49(9): 497-501].

The experimental characterization of hindered-rotation barriers and mapping the energetic heterogeneity of water molecules bound to the molecular “surface” of proteins is critical for understanding the functional interaction of proteins with their environment. Here, we show how to achieve this goal by an original wide-line NMR procedure, which is based on the spectral motional narrowing phenomenon following the melting (thawing) process of interfacial ice. The procedure highlights the differences between globular and intrinsically disordered proteins and it enables to delineate the effect of solvent on protein structure, making a distinction between point mutants, monomeric and oligomeric states, and characterizing the molecular interactions taking part in different cellular processes. We put this unique experimental approach introducing novel physical quantities and quantifying the heterogeneous distribution of motional activation energy of water in the interfacial landscape into a historical perspective, demonstrating its utility through a variety of globular and disordered proteins.

The propensity of α-synuclein to form amyloid plays an important role in Parkinson's disease. Three familial mutations, A30P, E46K, and A53T, correlate with Parkinson's disease. Therefore, unraveling the structural effects of these mutations has basic implications in understanding the molecular basis of the disease. Here, we address this issue through comparing details of the hydration of wild-type α-synuclein and its A53T mutant by a combination of wide-line NMR, differential scanning calorimetry, and molecular dynamics simulations. All three approaches suggest a hydrate shell compatible with a largely disordered state of both proteins. Its fine details, however, are different, with the mutant displaying a somewhat higher level of hydration, suggesting a bias to more open structures, favorable for protein-protein interactions leading to amyloid formation. These differences disappear in the amyloid state, suggesting basically the same surface topology, irrespective of the initial monomeric state.

The paper presents the experimental results of rigid-lattice proton magnetic resonance spectra and second moments in Pd–H, Pd 0.90 Ag 0.10 –H, Pd 0.80 Ag 0.20 –H, Pd 0.75 Ag 0.25 –H, and Pd 0.65 Ag 0.35 –H alloys at T = 2.4 K in a wide hydrogen concentration range. Free induction decay (FID), solid and inhomogeneous echoes were detected and interpreted. The interpretation based on dipole–dipole interaction of homogeneously distributed proton spin system is satisfactory at high hydrogen concentration, but an inhomogeneous field of paramagnetic origin (most probably coming from Fe impurities) plays the dominant role at small hydrogen content. An unexpected correlation was found between the macroscopic magnetic susceptibility and the second moment coming from the inhomogeneous internal field.

Crystalline Pd 1-x Ag x -H y (x = 0-0.35) alloys were studied as model systems representing a chemically disordered system for hydrogen-storage materials. Extensive 1 H relaxation rate (R 1 , R 1p and R 2 ) measurements were carried out in a wide temperature range of 2.4-400 K. Palladium-silver alloys of silver concentration as high as 35 at.% were studied by NMR spectroscopy for the first time. The relaxation mechanisms magnetic dipolar relaxation (hydrogen diffusion), Korringa relaxation, relaxation by paramagnetic impurity ions and cross-relaxation to quadrupolar metal nuclei were identified and characterized. The presence of cross-relaxation between 1 H and quadrupolar 105 Pd nuclear spins was assumed and demonstrated for the first time in Pd-H and Pd 1-x Ag x -H systems. More than one jump processes of diffusing hydrogen were found to be present in the studied palladium-silver alloys. Only lower estimated limit values of Korringa constants could be obtained since no frequency independent R 1 versus T region was found. These values are significantly higher than the ones reported by others.

Intrinsically unstructured/disordered proteins (IUPs) exist in a disordered and largely solvent-exposed, still functional, structural state under physiological conditions. As their function is often directly linked with structural disorder, understanding their structure-function relationship in detail is a great challenge to structural biology. In particular, their hydration and residual structure, both closely linked with their mechanism of action, require close attention. Here we demonstrate that the hydration of IUPs can be adequately approached by a technique so far unexplored with respect to IUPs, solid-state NMR relaxation measurements. This technique provides quantitative information on various features of hydrate water bound to these proteins. By freezing nonhydrate (bulk) water out, we have been able to measure free induction decays pertaining to protons of bound water from which the amount of hydrate water, its activation energy, and correlation times could be calculated. Thus, for three IUPs, the first inhibitory domain of calpastatin, microtubule-associated protein 2c, and plant dehydrin early responsive to dehydration 10, we demonstrate that they bind a significantly larger amount of water than globular proteins, whereas their suboptimal hydration and relaxation parameters are correlated with their differing modes of function. The theoretical treatment and experimental approach presented in this article may have general utility in characterizing proteins that belong to this novel structural class.

