Minimal Effects of Macromolecular Crowding on an Intrinsically Disordered Protein: A Small-Angle Neutron Scattering Study

Small-angle neutron scattering was used to study the effects of macromolecular crowding by two globular proteins, i.e., bovine pancreatic trypsin inhibitor and equine metmyoglobin, on the conformational ensemble of an intrinsically disordered protein, the N protein of bacteriophage λ. The λ N protein was uniformly labeled with ²H, and the concentrations of D₂O in the samples were adjusted to match the neutron scattering contrast of the unlabeled crowding proteins, thereby masking their contribution to the scattering profiles. Scattering from the deuterated λ N was recorded for samples containing up to 0.12 g/mL bovine pancreatic trypsin inhibitor or 0.2 g/mL metmyoglobin. The radius of gyration of the uncrowded protein was estimated to be 30 Å and was found to be remarkably insensitive to the presence of crowders, varying by <2 Å for the highest crowder concentrations. The scattering profiles were also used to estimate the fractal dimension of λ N, which was found to be ∼1.8 in the absence or presence of crowders, indicative of a well-solvated and expanded random coil under all of the conditions examined. These results are contrary to the predictions of theoretical treatments and previous experimental studies demonstrating compaction of unfolded proteins by crowding with polymers such as dextran and Ficoll. A computational simulation suggests that some previous treatments may have overestimated the effective volumes of disordered proteins and the variation of these volumes within an ensemble. The apparent insensitivity of λ N to crowding may also be due in part to weak attractive interactions with the crowding proteins, which may compensate for the effects of steric exclusion.     

Minimal Effects of Macromolecular Crowding on an Intrinsically Disordered Protein: A Small-Angle Neutron Scattering Study

Small-angle neutron scattering was used to study the effects of macromolecular crowding by two globular proteins, i.e., bovine pancreatic trypsin inhibitor and equine metmyoglobin, on the conformational ensemble of an intrinsically disordered protein, the N protein of bacteriophage λ. The λ N protein was uniformly labeled with ²H, and the concentrations of D₂O in the samples were adjusted to match the neutron scattering contrast of the unlabeled crowding proteins, thereby masking their contribution to the scattering profiles. Scattering from the deuterated λ N was recorded for samples containing up to 0.12 g/mL bovine pancreatic trypsin inhibitor or 0.2 g/mL metmyoglobin. The radius of gyration of the uncrowded protein was estimated to be 30 Å and was found to be remarkably insensitive to the presence of crowders, varying by <2 Å for the highest crowder concentrations. The scattering profiles were also used to estimate the fractal dimension of λ N, which was found to be ∼1.8 in the absence or presence of crowders, indicative of a well-solvated and expanded random coil under all of the conditions examined. These results are contrary to the predictions of theoretical treatments and previous experimental studies demonstrating compaction of unfolded proteins by crowding with polymers such as dextran and Ficoll. A computational simulation suggests that some previous treatments may have overestimated the effective volumes of disordered proteins and the variation of these volumes within an ensemble. The apparent insensitivity of λ N to crowding may also be due in part to weak attractive interactions with the crowding proteins, which may compensate for the effects of steric exclusion.     

Characterization of proteins with wide-angle X-ray solution scattering (WAXS)

X-ray solution scattering in both the small-angle (SAXS) and wide-angle (WAXS) regimes is making an increasing impact on our understanding of biomolecular complexes. The accurate calculation of WAXS patterns from atomic coordinates has positioned the approach for rapid growth and integration with existing Structural Genomics efforts. WAXS data are sensitive to small structural changes in proteins; useful for calculation of the pair-distribution function at relatively high resolution; provides a means to characterize the breadth of the structural ensemble in solution; and can be used to identify proteins with similar folds. WAXS data are often used to test structural models, identify structural similarities and characterize structural changes. WAXS is highly complementary to crystallography and NMR. It holds great potential for the testing of structural models of proteins; identification of proteins that may exhibit novel folds; characterization of unfolded or natively disordered proteins; and detection of structural changes associated with protein function.     

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.     

