Intrinsically Disordered Proteins at the Nano-scale
Tamara Ehm, Hila Shinar, Sagi Meir, Amandeep Sekhon, Vaishali Sethi, Ian L. Morgan, Gil Rahamim, Omar A. Saleh, Roy Beck
IIntrinsically Disordered Proteins at the Nano-scale
T. Ehm , , H. Shinar , S. Meir , A. Sekhon , V. Sethi ,I. L. Morgan , G. Rahamim , O. A. Saleh , and R. Beck The School of Physics and Astronomy, The Center for Nanoscience andNanotechnology, and the Center for Physics and Chemistry of Living Systems, TelAviv University, Israel Faculty of Physics and Center for NanoScience, Ludwig-Maximilians-Universit¨at,M¨unchen, Germany BMSE Program, University of California, Santa Barbara CA 93110 Materials Department, University of California, Santa Barbara CA 93110E-mail: [email protected]
Abstract.
The human proteome is enriched in proteins that do not fold into a stable 3Dstructure. These intrinsically disordered proteins (IDPs) spontaneously fluctuatebetween a large number of configurations in their native form. Remarkably, thedisorder does not lead to dysfunction as with denatured folded proteins. In fact, unlikedenatured proteins, recent evidences strongly suggest that multiple biological functionsstem from such structural plasticity. Here, focusing on the nanoscopic length-scale, wereview the latest advances in IDP research and discuss some of the future directionsin this highly promising field.
Keywords : Intrinsically disordered proteins, nanoscopic characterization, single-molecule force spectroscopy, SAXS, FRET, NMR, FCS
1. Introduction
The conventional paradigm in structural biology draws a direct connection between theamino-acid sequence of a protein to a singular 3D structure. The unique structureis considered essential to the protein’s biological function by permitting only specificinteractions.In the last two decades it has been recognized that up to 40-50% of the proteomedoes not fit this simplified convention (Fig. 1). Instead, intrinsically disordered proteinsand regions (IDP/IDR) fluctuate between a large number of conformations while stillretaining their biological functions [1–5]. An IDR is usually defined as an unstructuredamino-acid stretch as part of a (folded) protein, and an IDP as a complete protein thatdoes not fold to a stable 3D structure [2–5]. For brevity, we will also use the IDP termto describe proteins having IDRs. a r X i v : . [ phy s i c s . b i o - ph ] J a n ntrinsically disordered proteins Sequence
DisorderedFolded
Function
Order-to-Disorder TransitionLock & Key (un)Structure
Master-key Liquid Phase Separation Aggregation Passivation & Filtration
Figure 1.
Contemporary sequence-function paradigm. The folded and disorderedconformations, and the transition between the two, lead to biological function.
Typically, most IDP sequences are rich in structure-breaking charged and polaramino acids, and depleted in order-promoting hydrophobic residues (Fig. 2, andRefs. [2, 4–8]). Indeed, computer-based methods exploit the amino-acid propensity andsequence as a sign of a disorder [9]. Generally, IDPs fall into three distinct compositionalclasses that reflect the fraction of charged versus polar residues: polar, polyampholytes,and polyelectrolytes [10, 11]. In addition, the balance between solvent mediatedintra-chain attraction and repulsion directly influences the IDP’s compactness. Thecompactness, in-turn, determines the accessibility to interact with other biomolecules.Therefore, modeling the inter- and intra-molecular IDP interactions, as well as theresulting ensemble of conformations, is an extremely complex and relevant problem inthe research of nano-scale systems.It is possible to characterize the IDP ensemble average parameters such as theend-to-end distance ( R ee ) or the radius of gyration ( R G ) distributions. In addition,the IDP length, charged amino-acid distribution, and propensity to form transientbridges, dictate the ensemble physical properties and can be connected to polymerphysics theories. This could lead to new language that relates the biological functionto basic physical parameters. Thus, IDP research has the opportunity to gain a novelinsight into of the biological sequence-to-function problem (Fig. 1).
