Effects of Molecular Crowding on the Dynamics of Intrinsically Disordered Proteins

Inside cells, the concentration of macromolecules can reach up to 400 g/L. In such crowded environments, proteins are expected to behave differently than in vitro. It has been shown that the stability and the folding rate of a globular protein can be altered by the excluded volume effect produced by a high density of macromolecules. However, macromolecular crowding effects on intrinsically disordered proteins (IDPs) are less explored. These proteins can be extremely dynamic and potentially sample a wide ensemble of conformations under non-denaturing conditions. The dynamic properties of IDPs are intimately related to the timescale of conformational exchange within the ensemble, which govern target recognition and how these proteins function. In this work, we investigated the macromolecular crowding effects on the dynamics of several IDPs by measuring the NMR spin relaxation parameters of three disordered proteins (ProTα, TC1, and α-synuclein) with different extents of residual structures. To aid the interpretation of experimental results, we also performed an MD simulation of ProTα. Based on the MD analysis, a simple model to correlate the observed changes in relaxation rates to the alteration in protein motions under crowding conditions was proposed. Our results show that 1) IDPs remain at least partially disordered despite the presence of high concentration of other macromolecules, 2) the crowded environment has differential effects on the conformational propensity of distinct regions of an IDP, which may lead to selective stabilization of certain target-binding motifs, and 3) the segmental motions of IDPs on the nanosecond timescale are retained under crowded conditions. These findings strongly suggest that IDPs function as dynamic structural ensembles in cellular environments.     

Intrinsically Disordered Protein Exhibits Both Compaction and Expansion under Macromolecular Crowding

Conformational malleability allows intrinsically disordered proteins (IDPs) to respond agilely to their environments, such as nonspecifically interacting with in vivo bystander macromolecules (or crowders). Previous studies have emphasized conformational compaction of IDPs due to steric repulsion by macromolecular crowders, but effects of soft attraction are largely unexplored. Here we studied the conformational ensembles of the IDP FlgM in both polymer and protein crowders by small-angle neutron scattering. As crowder concentrations increased, the mean radius of gyration of FlgM first decreased but then exhibited an uptick. Ensemble optimization modeling indicated that FlgM conformations under protein crowding segregated into two distinct populations, one compacted and one extended. Coarse-grained simulations showed that compacted conformers fit into an interstitial void and occasionally bind to a surrounding crowder, whereas extended conformers snake through interstitial crevices and bind multiple crowders simultaneously. Crowder-induced conformational segregation may facilitate various cellular functions of IDPs.     

Chain collapse of an amyloidogenic intrinsically disordered protein

Natively unfolded or intrinsically disordered proteins (IDPs) are under intense scrutiny due to their involvement in both normal biological functions and abnormal protein misfolding disorders. Polypeptide chain collapse of amyloidogenic IDPs is believed to play a key role in protein misfolding, oligomerization, and aggregation leading to amyloid fibril formation, which is implicated in a number of human diseases. In this work, we used bovine κ-casein, which serves as an archetypal model protein for amyloidogenic IDPs. Using a variety of biophysical tools involving both prediction and spectroscopic techniques, we first established that monomeric κ-casein adopts a collapsed premolten-globule-like conformational ensemble under physiological conditions. Our time-resolved fluorescence and light-scattering data indicate a change in the mean hydrodynamic radius from ∼4.6 nm to ∼1.9 nm upon chain collapse. We then took the advantage of two cysteines separated by 77 amino-acid residues and covalently labeled them using thiol-reactive pyrene maleimide. This dual-labeled protein demonstrated a strong excimer formation upon renaturation from urea- and acid-denatured states under both equilibrium and kinetic conditions, providing compelling evidence of polypeptide chain collapse under physiological conditions. The implication of the IDP chain collapse in protein aggregation and amyloid formation is also discussed.     