RNA chaperoning and intrinsic disorder in the core proteins of Flaviviridae

RNA chaperone proteins are essential partners of RNA in living organisms and viruses. They are thought to assist in the correct folding and structural rearrangements of RNA molecules by resolving misfolded RNA species in an ATP-independent manner. RNA chaperoning is probably an entropy-driven process, mediated by the coupled binding and folding of intrinsically disordered protein regions and the kinetically trapped RNA. Previously, we have shown that the core protein of hepatitis C virus (HCV) is a potent RNA chaperone that can drive profound structural modifications of HCV RNA in vitro. We now examined the RNA chaperone activity and the disordered nature of core proteins from different Flaviviridae genera, namely that of HCV, GBV-B (GB virus B), WNV (West Nile virus) and BVDV (bovine viral diarrhoea virus). Despite low-sequence similarities, all four proteins demonstrated general nucleic acid annealing and RNA chaperone activities. Furthermore, heat resistance of core proteins, as well as far-UV circular dichroism spectroscopy suggested that a well-defined 3D protein structure is not necessary for core-induced RNA structural rearrangements. These data provide evidence that RNA chaperoning—possibly mediated by intrinsically disordered protein segments—is conserved in Flaviviridae core proteins. Thus, besides nucleocapsid formation, core proteins may function in RNA structural rearrangements taking place during virus replication.     

On the Calculation of SAXS Profiles of Folded and Intrinsically Disordered Proteins from Computer Simulations

Solution techniques such as small-angle X-ray scattering (SAXS) play a central role in structural studies of intrinsically disordered proteins (IDPs); yet, due to low resolution, it is generally necessary to combine SAXS with additional experimental sources of data and to use molecular simulations. Computational methods for the calculation of theoretical SAXS intensity profiles can be separated into two groups, depending on whether the solvent is modeled implicitly as continuous electron density or considered explicitly. The former offers reduced computational cost but requires the definition of a number of free parameters to account for, for example, the excess density of the solvation layer. Overfitting can thus be an issue, particularly when the structural ensemble is unknown. Here, we investigate and show how small variations of the contrast of the hydration shell, δρ, severely affect the outcome, analysis and interpretation of computed SAXS profiles for folded and disordered proteins. For both the folded and disordered proteins studied here, using a default δρ may, in some cases, result in the calculation of non-representative SAXS profiles, leading to an overestimation of their size and a misinterpretation of their structural nature. The solvation layer of the different IDP simulations also impacts their size estimates differently, depending on the protein force field used. The same is not true for the folded protein simulations, suggesting differences in the solvation of the two classes of proteins, and indicating that different force fields optimized for IDPs may cause expansion of the polypeptide chain through different physical mechanisms.     

Probing the Average Local Structure of Biomolecules Using Small-Angle Scattering and Scaling Laws

Small-angle neutron and x-ray scattering have become invaluable tools for probing the nanostructure of molecules in solution. It was recently shown that the definite integral of the scattering profile exhibits a scaling (power-law) behavior with respect to molecular mass. We derive the origin of this relationship, and discuss how the integrated scattering profile can be used to identify differing levels of disorder over local ≲30 Å length scales. We apply our analysis to globular and intrinsically disordered proteins.     

Refining conformational ensembles of flexible proteins against small-angle x-ray scattering data

Intrinsically disordered proteins and flexible regions in multidomain proteins display substantial conformational heterogeneity. Characterizing the conformational ensembles of these proteins in solution typically requires combining one or more biophysical techniques with computational modeling or simulations. Experimental data can either be used to assess the accuracy of a computational model or to refine the computational model to get a better agreement with the experimental data. In both cases, one generally needs a so-called forward model (i.e., an algorithm to calculate experimental observables from individual conformations or ensembles). In many cases, this involves one or more parameters that need to be set, and it is not always trivial to determine the optimal values or to understand the impact on the choice of parameters. For example, in the case of small-angle x-ray scattering (SAXS) experiments, many forward models include parameters that describe the contribution of the hydration layer and displaced solvent to the background-subtracted experimental data. Often, one also needs to fit a scale factor and a constant background for the SAXS data but across the entire ensemble. Here, we present a protocol to dissect the effect of the free parameters on the calculated SAXS intensities and to identify a reliable set of values. We have implemented this procedure in our Bayesian/maximum entropy framework for ensemble refinement and demonstrate the results on four intrinsically disordered proteins and a protein with three domains connected by flexible linkers. Our results show that the resulting ensembles can depend on the parameters used for solvent effects and suggest that these should be chosen carefully. We also find a set of parameters that work robustly across all proteins.     

An Extended Guinier Analysis for Intrinsically Disordered Proteins

Guinier analysis allows model-free determination of the radius of gyration (R_g) of a biomolecule from X-ray or neutron scattering data, in the limit of very small scattering angles. Its range of validity is well understood for globular proteins, but is known to be more restricted for unfolded or intrinsically disordered proteins (IDPs). We have used ensembles of disordered structures from molecular dynamics simulations to investigate which structural properties cause deviations from the Guinier approximation at small scattering angles. We find that the deviation from the Guinier approximation is correlated with the polymer scaling exponent ν describing the unfolded ensemble. We therefore introduce an empirical, ν-dependent, higher-order correction term, to augment the standard Guinier analysis. We test the new fitting scheme using all-atom simulation data for several IDPs and experimental data for both an IDP and a destabilized mutant of a folded protein. In all cases tested, we achieve an accuracy of the inferred R_g within ～ 3% of the true R_g. The method is straightforward to implement and extends the range of validity to a maximum qR_g of ～ 2 versus ～ 1.1 for Guinier analysis. Compared with the Guinier or Debye approaches, our method allows data from wider angles with lower noise to be used to analyze scattering data accurately. In addition to R_g, our fitting scheme also yields estimates of the scaling exponent ν in excellent agreement with the reference ν determined from the underlying molecular ensemble.     