In this work, the electrochemical hydrogen doping and withdrawal process of Pd metal was examined with respect to the applicability of Faraday’s law. It was found that after a suitable electrolytic pre-treatment, the normalized resistivity ( R / R 0 ) versus hydrogen concentration ( c F = H/M) curves where c F was determined from Faraday’s law by assuming that the 100% current efficiency is independent of the value of the applied charging current density in a wide range. In the α-Pd(H) phase (up to c (α max ) = 0.015H/M), a good quantitative agreement was found with previous resistivity studies by both electrolytic and gas-phase charging. However, for c > c (α max ) our R / R 0 versus c F data did not agree with the R / R 0 versus c H curve from previously reported electrochemical charging experiments of Pd metal where the doped amount of hydrogen, c H , was determined by direct methods. The two sets of resistivity data can be brought into good agreement by assuming that the current efficiency for the absorption process is η a = 1.55. Similarly, for the electrolytic desorption process, an agreement with the previously reported R / R 0 versus c H curve could be achieved by taking η d = 1.9. These current efficiency values higher than unity would imply that each hydrogen absorbed/desorbed by electrolytic charge/discharge processes is accompanied by the absorption/desorption of some excess H atoms by processes not requiring electric charge transfer (i.e., only neutral H atoms are involved). At present, we do not have a physical/chemical picture about the nature of such processes.

The partition of hydrogen into diffusible and residual parts was realized by pulse NMR spectroscopy, by gas chromatography and by prompt gamma activation analysis (PGAA). The total hydrogen content was determined by the two non-NMR methods and the diffusible (mobile) component by CPMG NMR pulse sequence. Results on amorphous Ni(Cu)–Zr–H systems of different compositions are shown. Partially crystallized samples were also studied as an extension. A method proposed by us directly gives the fractional population of hydrogen atoms in the free (mobile) state on the spin–spin relaxation time scale. On the other hand the least values of the residual hydrogen content correlate surprisingly well with the numbers of filled four Zr-type H-sites calculated by Batalla et al. [NATO ASI Ser. 136 (1985) 203] for 0.21-nm exclusion distance.

Reductive hydrogenation was applied to two types of single-walled carbon nanotubes with different diameter range. Alkali metal intercalation, followed by reaction with methanol, led to hydrogenated products. Both yield and selectivity of this reaction showed strong dependence on diameter, contrary to expectation based on simple curvature effects. The observed yield, as detected by thermogravimetry-mass spectroscopy and 1H-NMR, is drastically reduced in small-diameter tubes where the alkali dopant does not reach the inside of the bundles. Wide range optical transmission measurements were employed to determine the selectivity and indicate that besides higher yield, lower diameter selectivity occurs above a critical diameter., 14 pages, 4 figures

The paper presents the results of an experimental investigation of rigid-lattice proton magnetic resonance (PMR) spectra and second moments in Ni 0.67 Zr 0.33 –H, Ni 0.50 Zr 0.50 –H, Ni 0.33 Zr 0.67 –H binary glassy alloy–hydrogen systems in the range of hydrogen-per-metal ( H / M ) ratio of 0.14≤ H / M ≤1.9. The line shape can be described by the empirical Harper-Barnes function with exponent values ranging from 1.45 to 2.95 in the whole hydrogen concentration range. The generally assumed Gaussian form is valid in a narrow H / M interval only. The second moment as a function of H / M can be fitted by a power function of an exponent 3/4. The exponent is independent of the composition of the alloy. We found that a simple lattice gas model based on a homogeneous hydrogen distribution contradicts the experimental results. Instead, a short-range order model has been proposed in which the hydrogen atoms are located in the centres of distorted tetrahedra of metal atoms, that is in the basic building blocks of the amorphous alloys. The Switendick criterion was also taken into consideration. We have shown that few proton first neighbours (0–2) and a small number of second neighbours (1–8), that is, a cluster of hydrogen is sufficient to account for the measured value of the second moment. Ternary Zr 0.5 Ni 0.5− x Cu x –H alloys have been investigated, too. The addition of Cu nuclei did not lead to the second moment enhancement expected for a homogeneous distribution of components.