2. Biological function
The structural plasticity of IDPs is the key to their function. This plasticity, almostby definition, limits the strength of the IDPs’ interactions with other biomolecules andthe environment. Interactions larger than the thermal energy would ultimately lead toa specific configuration, which conflicts with the IDPs’ large ensemble of conformations[13, 14]. Therefore, we must ask ourselves: does structural plasticity compete with ntrinsically disordered proteins (B)(A) D i s o r de r P r open s i t y M ean ab s o l u t e c hange Mean hydrophobicity -0.751.000.750.50
Disordered Disordered
Hydrophilic
PositiveNegativeHydrophobic
Amino Acids
Figure 2.
The role of charge, hydrophobicity and amino acid residue in IDPs.(A) Nearly 250 folded (open circles) and nearly 90 natively unfolded proteins (blackdiamonds) demonstrate that IDPs are charged and hydrophilic. The solid lineempirically separates between IDPs and compact globular proteins. Panel is adaptedfrom [5]. (B) The contribution of each amino acid in promoting disorder. Disorderedpropensity is evaluated from the fractional difference of amino acids composition ofIDPs in the DisProt database and a completely ordered proteins from the protein-database (PDB). Panel is adapted from [12]. .specificity?Distinct from many structural proteins, IDPs can interact with multiple differentpartners. In analogy to the lock-and-key concept for structural proteins, IDPs serve as“master-keys”, each capable of openning many different “locks” [1, 3, 7]. However, givenIDP’s prevalence, several other functions have been reported including cellular signalingpathways, a regulator for protein-protein and protein-DNA networks or folding intoordered structures upon binding to other cellular counterparts, to name a few (Fig.1) [15–18].In several cases disorder-to-order transition of IDPs is the ensemble’s conforma-tional response to various stimuli. Such response can lead to collapse or expansionof the IDP, e.g. globule-to-coil or collapse transitions [7, 10, 19]. Recently, high-speedAFM measurements demonstrated constantly folded and disordered regions as well asdisorder-to-order transitions in IDPs [19].Unraveling the factors governing the IDPs’ ensemble conformation and theirdisorder-to-order transition is crucial to understand their biological functionalities [20].An illustrative example is the regulation of glucose homeostasis by human pancreaticglucokinase (GCK) enzyme. GCK catalyzes glucose catabolism in the pancreas. At lowglucose concentration, IDR of GCK associates with glucose and undergoes a disordered-to-ordered transition. Following glucose release, the IDR undergoes order-to-disordertransition on millisecond time-scale. This “time delay loop” allows slow turnover andkinetic cooperativity of the enzyme. At high glucose concentration, the delay loop isbypassed and excess glucose is catabolized [21]. ntrinsically disordered proteins has entered clinical use [17–19]. A recently proposed ap-proach to obtain drugs targeting disordered regions relies onthe computational docking of small-molecule fragmentsagainst an ensemble of representative conformations ofthe protein of interest [20]. Its application to a -synuclein,a disordered protein involved in Parkinson’s disease, iden-tified a compound that inhibits the aggregation of a -synu-clein [20]. However, it is still poorly understood whether thiscompound binds more preferentially the monomeric proteinthan its aggregated species. A clearer example of directtargeting of monomeric disordered proteins is the case ofthe oncoprotein c-Myc [21–23]. A recent high-throughputscreening yielded a series of compounds, which interact withits disordered regions and prevent binding to its partner,Max. However, the mechanism of these drug-binding inter-actions remains unclear and these compounds have not yetentered clinical use [21–24].Disordered proteins populate ensembles of many con-formations, each with its own occupation probability. Thebehaviour of disordered proteins is governed by theseensembles and can be drastically different from that ofany individual conformation. Upon interacting with othermolecules, such as protein-binding partners, disorderedproteins may pay an entropic cost because their conforma-tion space is restricted in the bound form, which can becompensated by an enthalpic gain [11,25]. Conversely, inan alternative scenario, a change in the behaviour ofdisordered proteins may be achieved through the use ofsmall molecules, such