Not Only Expansion: Proline Content and Density Also Induce Disordered Protein Conformation Compaction

Intrinsically disordered proteins (IDPs) adopt a wide array of different conformations that can be constrained by the presence of proline residues, which are frequently found in IDPs. To assess the effects of proline, we designed a series of peptides that differ with respect to the number of prolines in the sequence and their organization. Using high-resolution atomistic molecular dynamics simulations, we found that accounting for whether the proline residues are clustered or isolated contributed significantly to explaining deviations in the experimentally-determined gyration radii of IDPs from the values expected based on the Flory scaling-law. By contrast, total proline content makes smaller contribution to explaining the effect of prolines on IDP conformation. Proline residues exhibit opposing effects depending on their organizational pattern in the IDP sequence. Clustered prolines (i.e., prolines with ≤2 intervening non-proline residues) result in expanded peptide conformations whereas isolated prolines (i.e., prolines with >2 intervening non-proline residues) impose compacted conformations. Clustered prolines were estimated to induce an expansion of ∼20% in IDP dimension (via formation of PPII structural elements) whereas isolated prolines were estimated to induce a compaction of ∼10% in IDP dimension (via the formation of backbone turns). This dual role of prolines provides a mechanism for conformational switching that does not rely on the kinetically much slower isomerization of cis proline to the trans form. Bioinformatic analysis demonstrates high populations of both isolated and clustered prolines and implementing them in coarse-grained molecular dynamics models illustrates that they improve the characterization of the conformational ensembles of IDPs.     

Deuterium isotope shifts for backbone 1H, 15N and 13C nuclei in intrinsically disordered protein α-synuclein

Intrinsically disordered proteins (IDPs) are abundant in nature and characterization of their potential structural propensities remains a widely pursued but challenging task. Analysis of NMR secondary chemical shifts plays an important role in such studies, but the output of such analyses depends on the accuracy of reference random coil chemical shifts. Although uniform perdeuteration of IDPs can dramatically increase spectral resolution, a feature particularly important for the poorly dispersed IDP spectra, the impact of deuterium isotope shifts on random coil values has not yet been fully characterized. Very precise ²H isotope shift measurements for ¹³C^??, ¹³C^??, ¹³C??, ¹⁵N, and ¹H^N have been obtained by using a mixed sample of protonated and uniformly perdeuterated ??-synuclein, a protein with chemical shifts exceptionally close to random coil values. Decomposition of these isotope shifts into one-bond, two-bond and three-bond effects as well as intra- and sequential residue contributions shows that such an analysis, which ignores conformational dependence, is meaningful but does not fully describe the total isotope shift to within the precision of the measurements. Random coil ²H isotope shifts provide an important starting point for analysis of such shifts in structural terms in folded proteins, where they are known to depend strongly on local geometry.     

Macromolecular Crowding Induces a Binding Competent Transient Structure in Intrinsically Disordered Gab1

Intrinsically disordered proteins (IDPs) are an important class of proteins which lack tertiary structure elements. Their dynamic properties can depend on reversible post-translational modifications and the complex cellular milieu, which provides a crowded environment. Both influences the thermodynamic stability and folding of globular proteins as well as the conformational plasticity of IDPs. Here we investigate the intrinsically disordered C-terminal region (amino acids 613–694) of human Grb2-associated binding protein 1 (Gab1), which binds to the disease-relevant Src homolog region 2 (SH2) domain-containing protein tyrosine phosphatase SHP2 (PTPN11). This binding is mediated by phosphorylation at Tyr 627 and Tyr 659 in Gab1. We characterize induced structure in Gab1_613–694 and binding to SHP2 by NMR, CD and ITC under non-crowding and crowding conditions, employing chemical and biological crowding agents and compare the results of the non-phosphorylated and tyrosine phosphorylated C-terminal Gab1 fragment. Our results show that under crowding conditions pre-structured motifs in two distinct regions of Gab1 are formed whereas phosphorylation has no impact on the dynamics and IDP character. These structured regions are identical to the binding regions towards SHP2. Therefore, biological crowders could induce some SHP2 binding capacity. Our results therefore indicate that high concentrations of macromolecules stabilize the preformed or excited binding state in the C-terminal Gab1 region and foster the binding to the SH2 tandem motif of SHP2, even in the absence of tyrosine phosphorylation.     

Transient Tertiary Structures of Disordered Dynein Intermediate Chain Regulate its Interactions with Multiple Partners