Results of electrical resistance measurements are given on Pd80 Ag20 alloy during hydrogen uptake from gaseous H2 atmosphere and from liquid electrolyte by cathodic charging. For both cases in the course of a monotonous increase of the hydrogen concentration in the alloy, the electrical resistivity shows anomalous behavior. These kinetic curves reflect the effect of phase changes in the non-equilibrium state. The ‘non-equilibrium’ expression is used in this entire work in the sense that no care was taken in waiting for the stable value of resistivity after any partial filling, but the resistivity was frequently measured while the sample was continuously charged with hydrogen. A model has been proposed for the interpretation of the experimental findings.

The hydrogen charging and discharging processes are investigated in Ni67−xCuxZr33 glassy alloys by in situ resistance measurements in order to clarify the surface and bulk contribution to the activation process. The process has been monitored by conventional DC method using a computer-controlled system, exposing the samples to hydrogen at pressure from 0.1–8.6 MPa at temperatures of 293 K and 393 K. The H-contents (H/M) were determined by weight and by pulse NMR measurements. The NMR data are obtained by the Carr–Purcell–Meiboom–Gill (CPMG) method. The charging is extremely long and the resulting H-distribution is inhomogenous during the first cycle, especially at 293 K. This fact shows the significance of the surface-activation during the H-uptake from the gas phase. The duration of the second saturation decreases significantly, and the resistance–time curves obtained: on two parallel samples are very similar. The saturation value for the normalized resistance (R/R0) remains almost the same during several cycles. Increasing the temperature of the charging (393 K), the equilibrium value of the resistance decreases. The resistance of the samples at the end of the discharging process is always higher than before charging. This indicates the partial irreversibility of the H-absorption at constant temperature. This observation is correlated with the NMR results.

1 H spin–spin relaxation time ( T 2 ) and complementary hydrogen content, PMR spectrum width and spin–lattice relaxation time ( T 1 ) have been measured in Zr y (Ni 1− x Cu x ) 1− y –H ternary amorphous alloys of different hydrogen content at 0≤ x ≤33 at.% Cu and y =33, 50 and 67 at.% Zr concentrations using Carr–Purcell–Meiboom–Gill (CPMG), solid-echo and saturation recovery pulse sequences, respectively. At high hydrogen contents the T 2 measured by the slope of the CPMG echo train depends on both the Zr and Cu content, but is independent of the hydrogen content. The differences in the spin–spin relaxation behaviours can be attributed to the substantial change of correlation time and not to the change of activation energy or local fields. The measurements were made in the “motional narrowing” state, consequently our E a and τ ∞ quantities are averaged to the diffusion motion of protons taking part in this process. At small hydrogen contents T 2 depends on hydrogen content and the T 2 vs. 1/ T curves cannot be fitted by single Arrhenius plots. The hydrogen content measured by echo train has turned out to be systematically smaller than that measured by weight increase, demonstrating that not all the hydrogen takes part in the diffusion process.

The mobility of the proton-proton radial vectors is introduced as a quantitative measure for the structural dynamics of organic materials, especially protein molecules. As defined for the entire molecule, the hydrogen mobility (HM) is proposed as an “order parameter,” which describes the effect of motional narrowing on inter-proton dipole-dipole interactions. HM satisfies all requirements of an order parameter in the Landau molecular field theory of phase transitions. The wide-line NMR second moments needed to obtain HM are exactly defined and measurable physical quantities, which are not produced by mathematical fitting and do not carry the limitations and restrictions of any model (theoretical formalism). We first demonstrate the usefulness of HM on small organic molecules with data taken form the literature. We outline its link with structural and functional characteristics on a range of proteins: HM provides a model-free parameter based on first principles that can clearly distinguish between globular and intrinsically disordered proteins, and can also provide insight into the behavior of disease-related mutants.