that the conformational space of adisordered protein is not restricted, but rather entropicallyexpanded by new, transiently bound states. In the follow-ing, we discuss these and other potential mechanismsthrough which small molecules could be effective at target-ing monomeric disordered proteins. Thermodynamics of protein-ligand binding The binding of two molecules occurs spontaneously when itis associated with an overall decrease in free energy ( D G < where D G indicates the difference between the freeenergy G of the final state and that of the initial state. This difference can be expressed as the sum of enthalpic andentropic contributions (Equation D G ¼ D H " T D S [1]where the change in enthalpy ( D H ) is determined by avariety of interatomic forces, including electrostatic, vander Waals, and hydrogen-bonding interactions, and theentropic contribution D S represents the change in the sizeof the conformational space available to the overall system,including the protein, ligand, and solvent molecules.Enthalpic and entropic factors can either contributefavourably or unfavourably to D G , resulting in the fourpossible modes: (i) D H > D S < (ii) D H < D S < (iii) D H < D S > and (iv) D H > D S > Only modes (ii–iv)yield negative D G values, thus lead to binding. Protein-ligand binding systems can be characterised experimen-tally into one of these four modes using, for example,isothermal titration calorimetry (ITC) [26]. ITC experi-ments allow direct, in-solution, label-free determinationof both D G and D H for a protein-ligand binding system,including contributions from the solvent. The difference ofthese observed values can be used to calculate – T D S usingEquation [26–28]. While many protein-ligand bindingevents are driven by enthalpic factors, in some casesentropy can contribute favourably towards a negativechange in free energy and, thus, result in binding.To better understand the role of entropy in protein-ligand binding interactions, we reviewed all entries inthe Binding
Database (BindingDB) for which there arethermodynamic data (139 unique, non-mutant entries).
Wecategorised these entries according to the magnitude of theentropic contributions [27,29–32] (Figure Someenthalpically favourable interactions come at an entropiccost (black points in Figure
This compromise is com-monly referred to as enthalpy–entropy compensation.In rational drug design, it is possible to optimise enthal-pic contributions to promote binding to a target, andoccasionally the entropy is also optimised. This emphasisis reflected by the distribution of the entropic contributionsto binding across the BindingDB (Figure We note that D i s o r d e r c o n t e n t ( % ) Disordered Structured F r a c ! o n o f hu m a n g e n o m e C u m u l a ! v e d i s t r i bu ! o n f un c ! o n Disease*(7508)Druggable(3734 ) Disordered(8061) (A) Cumula ! ve protein frac ! on Human genomeCardiovasculardiseasesDiabetesNeurodegenera ! onDruggable genomeCancer (C) Disorder content (%) (B) T i BS Figure . Prevalence of protein disorder in some common human diseases. (A) Venn diagram of three subsets of the human proteome. Proteins are defined as ‘disordered’if they contain more than of their residues in regions of at least consecutive disordered amino acids, as ‘druggable’ if they are known or predicted to interact withdrugs [1], and as ‘disease-related’ or ‘disease-modifying’ (disease*) if they are involved in cancer, diabetes, neurodegeneration, or cardiovascular diseases (proteins inthese groups were determined with a keyword method adapted from [55,56]). (B) Fraction of proteins encoded by the human genome (right axis) binned according to theircontent of structural disorder (x-axis). Green bins represent highly disordered proteins, and orange bins structured ones.
The black line is the cumulative distributionfunction (left axis). Cartoons illustrate ensembles of three proteins with varying disorder content. (C) Comparison of the amount of protein disorder encoded by the humangenome, by the druggable genome, and in disease-related proteins. Proteins are binned horizontally by disordered content (colour bar). Black boxes represent the fractionof disordered proteins as defined in (A). The analysis of disorder was performed using the s2D method [10]; an individual residue was considered disordered if its a -helicaland b -strand populations are smaller than Opinion
Trends in Biochemical
Sciences
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Vol.
No. Figure 3.