Unraveling double stranded alpha-helical coiled coils: an x-ray diffraction study on hard alpha-keratin fibers

Transformations of proteins secondary and tertiary structures are generally studied in globular proteins in solution. In fibrous proteins, such as hard alpha-keratin, that contain long and well-defined double stranded alpha-helical coiled coil domains, such study can be directly done on the native fibrous tissue. In order to assess the structural behavior of the coiled coil domains under an axial mechanical stress, wide angle x-ray scattering and small angle x-ray scattering experiments have been carried out on stretched horse hair fibers at relative humidity around 30%. Our observations of the three major axial spacings as a function of the applied macroscopic strain have shown two rates. Up to 4% macroscopic strain the coiled coils were slightly distorted but retained their overall conformation. Above 4% the proportion of coiled coil domains progressively decreased. The main and new result of our study is the observation of the transition from alpha-helical coiled coils to disordered chains instead of the alpha-helical coiled coil to beta-sheet transition that occurs in wet fibers.     

Molecular crowding inhibits intramolecular breathing motions in proteins

In aqueous solution some proteins undergo large-scale movements of secondary structures, subunits or domains, referred to as protein “breathing”, that define a native-state ensemble of structures. These fluctuations are sensitive to the nature and concentration of solutes and other proteins and are thereby expected to be different in the crowded interior of a cell than in dilute solution. Here we use a combination of wide angle X-ray scattering (WAXS) and computational modeling to derive a quantitative measure of the spatial scale of conformational fluctuations in a protein solution. Concentration-dependent changes in the observed scattering intensities are consistent with a model of structural fluctuations in which secondary structures undergo rigid-body motions relative to one another. This motion increases with decreasing protein concentration or increasing temperature. Analysis of a set of five structurally and functionally diverse proteins reveals a diversity of kinetic behaviors. Proteins with multiple disulfide bonds exhibit little or no increase in breathing in dilute solutions. The spatial extent of structural fluctuations appears highly dependent on both protein structure and concentration and is universally suppressed at very high protein concentrations.     

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.     

Ensemble modeling of protein disordered states: Experimental restraint contributions and validation

Disordered states of proteins include the biologically functional intrinsically disordered proteins and the unfolded states of normally folded proteins. In recent years, ensemble‐modeling strategies using various experimental measurements as restraints have emerged as powerful means for structurally characterizing disordered states. However, these methods are still in their infancy compared with the structural determination of folded proteins. Here, we have addressed several issues important to ensemble modeling using our ENSEMBLE methodology. First, we assessed how calculating ensembles containing different numbers of conformers affects their structural properties. We find that larger ensembles have very similar properties to smaller ensembles fit to the same experimental restraints, thus allowing a considerable speed improvement in our calculations. In addition, we analyzed the contributions of different experimental restraints to the structural properties of calculated ensembles, enabling us to make recommendations about the experimental measurements that should be made for optimal ensemble modeling. The effects of different restraints, most significantly from chemical shifts, paramagnetic relaxation enhancements and small‐angle X‐ray scattering, but also from other data, underscore the importance of utilizing multiple sources of experimental data. Finally, we validate our ENSEMBLE methodology using both cross‐validation and synthetic experimental restraints calculated from simulated ensembles. Our results suggest that secondary structure and molecular size distribution can generally be modeled very accurately, whereas the accuracy of calculated tertiary structure is dependent on the number of distance restraints used. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