The N-terminal domain of dynein intermediate chain (N–IC) is central to the cytoplasmic dynein ‘cargo attachment subcomplex’ and regulation of motor activity. It is a prototypical intrinsically disordered protein (IDP), serving as a primarily disordered polybivalent molecular scaffold for numerous binding partners, including three dimeric dynein light chains and coiled coil domains of dynein partners dynactin p150^Glued and NudE. At the very N-terminus, a 40 amino acid single alpha helix (SAH) forms the major binding site for both p150^Glued and NudE, while a shorter nascent helix (H2) separated from SAH by a disordered linker, is necessary for tight binding to dynactin p150^Glued but not to NudE. Here we demonstrate that transient tertiary interactions in this highly dynamic protein underlie the differences in its interactions with p150^Glued and NudE. NMR paramagnetic relaxation enhancement experiments and restrained molecular dynamics simulations identify interactions between the two non-contiguous SAH and H2 helical regions, the extent of which correlates with the length and stability of H2, showing clearly that tertiary and secondary structure formation are coupled in IDPs. These interactions are significantly attenuated when N–IC is bound to NudE, suggesting that NudE binding shifts the conformational ensemble to one that is more extended and with less structure in H2. While the intrinsic disorder and flexibility in N–IC modulate its ability to serve as a binding platform for numerous partners, deviations of this protein from random-coil behavior provide a process for regulating these binding interactions and potentially the dynein motor.     

Studying backbone torsional dynamics of intrinsically disordered proteins using fluorescence depolarization kinetics

Intrinsically disordered proteins (IDPs) do not autonomously adopt a stable unique 3D structure and exist as an ensemble of rapidly interconverting structures. They are characterized by significant conformational plasticity and are associated with several biological functions and dysfunctions. The rapid conformational fluctuation is governed by the backbone segmental dynamics arising due to the dihedral angle fluctuation on the Ramachandran ?–ψ conformational space. We discovered that the intrinsic backbone torsional mobility can be monitored by a sensitive fluorescence readout, namely fluorescence depolarization kinetics, of tryptophan in an archetypal IDP such as α-synuclein. This methodology allows us to map the site-specific torsional mobility in the dihedral space within picosecond-nanosecond time range at a low protein concentration under the native condition. The characteristic timescale of ~?1.4 ns, independent of residue position, represents collective torsional dynamics of dihedral angles (? and ψ) of several residues from tryptophan and is independent of overall global tumbling of the protein. We believe that fluorescence depolarization kinetics methodology will find broad application to study both short-range and long-range correlated motions, internal friction, binding-induced folding, disorder-to-order transition, misfolding and aggregation of IDPs.     

Soft interactions and volume exclusion by polymeric crowders can stabilize or destabilize transient structure in disordered proteins depending on polymer concentration

The effects of macromolecular crowding on the transient structure of intrinsically disordered proteins is not well‐understood. Crowding by biological molecules inside cells could modulate transient structure and alter IDP function. Volume exclusion theory and observations of structured proteins suggest that IDP transient structure would be stabilized by macromolecular crowding. Amide hydrogen exchange (HX) of IDPs in highly concentrated polymer solutions would provide valuable insights into IDP transient structure under crowded conditions. Here, we have used mass spectrometry to measure HX by a transiently helical random coil domain of the activator of thyroid and retinoid receptor (ACTR) in solutions containing 300 g L^?1 and 400 g L^?1 of Ficoll, a synthetic polysaccharide, using a recently‐developed strong cation exchange‐based cleanup method [Rusinga, et al., Anal Chem 2017;89:1275–1282]. Transiently helical regions of ACTR exchanged faster in 300 g L^?1 Ficoll than in dilute buffer. In contrast, one transient helix exchanged more slowly in 400 g L^?1 Ficoll. Nonspecific interactions destabilize ACTR helicity in 300 g L^?1 Ficoll because ACTR engages with the Ficoll polymer mesh. In contrast, 400 g L^?1 Ficoll is a semi‐dilute solution where ACTR cannot engage the Ficoll mesh. At this higher concentration, volume exclusion stabilizes ACTR helicity because ACTR is compacted in interstitial spaces between Ficoll molecules. Our results suggest that the interplay between nonspecific interactions and volume exclusion in different cellular compartments could modulate IDP function by altering the stability of IDP transient structures. Proteins 2017; 85:1468–1479. © 2017 Wiley Periodicals, Inc.     

Identification of minimally interacting modules in an intrinsically disordered protein

The conformational characterization of intrinsically disordered proteins (IDPs) is complicated by their conformational heterogeneity and flexibility. If an IDP could somehow be divided into smaller fragments and reconstructed later, theoretical and spectroscopic studies could probe its conformational variability in detail. Here, we used replica molecular-dynamics simulations and network theory to explore whether such a divide-and-conquer strategy is feasible for α-synuclein, a prototypical IDP. We characterized the conformational variability of α-synuclein by conducting >100 unbiased all-atom molecular-dynamics simulations, for a total of >10 μs of trajectories. In these simulations, α-synuclein formed a heterogeneous ensemble of collapsed coil states in an aqueous environment. These states were stabilized by heterogeneous contacts between sequentially distant regions. We find that α-synuclein contains residual secondary structures in the collapsed states, and the heterogeneity in the collapsed state makes it feasible to split α-synuclein into sequentially contiguous minimally interacting fragments. This study reveals previously unknown characteristics of α-synuclein and provides a new (to our knowledge) approach for studying other IDPs.     