Disordered proteins links to human diseases. (A) Venn diagram ofthree types of proteins: Those that interact with drugs; those that are related todisease; and those that are disordered. (B) Fraction of humans proteins (right axis)binned according to their content of structural disorder. Black line is the cumulativedistribution function (left axis). (C) Amount of disorder in different protein categories.Figure adapted from Ref. [22].
Obviously, the function of IDP is driven by their amino-acid sequence withmutations resulting in numerous diseases (Fig. 3). Known examples are the disorderedregions found in the Alzheimer and Parkinson associated proteins Amyloid- β and α -synuclein, that can form toxic oligomers, amyloid fibrils, and other types of aggregates[23,24]. Other diseases, such as the recently emerged novel coronavirus (SARS-CoV-19),are intimately related to protein disorder. The SARS-CoV-19 genome sequences encodean IDR in its nucleocapsid, an essential structural component that binds to RNA andinteracts with several proteins in its multitasking role [25, 26].IDPs are also present in many biological receptors and play a functional role which isoften poorly understood [27, 28]. For example, the role of the disordered acidic domainsof the Toc159 chloroplast preprotein, which binds to transit peptides, is still unknown[27]. Many RNA binding proteins (RBP) also contain disordered regions. There, thedisordered domains play a central role in regulatory processes (e.g., stability) [29]. Onefascinating class of IDPs comprises neuronal-specific cytoskeleton proteins, includingneurofilament (NF) proteins, α -internexin, vimentin, microtubule-associated protein 2(MAP2), and tau [6, 30–34]. A key motif displayed by these proteins involves long IDRsmediating the assembly of filaments into a hydrogel network [6, 34, 35]. This network isresponsible, for example, for defining the structure, size, and mechanics of axons, withdirect effects on electrical conduction [31, 34, 36–38]. Interestingly, the basic structuralproperties of these networks can be understood using the polymer-physics theoreticalarsenal. Nonetheless, the full functional behaviour is sequence specific, and so can notbe coarse-grained [6, 34, 37].To cope with the enormous traffic of molecular exchange across the nuclear envelope,nuclear pore complexes (NPCs) have evolved to exchange cargo rapidly and with highselectivity. NPCs are composed of 30 different nucleoporins that assemble into sub-complexes to build the NPC’s distinct modules. Though it is not entirely clear howthe cytoplasmic filaments are anchored to the NPC core, the central, disordered proteinsegments of Nup145N, Nup100 and Nup116 are known integral parts of this interaction ntrinsically disordered proteins µ m to 10 µ m andthus can be observed with optical microscopy. For example, the disordered regions ofDdx4 [42], LAF-1 [43], FUS [44], which are rich in glycine, form droplets of 1 µ m.Even folded proteins that have IDRs, such as hnRNPA1 [45] and TDP-43 [46],form droplets, designated as stress granules [47]. Assembly and disassembly of thesegranules is a highly regulated process involving the IDR interactions with multipleproteins and mRNAs. Recently, using multi-bait engineered ascorbate peroxidase(APEX) proximity labeling technique, over a hundred proteins were discovered thateither promote assembly or induced disassembly of stress granules [16]. Importantly,APEX revealed that most of these proteins are indeed disordered. Such studies areextremely relevant for in-depth understanding of the compositional changes in stressgranules’ proteins in neurodegenerative disorders.