Ca2+ attenuates nucleation activity of leiomodin

A transient increase in Ca²⁺ concentration in sarcomeres is essential for their proper function. Ca²⁺ drives striated muscle contraction via binding to the troponin complex of the thin filament to activate its interaction with the myosin thick filament. In addition to the troponin complex, the myosin essential light chain and myosin‐binding protein C were also found to be Ca²⁺ sensitive. However, the effects of Ca²⁺ on the function of the tropomodulin family proteins involved in regulating thin filament formation have not yet been studied. Leiomodin, a member of the tropomodulin family, is an actin nucleator and thin filament elongator. Using pyrene‐actin polymerization assay and transmission electron microscopy, we show that the actin nucleation activity of leiomodin is attenuated by Ca²⁺. Using circular dichroism and nuclear magnetic resonance spectroscopy, we demonstrate that the mostly disordered, negatively charged region of leiomodin located between its first two actin‐binding sites binds Ca²⁺. We propose that Ca²⁺ binding to leiomodin results in the attenuation of its nucleation activity. Our data provide further evidence regarding the role of Ca²⁺ as an ultimate regulator of the ensemble of sarcomeric proteins essential for muscle function.Summary StatementCa²⁺ fluctuations in striated muscle sarcomeres modulate contractile activity via binding to several distinct families of sarcomeric proteins. The effects of Ca²⁺ on the activity of leiomodin—an actin nucleator and thin filament length regulator—have remained unknown. In this study, we demonstrate that Ca²⁺ binds directly to leiomodin and attenuates its actin nucleating activity. Our data emphasizes the ultimate role of Ca²⁺ in the regulation of the sarcomeric protein interactions.     

NMR illuminates intrinsic disorder

Nuclear magnetic resonance (NMR) has long been instrumental in the characterization of intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs). This method continues to offer rich insights into the nature of IDPs in solution, especially in combination with other biophysical methods such as small-angle scattering, single-molecule fluorescence, electron paramagnetic resonance (EPR), and mass spectrometry. Substantial advances have been made in recent years in studies of proteins containing both ordered and disordered domains and in the characterization of problematic sequences containing repeated tracts of a single or a few amino acids. These sequences are relevant to disease states such as Alzheimer's, Parkinson's, and Huntington's diseases, where disordered proteins misfold into harmful amyloid. Innovative applications of NMR are providing novel insights into mechanisms of protein aggregation and the complexity of IDP interactions with their targets. As a basis for understanding the solution structural ensembles, dynamic behavior, and functional mechanisms of IDPs and IDRs, NMR continues to prove invaluable.     

Purification and SAXS Analysis of the Integrin Linked Kinase,PINCH, Parvin (IPP) Heterotrimeric Complex

The heterotrimeric protein complex containing the integrin linked kinase (ILK), parvin, and PINCH proteins, termed the IPP complex, is an essential component of focal adhesions, where it interacts with many proteins to mediate signaling from integrin adhesion receptors. Here we conduct a biochemical and structural analysis of the minimal IPP complex, comprising full-length human ILK, the LIM1 domain of PINCH1, and the CH2 domain of α-parvin. We provide a detailed purification protocol for IPP and show that the purified IPP complex is stable and monodisperse in solution. Using small-angle X-ray scattering (SAXS), we also conduct the first structural characterization of IPP, which reveals an elongated shape with dimensions 120×60×40 Å. Flexibility analysis using the ensemble optimization method (EOM) is consistent with an IPP complex structure with limited flexibility, raising the possibility that inter-domain interactions exist. However, our studies suggest that the inter-domain linker in ILK is accessible and we detect no inter-domain contacts by gel filtration analysis. This study provides a structural foundation to understand the conformational restraints that govern the IPP complex.     

Analytical methods for structural ensembles and dynamics of intrinsically disordered proteins

Intrinsically disordered proteins, proteins that do not have a well-defined three-dimensional structure, make up a significant proportion of our proteome and are particularly prevalent in signaling and regulation. Although their importance has been realized for two decades, there is a lack of high-resolution experimental data. Molecular dynamics simulations have been crucial in reaching our current understanding of the dynamical structural ensemble sampled by intrinsically disordered proteins. In this review, we discuss enhanced sampling simulation methods that are particularly suitable to characterize the structural ensemble, along with examples of applications and limitations. The dynamics within the ensemble can be rigorously analyzed using Markov state models. We discuss recent developments that make Markov state modeling a viable approach for studying intrinsically disordered proteins. Finally, we briefly discuss challenges and future directions when applying molecular dynamics simulations to study intrinsically disordered proteins.     

The evidence of large-scale DNA-induced compaction in the mycobacterial chromosomal ParB

The bacterial chromosome trafficking apparatus or the segrosome participates in the mitotic-like segregation of the chromosomes prior to cell division in several bacteria. ParB, which is the parS DNA-binding component of the segrosome, polymerizes on the parS-adjacent chromosome to form a nucleoprotein filament of unknown nature for the segregation function. We combined static light scattering, circular dichroism and small-angle X-ray scattering to present evidence that the apo form of the mycobacterial ParB forms an elongated dimer with intrinsically disordered regions as well as folded domains in solution. A comparison of the solution scattering of the apo and the parS-bound ParBs indicates a rather drastic compaction of the protein upon DNA binding. We propose that this binding-induced conformational transition is priming the ParB for polymerization on the DNA template.