Structural Characterization of N-WASP Domain V Using MD Simulations with NMR and SAXS Data

Because of their large conformational heterogeneity, structural characterization of intrinsically disordered proteins (IDPs) is very challenging using classical experimental methods alone. In this study, we use NMR and small-angle x-ray scattering (SAXS) data with multiple molecular dynamics (MD) simulations to describe the conformational ensemble of the fully disordered verprolin homology domain of the neural Aldrich syndrome protein involved in the regulation of actin polymerization. First, we studied several back-calculation software of SAXS scattering intensity and optimized the adjustable parameters to accurately calculate the SAXS intensity from an atomic structure. We also identified the most appropriate force fields for MD simulations of this IDP. Then, we analyzed four conformational ensembles of neural Aldrich syndrome protein verprolin homology domain, two generated with the program flexible-meccano with or without NMR-derived information as input and two others generated by MD simulations with two different force fields. These four conformational ensembles were compared to available NMR and SAXS data for validation. We found that MD simulations with the AMBER-03w force field and the TIP4P/2005s water model are able to correctly describe the conformational ensemble of this 67-residue IDP at both local and global level.     

Intrinsically Disordered Proteins and Their Environment: Effects of Strong Denaturants, Temperature, pH, Counter Ions, Membranes, Binding Partners, Osmolytes, and Macromolecular Crowding

Intrinsically disordered proteins (IDPs) differ from “normal” ordered proteins at several levels, structural, functional and conformational. Amino acid biases characteristic for IDPs determine their structural variability and lack of rigid well-folded structure. This structural plasticity is necessary for the unique functional repertoire of IDPs, which is complementary to the catalytic activities of ordered proteins. Amino acid biases also drive atypical responses of IDPs to changes in their environment. The conformational behavior of IDPs is characterized by the low cooperativity (or the complete lack thereof) of the denaturant-induced unfolding, lack of the measurable excess heat absorption peak(s) characteristic for the melting of ordered proteins, “turned out” response to heat and changes in pH, the ability to gain structure in the presence of various counter ions, osmolytes, membranes and binding partners, and by the unique response to macromolecular crowding. This review describes some of the most characteristic features of the IDP conformational behavior and the unique response of IDPs to changes in their environment.     

Conformational response to charge clustering in synthetic intrinsically disordered proteins

Background

Recent theoretical and computational studies have shown that the charge content and, most importantly, the linear distribution of opposite charges are major determinants of conformational properties of intrinsically disordered proteins (IDPs). Charge segregation in a sequence can be measured through κ, which represents a normalized measure of charge asymmetry. A strong inverse correlation between κ and radius of gyration has been previously demonstrated for two independent sets of permutated IDP sequences.

Using a FRET Library with Multiple Probe Pairs To Drive Monte Carlo Simulations of α-Synuclein

We describe a strategy for experimentally-constraining computational simulations of intrinsically disordered proteins (IDPs), using α-synuclein, an IDP with a central role in Parkinson’s disease pathology, as an example. Previously, data from single-molecule Förster Resonance Energy Transfer (FRET) experiments have been effectively utilized to generate experimentally constrained computational models of IDPs. However, the fluorophores required for single-molecule FRET experiments are not amenable to the study of short-range (<30 Å) interactions. Using ensemble FRET measurements allows one to acquire data from probes with multiple distance ranges, which can be used to constrain Monte Carlo simulations in PyRosetta. To appropriately employ ensemble FRET data as constraints, we optimized the shape and weight of constraining potentials to afford ensembles of structures that are consistent with experimental data. We also used this approach to examine the structure of α-synuclein in the presence of the compacting osmolyte trimethylamine-N-oxide. Despite significant compaction imparted by 2 M trimethylamine-N-oxide, the underlying ensemble of α-synuclein remains largely disordered and capable of aggregation, also in agreement with experimental data. These proof-of-concept experiments demonstrate that our modeling protocol enables one to efficiently generate experimentally constrained models of IDPs that incorporate atomic-scale detail, allowing one to study an IDP under a variety of conditions.     