3. Artificial IDPs and polymer physics
Disordered proteins’ ability to engage in a variety of manners often results in a richensemble of phase transition behaviors [48, 49]. These phase behaviors can be easilymanipulated by slightly changing the physio-chemical properties of the IDPs. Recently,the Chilkoti group studied a simplified version of artificial IDPs, all made of a repetitive8-mer peptide sequence originated from Drosophilia melanogaster Rec-1 resilin [50].Interestingly, changing a single amino acid in the repeat, or increasing the numberof repeats, results in a drastic and robust change in the LLPS temperature. TheIDP sequence also controls the temperature-ramp hysteresis phenomena. Notably, thishysteresis changes when reversing the amino acid sequence originating from N- to C-terminal [51]. These findings demonstrate the ability to fine-tune the phase behaviorsand indicates to the significance of IDP’s sequence, as in structural proteins.Given IDPs’ structural plasticity, it is tempting to investigate them as polymers,at least to first order (Fig. 4). By doing so, the power of statistical-mechanics can beharnessed to quantify the volumetric dimension of IDPs. Then, coarse-grained structural ntrinsically disordered proteins B NUS Native C NUS Denatured R g R ee A Figure 4. (A) Ensemble structural characterization of IDP that includes the endto end distance ( R ee ), typically measured by FRET, and radius of gyration ( R g ),measured by SAXS. Representative ensembles for NUS protein in its (B) nativecondition and (C) in denatured condition. Both ensembles recapture FRET and SAXSdata at these conditions and show larger asphericity in denatured conditions. Panels(B) and (C) are adapted from Ref. [52]. descriptors are obtained from the scaling laws of polymer physics [53]. Specifically, thedegree of IDP collapse or expansion can be quantified in terms of chain’s length andits relation to the ensemble’s radius of gyration: R g ∝ N ν . Here, N is the number ofmonomers, and ν is the Flory scaling exponent [6, 53].Qualitatively, ν determines the balance between intra-chain and solvent-chaininteractions, also referred to as solvent quality. For good ( ν ≈ . θ ( ν =0.5), or poor( ν ≈ .
3) solvents the conditions are respectively less, equal or favorable intra-chaininteractions respective to solvent/chain ones. Thus, the value of the scaling exponent ν can act as a physical descriptor for the collapse transition as a function of differentenvironmental conditions [54]. In the following, we will introduce several techniquestargeting the nanoscopic dimensions of IDPs.
4. Nanoscopic techniques
The detection and characterization of disordered proteins and regions are ofgreat interest in order to determine their function. IDPs’ structural plasticitydictate techniques and analyses targeting representative parameters of the protein’sconformational ensemble [55]. Naturally, we will not be able to discuss the entire breadthof techniques; instead, we will focus on some of those targeting the nano-scale regime,which is, in most cases, the relevant length-scale.
To further understand the conformational dynamics of IDPs, advanced moleculardynamics (MD) simulations are used. However, the inherently large number of degrees-of-freedom, the inaccuracies in the simulation models, and the need for long simulationshave stymied progress. To overcome these limitations, alternative approaches have ntrinsically disordered proteins ν = 0 .
6) behavior when considering R g vs. N . Yet, the simulated structural ensemblesshowed significant heterogeneity: certain IDPs showed elevated intra-segment contactsand transient sub-segment compactions. However, this was not universal: other IDPsin the study behaved as homogeneous random-walk chains. The authors concluded thatsequence-specific structure could be hidden behind polymeric exponents [62]. A common and powerful technique suited for IDP structural characterization is SAXS.The technique’s key advantage is measuring the ensemble conformations while kept insolution without the need for crystallization or external tagging. A simple SAXS analysisimmediately provides R g and the pair distance distribution [63, 64]. Additionally,traditional Kratky plots ( q I ( q ) vs. q ), directly obtained from the SAXS profiles ofIDPs, provide a qualitative picture of the presence of globular or unfolded conformations[65, 66]. Here, I is the scattering intensity, and q is the scattered wave-vectorproportional to the scattering angle.Moreover, the scattering profile can be used to extract the Flory exponent ( ν )mentioned above [52, 67]. More advanced analysis techniques are regularly used toextract the ensemble’s dominant conformations from the SAXS signal (Fig. 4). Theensemble optimization method [68, 69], and molecular form-factor [54] are just a fewexamples for such techniques resulting in representative conformations that fit theexperimental data. We note that the combination of SAXS with other complementarymethods (Fig. 4) such as simulations, NMR, and F¨orster resonance energy transfer(FRET) provides a more comprehensive picture of IDPs, and their conformations insolution [6, 37, 52, 55, 63, 70, 71]. The distance-dependent dipole-dipole interactions between probes bound to the sidechains of IDPs provide the basis for determining long-range intra-molecular distancesbetween selected sites. Experiments based on single-molecule [72] or ensemble [73]measurements are characterized by 1 −
