A J-modulated protonless NMR experiment characterizes the conformational ensemble of the intrinsically disordered protein WIP

Intrinsically disordered proteins (IDPs) are multi-conformational polypeptides that lack a single stable three-dimensional structure. It has become increasingly clear that the versatile IDPs play key roles in a multitude of biological processes, and, given their flexible nature, NMR is a leading method to investigate IDP behavior on the molecular level. Here we present an IDP-tailored J-modulated experiment designed to monitor changes in the conformational ensemble characteristic of IDPs by accurately measuring backbone one- and two-bond J(¹⁵N,¹³Cα) couplings. This concept was realized using a unidirectional (H)NCO ¹³C-detected experiment suitable for poor spectral dispersion and optimized for maximum coverage of amino acid types. To demonstrate the utility of this approach we applied it to the disordered actin-binding N-terminal domain of WASp interacting protein (WIP), a ubiquitous key modulator of cytoskeletal changes in a range of biological systems. One- and two-bond J(¹⁵N,¹³Cα) couplings were acquired for WIP residues 2–65 at various temperatures, and in denaturing and crowding environments. Under native conditions fitted J-couplings identified in the WIP conformational ensemble a propensity for extended conformation at residues 16–23 and 45–60, and a helical tendency at residues 28–42. These findings are consistent with a previous study of the based upon chemical shift and RDC data and confirm that the WIP^2–65 conformational ensemble is biased towards the structure assumed by this fragment in its actin-bound form. The effects of environmental changes upon this ensemble were readily apparent in the J-coupling data, which reflected a significant decrease in structural propensity at higher temperatures, in the presence of 8 M urea, and under the influence of a bacterial cell lysate. The latter suggests that crowding can cause protein unfolding through protein–protein interactions that stabilize the unfolded state. We conclude that J-couplings are a useful measureable in characterizing structural ensembles in IDPs, and that the proposed experiment provides a practical method for accurately performing such measurements, once again emphasizing the power of NMR in studying IDP behavior.     

Thermodynamic and sequential characteristics of phase separation and droplet formation for an intrinsically disordered region/protein ensemble

Liquid–liquid phase separation (LLPS) of some IDPs/IDRs can lead to the formation of the membraneless organelles in vitro and in vivo, which are essential for many biological processes in the cell. Here we select three different IDR segments of chaperon Swc5 and develop a polymeric slab model at the residue-level. By performing the molecular dynamics simulations, LLPS can be observed at low temperatures even without charge interactions and disappear at high temperatures. Both the sequence length and the charge pattern of the Swc5 segments can influence the critical temperature of LLPS. The results suggest that the effects of the electrostatic interactions on the LLPS behaviors can change significantly with the ratios and distributions of the charged residues, especially the sequence charge decoration (SCD) values. In addition, three different forms of swc conformation can be distinguished on the phase diagram, which is different from the conventional behavior of the free IDP/IDR. Both the packed form (the condensed-phase) and the dispersed form (the dilute-phase) of swc chains are found to be coexisted when LLPS occurs. They change to the fully-spread form at high temperatures. These findings will be helpful for the investigation of the IDP/IDR ensemble behaviors as well as the fundamental mechanism of the LLPS process in bio-systems.     

A preformed binding interface in the unbound ensemble of an intrinsically disordered protein: evidence from molecular simulations

Intrinsically disordered proteins play an important role in cellular signalling, mediated by their interactions with other biomolecules. A key question concerns the nature of their binding mechanism, and whether the bound structure is induced only by proximity to the binding partner. This is difficult to answer through experiment alone because of the very heterogeneous nature of the unbound ensemble, and the probable rapid interconversion of the various unbound structures. Here we report the most extensive set of simulations on NCBD to date: we use large-scale replica exchange molecular dynamics to explore the unbound state. An important feature of the study is the use of an atomistic force field that has been parametrised against experimental data for weakly structured peptides, together with an accurate explicit water model. Neither the force field nor the starting conformations are biased towards a particular structure. The regions of NCBD that have high helical propensity in the simulations correspond closely to helices in the 'core' unbound conformation determined by NMR, although no single member of the simulated unbound ensemble closely resembles the core conformation, or either of the two known bound conformations. We have validated the results against NMR spectroscopy and SAXS measurements, obtaining reasonable agreement. The two helices which most stabilise the binding of NCBD with ACTR are formed readily; the third helix, which is less important for binding but is involved in most of the intraprotein contacts of NCBD in the bound conformation, is formed more rarely, and tends not to coexist with the other helices. These results support a mechanism by which NCBD gains the advantages of disorder, while forming binding-competent structures in the unbound state. We obtain support for this mechanism from coarse-grained simulations of NCBD with, and without, its binding partner.     