Spin-spin relaxation time ( T 2 ) and complementary hydrogen content (H/M), PMR spectrum width (FWHM) and spin lattice relaxation time ( T 1 ) have been measured in (Ni 1 − x Cu x ) 0.5 Zr 0.5 -H y ternary amorphous alloys at 0 ⩽ x ⩽ 29 at.% concentrations using Carr-Purcell-Meiboom-Gill (CPMG), solid-echo and saturation recovery pulse sequences respectively. The T 2 relaxation time measured by the slope of the CPMG echo train depends on both the hydrogen and Cu content. The dependence can be attributed to the change of correlation time and not to the change of activation energy or local field. The measurements were made in the ‘motional narrowing’ state; consequently our E a and τ ∞ quantities are averaged to the diffusional motion of protons taking part in this process. The extrapolated amplitude of the echo train measured on the alloy-hydrogen system and normalized to the same quantity in water, gives the value of proton magnetization in thermal equilibrium, that is the hydrogen content free from any perturbing effects. The hydrogen content measured by CPMG echo train has turned out to be systematically smaller than that measured by weight increase.

Equilibrium pressure and potential measurements at room temperature as a function of hydrogen concentration are reported for Ni 1− x Zr x amorphous alloys with 0.33 ⩽ x ⩽ 0.45. The full range of charge of the samples was achieved by taking into account the hydrogen gas evolved in the closed electrochemical cell. The change in slope of the equilibrium potential with the state of charge shows that different types of site are filled. On the basis of the pressure-composition (P-C) diagrams, the samples with x = 0.33, 0.36 and 0.39 were selected for further electrochemical investigations.

The aim of this paper is to reinvestigate the proton magnetic resonance spectra in Zr 0.5 Ni 0.5− y Cu y H x metallic glasses. Solid-echo pulse sequences were used to analyze the line shape. The measurements were made in the temperature range 2.2–300 K and at a frequency of 87.6 MHz. The reinvestigation of the line shape mostly at low temperatures was initiated after having realized that the splitting reported earlier on our samples was a consequence of the relatively long recovery time of our spectrometer. The present solid-echo measurements yielded simple spectra describable by a Harper-Barnes line shape close to gaussian.

The proton NMR line shift with respect to water, the bulk susceptibility and in a few cases the Korringa contribution to the spin lattice relaxation time were measured in Zr0.33Ni0.67 Hx amorphous alloys with hydrogen concentration 0.1 ⩽ x ⩽ 0.7and at 343 K. All the parameters, including the hydrogen content and the bulk susceptibility, were measured simultaneously in situ, and in the shift measurements a resolution of ±1 ppm was attained. The residual Knight shift depends on the hydrogen content and is positive relative to water. Preliminary low temperature spin lattice relaxation time measurements indicate that the positive shift is the consequence of the balance of s- and d-contributions.