10 nm distance resolution range and an abilityto recover distributions of intra-molecular distances in the transient ensembles of IDP. ntrinsically disordered proteins α -synuclein deviates from ideal chain behavior atsegments labeled in the NAC domain and N-termini using the ensemble method. It wassuggested that those conformations bias might be related to the initiation of amyloidtransition [73]. Another example, using a single-molecule method, showed that thepreviously mentioned scaling exponents ( ν ) in water strongly depend on the sequencecomposition. Two of the total examined IDPs did not reach the θ -point under anysolvent conditions. This may reflect their biological functional need for an expandedstate optimized for interactions with cellular partners [72]. NMR spectroscopy offers a unique platform in deciphering IDP dynamics and structure[74]. The traditional NMR hydrogen chemical shift signal is, in many cases, insufficientfor IDP characterization. Fortunately, new tools have been developed to overcome lowsignal to noise ratio difficulties such as paramagnetic relaxation enhancement (PRE) [75]and proline based 2D H-N residue correlations [76].In PRE, the introduction of paramagnetic spin labels in proteins affects the chemicalshift and transverse relaxation rate signal between the unpaired electron and NMR activenuclei on the basis of distance between them [75]. For example, the PRE signal and N relaxation data was analyzed to quantify the interaction between the IDP osteopontinand heparin [77]. There, it was found that on binding with heparin, osteopontin largelyremains in a disordered state and undergoes structural/dynamical adaption which ismainly mediated by electrostatic interactions.A recent development using in-cell NMR spectroscopy provides the opportunityto explore the structural plasticity of an IDP in its native environment [78, 79]. Botheukaryotic and prokaryotic IDPs have been investigated using in-cell NMR such as α -synuclein, prokaryotic ubiquitin-like protein, Pup, tubulin-related neuronal protein, tau,FG-Nups in nucleoporins, and the negative regulator of flagellin synthesis, FlgM. Thesestudies revealed that the cellular conformational dynamics may differ significantly fromthese observed in-vitro [80]. Due to diffusion and the protein structural plasticity, the emission of fluorescently-labeled molecules fluctuate. In fluorescence correlation spectroscopy (FCS), thesefluctuations are recorded within an illuminated confocal volume [81–83]. The timescalefor conformational fluctuation (i.e., chemical kinetics within the biomolecule) lies in thenano- to microseconds range, whereas for translational diffusion from microsecond tomilliseconds. Information about the diffusion coefficient and chemical kinetics can beinferred from the fluorescence fluctuations autocorrelation and cross-correlation signal.Furthermore, from the diffusion coefficient, it is possible to determine the molecular sizeand hydrodynamic radius of the investigated biomolecules. ntrinsically disordered proteins fL SingleNFL tailNFL tailpolymerA B CFoldons
Figure 5.
Mechanical perturbation reveals subtle internal IDP structure. (A) Underconstant tension f , the length L of a polymer of NFL tails is measured. (B) A singleNFL tail contains multiple, independent regions with internal structure (foldons).(C) An initial large tension breaks apart internal structure in the NFL tails. Afterrapidly lowering the tension, at constant tension, the NFL polymer shows a logarithmicdecrease in length over time t , indicating multiple regions of internal structure arereforming. Panel (C) is adapted from Ref. [86]. FCS is also sensitive to fluorophore quenching reactions. These measurementscan provide information about the internal dynamics of the IDPs [84]. Fluorescenceself-quenching of tetramethyl rhodamine, which is chemically labelled at two differentresidual positions, was analyzed to study the conformational kinetics of unfoldedintestinal fatty acid binding protein [85] and amyloid forming yeast prion protein,Sup35 [84].