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.     

Nonspecific shielding of unfavorable electrostatic intramolecular interactions in the erythropoietin native-state increase conformational stability and limit non-native aggregation

Previous equilibrium and kinetic folding studies of the glycoprotein erythropoietin indicate that sodium chloride increases the conformational stability of this therapeutically important cytokine, ostensibly by stabilizing the native-state [Banks DD, (2011) The Effect of Glycosylation on the Folding Kinetics of Erythropoietin. J Mol Biol 412:536–550]. The focus of the current report is to determine the underlying cause of the salt dependent increase in erythropoietin conformational stability and to understand if it has any impact on aggregation, an instability that remains a challenge to the biotech industry in maintaining the efficacy and shelf-life of protein therapeutics. Isothermal urea denaturation experiments conducted at numerous temperatures in the absence and presence of sodium chloride indicated that salt stabilizes erythropoietin primarily by increasing the difference in enthalpy between the native and unfolded sates. This result, and the finding that the salt induced increases in erythropoietin melting temperatures were independent of the identity of the salt cation and anion, indicates that salt likely increases the conformational stability of erythropoietin at neutral pH by nonspecific shielding of unfavorable electrostatic interaction(s) in the native-state. The addition of salt (even low concentrations of the strong chaotrope salt guanidinium hydrochloride) also exponentially decreased the initial rate of soluble erythropoietin non-native aggregation at 37 °C storage.     

Atomistic ensemble modeling and small-angle neutron scattering of intrinsically disordered protein complexes: applied to minichromosome maintenance protein

The minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in archaea and eukarya. In this work we determined the solution structure of the N-terminal portion of the MCM complex from the archaeon Methanothermobacter thermautotrophicus (N-mtMCM) in the presence and absence of DNA using small-angle neutron scattering (SANS). N-mtMCM is a multimeric protein complex that consists of 12 monomers, each of which contains three distinct domains and two unstructured regions. Using an all-atom approach incorporating modern force field and Monte Carlo methods to allow the unstructured regions of each monomer to be varied independently, we generated an ensemble of biologically relevant structures for the complex. An examination of the subsets of structures that were most consistent with the SANS data revealed that large movements between the three domains of N-mtMCM can occur in solution. Furthermore, changes in the SANS curves upon DNA binding could be correlated to the motion of a particular N-mtMCM domain. These results provide structural support to the previously reported biochemical observations that large domain motions are required for the activation of the MCM helicase in archaea and eukarya. The methods developed here for N-mtMCM solution structure modeling should be suitable for other large protein complexes with unstructured flexible regions.     

Segmental isotope-labeling of the intrinsically disordered protein PQBP1

Polyglutamine tract-binding protein 1 (PQBP1) is an intrinsically disordered protein abundantly expressed in the brain. Mutations in the PQBP1 gene are causative for X-linked mental retardation disorders. Here, we investigated the structure of the C-terminal segment within the context of full-length PQBP1. We produced a segmentally isotope-labeled PQBP1 composed of a non-labeled segment (residues 1–219; N-segment) and a ¹³C/¹⁵N-labeled segment (residues 220–265; C-segment). Our results demonstrate that the segmental isotope-labeling combined with NMR spectroscopy is useful for detecting a very weak intra-molecular interaction in an intrinsically disordered protein.     

Calculation of the total electrostatic energy of a macromolecular system: solvation energies, binding energies, and conformational analysis

In this report we describe an accurate numerical method for calculating the total electrostatic energy of molecules of arbitrary shape and charge distribution, accounting for both Coulombic and solvent polarization terms. In addition to the solvation energies of individual molecules, the method can be used to calculate the electrostatic energy associated with conformational changes in proteins as well as changes in solvation energy that accompany the binding of charged substrates. The validity of the method is examined by calculating the hydration energies of acetate, methyl ammonium, ammonium, and methanol. The method is then used to study the relationship between the depth of a charge within a protein and its interaction with the solvent. Calculations of the relative electrostatic energies of crystal and misfolded conformations of Themiste dyscritum hemerythrin and the VL domain of an antibody are also presented. The results indicate that electrostatic charge-solvent interactions strongly favor the crystal structures. More generally, it is found that charge-solvent interactions, which are frequently neglected in protein structure analysis, can make large contributions to the total energy of a macromolecular system.     