CASK-interactive protein1 is a newly recognized post-synaptic density protein in mammalian neurons. Although its N-terminal region contains several well-known functional domains, its entire C-terminal proline-rich region of 800 amino acids lacks detectable sequence homology to any previously characterized protein. We used multiple techniques for the structural characterization of this region and its three fragments. By bioinformatics predictions, CD spectroscopy, wide-line and 1H-NMR spectroscopy, limited proteolysis and gel filtration chromatography, we provided evidence that the entire proline-rich region of CASK-interactive protein1 is intrinsically disordered. We also showed that the proline-rich region is biochemically functional, as it interacts with the adaptor protein Abl-interactor-2. To extend the finding of a high level of disorder in this scaffold protein, we collected 74 scaffold proteins (also including proteins denoted as anchor and docking), and predicted their disorder by three different algorithms. We found that a very high fraction (53.6% on average) of the residues fall into local disorder and their ordered domains are connected by linker regions which are mostly disordered (64.5% on average). Because of this high frequency of disorder, the usual design of scaffold proteins of short globular domains (86 amino acids on average) connected by longer linker regions (140 amino acids on average) and the noted binding functions of these regions in both CASK-interactive protein1 and the other proteins studied, we suggest that structurally disordered regions prevail and play key recognition roles in scaffold proteins. Structured digital abstract • MINT-7034649: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with L1CAM (uniprotkb:P32004) by two hybrid (MI:0018) • MINT-7034677: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with NCK1 (uniprotkb:P16333) by two hybrid (MI:0018) • MINT-7034706: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with Stathmin-3 (uniprotkb:Q9NZ72) by two hybrid (MI:0018) • MINT-7034579: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with ABI2 (uniprotkb:Q9NYB9) by two hybrid (MI:0018) • MINT-7034720: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with Synaptotagmin (uniprotkb:P21579) by two hybrid (MI:0018) • MINT-7034691: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with Neurexin-2 (uniprotkb:Q9P2S2) by two hybrid (MI:0018) • MINT-7034617: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with CASK (uniprotkb:P07498) by two hybrid (MI:0018) • MINT-7034748: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with SIAH1 (uniprotkb:Q8IUQ4) by two hybrid (MI:0018) • MINT-7034663: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with Myosin-Ib (uniprotkb:O43795) by two hybrid (MI:0018) • MINT-7034734: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with Septin-4 (uniprotkb:O43236) by two hybrid (MI:0018) • MINT-7034634: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with EPHA2 (uniprotkb:P29317) by two hybrid (MI:0018) • MINT-7034765, MINT-7034783: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with ABI2 (uniprotkb:Q9NYB9) by pull down (MI:0096)

Results of resistivity measurements during electrolytical hydrogen charging on some Fe, Co and Ni based alloy ribbons in amorphous, nanocrystalline and body-centred cubic state are presented. The peculiarities of the H uptake are significantly different for the different structural modifications of alloys with the same chemical composition. In the limited concentration interval of equality of the transferred charge and the number of actually absorbed hydrogen atoms, the concentration variation of the resistivity increment caused by unit hydrogen concentration was determined.

The proton content in Zr 0.33 Ni 0.67 H x amorphous alloy was monitored by changes in electrical resistivity and with the extraplated spin echo amplitude measured by the Carr-Purcell-Meiboom-Gill (CPMG) nuclear magnetic resonance pulse sequence in the temperature interval 300 to 450 K. Both methods show that the hydrogen desorption processes can be approximately divided into two ranges. The first is a fast process which is at present not interpretable. In the second stage, the process is thermally activated, and the activation energy calcualted from the resistivity changes is equal to 0.32 ± 0.04 eV. The CPMG spin echo measurements also give the spin-spin relaxation time of diffusing protons and the activation energy of this process, 0.34 ± 0.02 eV, is exactly the same as extracted from the resistivity measurements.

Thymosine β4 (Tß4) is a 43 amino acid long intrinsically disordered protein (IDP), which was initially identified as an actin-binding and sequestering molecule. Later it was described to have multiple other functions, such as regulation of endothelial cell differentiation, blood vessel formation, wound repair, cardiac cell migration, and survival.1 The various functions of Tβ4 are mediated by interactions with distinct and structurally unrelated partners, such as PINCH, ILK, and stabilin-2, besides the originally identified G-actin. Although the cellular readout of these interactions and the formation of these complexes have been thoroughly described, no attempt was made to study these interactions in detail, and to elucidate the thermodynamic, kinetic, and structural underpinning of this range of moonlighting functions. Because Tβ4 is mostly disordered, and its 4 described partners are structurally unrelated (the CTD of stabilin-2 is actually fully disordered), it occurred to us that this system might be ideal to characterize the structural adaptability and ensuing moonlighting functions of IDPs. Unexpectedly, we found that Tβ4 engages in multiple weak, transient, and fuzzy interactions, i.e., it is capable of mediating distinct yet specific interactions without adapting stable folded structures.