Single-molecule force spectroscopy (SMFS) uses the thermodynamic effects of appliedtension to gain insight into the nanoscopic conformation of IDPs. SMFS consists oftethering an IDP between a static surface and a force probe and measuring the chainextension, with nanometer accuracy, as a function of force. This nanomechanical assay isa high-precision differential measurement of the effect of varying perturbations (tensions)on IDP interactions and conformations. For example, AFM-based experiments onseveral amyloid precursor IDPs show a sawtooth pattern that indicates the mechanicalunfolding of multiple different conformations [87]. Similarly, optical tweezer experimentson α -synuclein show it has several marginally stable and rapidly fluctuating subsegments[88]. A noteworthy recent work demonstrated the power of SMFS-based perturbationin revealing more subtle IDP structural behaviors [86]. In the work, a polyproteinof the disordered Neurofilament light-chain tail (NFLt) domain was subjected to force-quench experiments, in which a large tension pulled the chain straight, and was followedby a sudden jump to a small tension that permitted structure in the IDP to re-form(Fig. 5). The experiment was carried out in a magnetic tweezer, an SMFS instrument ntrinsically disordered proteins ≈ Intermolecular interactions of biomolecules play an essential role in the proper functionand growth of biological cells [89–91]. These interactions can be specific or non-specific.Non-specific interactions are transient and weak, mainly governed by steric repulsion orionic bridging. IDPs’ function is largely dominated by these weak interactions. Thiswas demonstrated, for example, in a recent AFM-based SMFS study, in which theinteractions between nuclear transport factors and disordered FG repeats was foundto display unexpected complexities due to weak, multivalent contacts between theinteracting partners [92].Improved protein-protein interaction resolution can be achieved using bulk, low-throughput spectroscopic methods such as FRET, surface plasmon resonance (SPR),and nuclear magnetic resonance (NMR), to name only a few. For example, theinteraction between the disordered region in transcription factors Sp1 and TAF4 wasestimated by NMR to be K d = 69 µ M [93]. IDP Ntr2 modulates the RNA HelicaseBrr2 with K d = 14 µ M and 7 µ M determined by SPR and NMR, respectively [94]. SPRwas also used to detect and characterize IDP–ligand interactions with tau protein as amodel IDP [95].Another recently introduced technique aiming to overcome the technical difficultiesto measure weak and transient protein-protein interaction is the nanoparticle mobilityassay [13]. There, nanoparticles grafted with IDPs were imaged while diffusing over asurface grafted with a second set of IDPs. A similar approach was used to study strongDNA-DNA interactions [96], delocalized long-range polymer-surface interactions [97]and bulk-mediated diffusion on supported lipid-bilayers [98]. By carefully analyzing theparticles’ diffusive nature, the authors detected an altered interaction caused by a singlemutation on the polypeptide sequence [13].
5. Summary and perspective
Two decades of IDP research place them at the forefront of proteomics [1]. Apparently,structural plasticity is valuable primarily because it enables the IDPs to adjust to theirenvironment. In addition, IDPs are also used in various nano-biomedical technologies.For example,
PASylation technology involves conjugating pharmacologically activecompounds with natively disordered biosynthetic polymers made of the small L-amino ntrinsically disordered proteins function could have been understood from coarse metrics suchas the IDP’s length, total charge, net hydrophobicity, etc. However, as with foldedproteins, the sequence of amino acids, and not just the net composition, does dictatethe resulting function, at least to some extent. Yet building a coarse-grained model forIDPs is a highly complicated and fascinating problem due to their unique properties.The most fundamental difficulty is somewhat technical - the large ensemble of IDPconformations is often impossible to sample with modern computers [104]. Anotherproblem evolves from the extended amount of electrostatic interactions that frequentlyexist in IDP systems. Those interactions tend to span over a broad range of length andtime scales, and hence are hard to quantify by a finite force field [105], although newIDP-specific force fields have been suggested [106–109]. Nevertheless, further researchis highly needed, especially since many phenomena involving IDPs are still not fullyunderstood [110].
Acknowledgments
This work was supported by the National Science Foundation under Grant No. 1715627,the United States-Israel Bi-national Science Foundation under Grant No. 2016696, andthe Israel Science Foundation under Grants No. 1454/19.
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