Interplay between the electrostatic membrane potential and conformational changes in membrane proteins

Transmembrane electrostatic membrane potential is a major energy source of the cell. Importantly, it determines the structure as well as function of charge‐carrying membrane proteins. Here, we discuss the relationship between membrane potential and membrane proteins, in particular whether the conformation of these proteins is integrally connected to the membrane potential. Together, these concepts provide a framework for rationalizing the types of conformational changes that have been observed in membrane proteins and for better understanding the electrostatic effects of the membrane potential on both reversible as well as unidirectional dynamic processes of membrane proteins.     

Influence of drying on the secondary structure of intrinsically disordered and globular proteins

Circular dichroism (CD) spectroscopy of five Arabidopsis late embryogenesis abundant (LEA) proteins constituting the plant specific families LEA_5 and LEA_6 showed that they are intrinsically disordered in solution and partially fold during drying. Structural predictions were comparable to these results for hydrated LEA_6, but not for LEA_5 proteins. FTIR spectroscopy showed that verbascose, but not sucrose, strongly affected the structure of the dry proteins. The four investigated globular proteins were only mildly affected by drying in the absence, but strongly in the presence of sugars. These data highlight the larger structural flexibility of disordered compared to globular proteins and the impact of sugars on the structure of both disordered and globular proteins during drying.     

Compaction properties of an intrinsically disordered protein: Sic1 and its kinase-inhibitor domain

IDPs in their unbound state can transiently acquire secondary and tertiary structure. Describing such intrinsic structure is important to understand the transition between free and bound state, leading to supramolecular complexes with physiological interactors. IDP structure is highly dynamic and, therefore, difficult to study by conventional techniques. This work focuses on conformational analysis of the KID fragment of the Sic1 protein, an IDP with a key regulatory role in the cell-cycle of Saccharomyces cerevisiae. FT-IR spectroscopy, ESI-MS, and IM measurements are used to capture dynamic and short-lived conformational states, probing both secondary and tertiary protein structure. The results indicate that the isolated Sic1 KID retains dynamic helical structure and populates collapsed states of different compactness. A metastable, highly compact species is detected. Comparison between the fragment and the full-length protein suggests that chain length is crucial to the stabilization of compact states of this IDP. The two proteins are compared by a length-independent compaction index.     

Importance of electrostatic interactions in the association of intrinsically disordered histone chaperone Chz1 and histone H2A.Z-H2B

Histone chaperones facilitate assembly and disassembly of nucleosomes. Understanding the process of how histone chaperones associate and dissociate from the histones can help clarify their roles in chromosome metabolism. Some histone chaperones are intrinsically disordered proteins (IDPs). Recent studies of IDPs revealed that the recognition of the biomolecules is realized by the flexibility and dynamics, challenging the century-old structure-function paradigm. Here we investigate the binding between intrinsically disordered chaperone Chz1 and histone variant H2A.Z-H2B by developing a structure-based coarse-grained model, in which Debye-Hückel model is implemented for describing electrostatic interactions due to highly charged characteristic of Chz1 and H2A.Z-H2B. We find that major structural changes of Chz1 only occur after the rate-limiting electrostatic dominant transition state and Chz1 undergoes folding coupled binding through two parallel pathways. Interestingly, although the electrostatic interactions stabilize bound complex and facilitate the recognition at first stage, the rate for formation of the complex is not always accelerated due to slow escape of conformations with non-native electrostatic interactions at low salt concentrations. Our studies provide an ionic-strength-controlled binding/folding mechanism, leading to a cooperative mechanism of "local collapse or trapping" and "fly-casting" together and a new understanding of the roles of electrostatic interactions in IDPs' binding.     