Side chain electrostatic interactions and pH‐dependent expansion of the intrinsically disordered,highly acidic carboxyl‐terminus of γ‐tubulin

Intramolecular electrostatic attraction and repulsion strongly influence the conformational sampling of intrinsically disordered proteins and domains (IDPs). In order to better understand this complex relationship, we have used nuclear magnetic resonance to measure side chain pK_a values and pH‐dependent translational diffusion coefficients for the unstructured and highly acidic carboxyl‐terminus of γ‐tubulin (γ‐CT), providing insight into how the net charge of an IDP relates to overall expansion or collapse of the conformational ensemble. Many of the pK_a values in the γ‐CT are shifted upward by 0.3–0.4 units and exhibit negatively cooperative ionization pH profiles, likely due to the large net negative charge that accumulates on the molecule as the pH is raised. pK_a shifts of this magnitude correspond to electrostatic interaction energies between the affected residues and the rest of the charged molecule that are each on the order of 1 kcal mol^?1. Diffusion of the γ‐CT slowed with increasing net charge, indicative of an expanding hydrodynamic radius (r_H). The degree of expansion agreed quantitatively with what has been seen from comparisons of IDPs with different charge content, yielding the general trend that every 0.1 increase in relative charge (|Q|/res) produces a roughly 5% increase in r_H. While γ‐CT pH titration data followed this trend nearly perfectly, there were substantially larger deviations for the database of different IDP sequences. This suggests that other aspects of an IDP's primary amino acid sequence beyond net charge influence the sensitivity of r_H to electrostatic interactions.     

How multisite phosphorylation impacts the conformations of intrinsically disordered proteins

Phosphorylation of intrinsically disordered proteins (IDPs) can produce changes in structural and dynamical properties and thereby mediate critical biological functions. How phosphorylation effects intrinsically disordered proteins has been studied for an increasing number of IDPs, but a systematic understanding is still lacking. Here, we compare the collapse propensity of four disordered proteins, Ash1, the C-terminal domain of RNA polymerase (CTD2’), the cytosolic domain of E-Cadherin, and a fragment of the p130Cas, in unphosphorylated and phosphorylated forms using extensive all-atom molecular dynamics (MD) simulations. We find all proteins to show V-shape changes in their collapse propensity upon multi-site phosphorylation according to their initial net charge: phosphorylation expands neutral or overall negatively charged IDPs and shrinks positively charged IDPs. However, force fields including those tailored towards and commonly used for IDPs overestimate these changes. We find quantitative agreement of MD results with SAXS and NMR data for Ash1 and CTD2’ only when attenuating protein electrostatic interactions by using a higher salt concentration (e.g. 350 mM), highlighting the overstabilization of salt bridges in current force fields. We show that phosphorylation of IDPs also has a strong impact on the solvation of the protein, a factor that in addition to the actual collapse or expansion of the IDP should be considered when analyzing SAXS data. Compared to the overall mild change in global IDP dimension, the exposure of active sites can change significantly upon phosphorylation, underlining the large susceptibility of IDP ensembles to regulation through post-translational modifications.     

Monitoring the conformational changes of an intrinsically disordered peptide using a quartz crystal microbalance

Intrinsically disordered peptides (IDPs) have recently garnered much interest because of their role in biological processes such as molecular recognition and their ability to undergo stimulus-responsive conformational changes. The block V repeat-in-toxin motif of the Bordetella pertussis adenylate cyclase is an example of an IDP that undergoes a transition from a disordered state to an ordered beta roll conformation in the presence of calcium ions. In solution, a C-terminal capping domain is necessary for this transition to occur. To further explore the conformational behavior and folding requirements of this IDP, we have cysteine modified three previously characterized constructs, allowing for attachment to the gold surface of a quartz crystal microbalance (QCM). We demonstrate that, while immobilized, the C-terminally capped peptide exhibits similar calcium-binding properties to what have been observed in solution. In addition, immobilization on the solid surface appears to enable calcium-responsiveness in the uncapped peptides, in contrast to the behavior observed in solution. This work demonstrates the power of QCM as a tool to study the conformational changes of IDPs immobilized on surfaces and has implications for a range of potential applications where IDPs may be engineered and used including protein purification, biosensors, and other bionanotechnology applications.