The effects of truncating long-range forces on protein dynamics

This paper considers the effects of truncating long-range forces on protein dynamics. Six methods of truncation that we investigate as a function of cutoff criterion of the long-range potentials are (1) a shifted potential; (2) a switching function; (3) simple atom-atom truncation based on distance; (4) simple atom-atom truncation based on a list which is updated periodically (every 25 steps); (5) simple group-group truncation based on distance; and (6) simple group-group truncation based on a list which is updated periodically (every 25 steps). Based on 70 calculations of carboxymyoglobin we show that the method and distance of long range cutoff have a dramatic effect on overall protein behavior. Evaluation of the different methods is based on comparison of a simulation's rms fluctuation about the average coordinates, the rms deviation from the average coordinates of a no cutoff simulation and from the X-ray structure of the protein. The simulations in which long-range forces are truncated by a shifted potential shows large rms deviations for cutoff criteria less than 14 A, and reasonable deviations and fluctuations at this cutoff distance or larger. Simulations using a switching function are investigated by varying the range over which electrostatic interactions are switched off. Results using a short switching function that switches off the potential over a short range of distances are poor for all cutoff distances. A switching function over a 5-9 A range gives reasonable results for a distance-dependent dielectric, but not using a constant dielectric. Both the atom-atom and group-group truncation methods based on distance shows large rms deviation and fluctuation for short cutoff distances, while for cutoff distances of 11 A or greater, reasonable results are achieved. Although comparison of these to distance-based truncation methods show surprisingly larger rms deviations for the group-group truncation, contrary to simulation studies of aqueous ionic solutions. The results of atom-atom or group-group list-based simulations generally appear to be less stable than the distance-based simulations, and require more frequent velocity scaling or stronger coupling to a heat bath.     

白藜芦醇对AngⅡ诱导的血管平滑肌细胞CaN的影响

目的：观察白藜芦醇（Res）对血管紧张素Ⅱ（AngⅡ）诱导的血管平滑肌细胞（VSMCs）增殖及细胞中钙调蛋白（CaM）和钙调神经磷酸酶（CaN）活性的影响,并探讨其机制。方法：体外培养兔主动脉VSMCs,用免疫细胞化学方法鉴定。建立AngⅡ诱导的VSMCs增殖模型。取生长良好的第4～8代VSMCs,随机分为对照组,AnsⅡ组（0．1μmol／L）,AngⅡ＋Res组（20,40,80,160）μmol／L。应用MTT法检测细胞增殖程度,考马斯亮兰法进行CaM定量,定磷法进行CaN活性测定。结果：成功培养兔VSMCs并传代,免疫细胞化学染色均呈阳性表达。AngⅡ组VSMCs的增殖程度、CaM和CaN活性较对照组增高（P〈0．05,P〈0．01）。AngⅡ＋Res组各组VSMCs的CaM和CaN活性较AngⅡ组显著下降（P〈0．01）。结论：在一定范围内,Res可降低AngⅡ诱导的VSMCs增殖程度,其机制可能与干预CaN依赖的信号转导途径有关。     

The Dad1 subunit of the yeast kinetochore Dam1 complex is an intrinsically disordered protein

The Dam1 complex is an important part of the yeast kinetochore. It mediates attachment of the chromosome to the mitotic spindle and is involved in chromatid separation initiated at anaphase. It is comprised of 10 individual subunits and has been observed to oligomerize in various ways as it interacts with microtubules, including forming a ring. This work explores the biochemical and biophysical properties of Dad1, one of the Dam1 complex subunits from the human fungal pathogen Candida albicans. Unlike its Saccharomyces cerevisiae counterpart, C. albicans Dad1 can be expressed as a soluble protein in Escherichia coli. Analysis of this protein’s hydrodynamic properties, thermostability and primary sequence have been conducted. As a result, we conclude that isolated Dad1 is an intrinsically disordered protein.     

Structural analysis of the complex between calmodulin and full-length myelin basic protein, an intrinsically disordered molecule

Myelin basic protein (MBP) is present between the cytoplasmic leaflets of the compact myelin membrane in both the peripheral and central nervous systems, and characterized to be intrinsically disordered in solution. One of the best-characterized protein ligands for MBP is calmodulin (CaM), a highly acidic calcium sensor. We pulled down MBP from human brain white matter as the major calcium-dependent CaM-binding protein. We then used full-length brain MBP, and a peptide from rodent MBP, to structurally characterize the MBP–CaM complex in solution by small-angle X-ray scattering, NMR spectroscopy, synchrotron radiation circular dichroism spectroscopy, and size exclusion chromatography. We determined 3D structures for the full-length protein–protein complex at different stoichiometries and detect ligand-induced folding of MBP. We also obtained thermodynamic data for the two CaM-binding sites of MBP, indicating that CaM does not collapse upon binding to MBP, and show that CaM and MBP colocalize in myelin sheaths. In addition, we analyzed the post-translational modifications of rat brain MBP, identifying a novel MBP modification, glucosylation. Our results provide a detailed picture of the MBP–CaM interaction, including a 3D model of the complex between full-length proteins.