67 research outputs found
Evaluating the effect of phosphorylation on the structure and dynamics of Hsp27 dimers by means of ion mobility mass spectrometry
No full textThe quaternary structure and dynamics of the human small heat-shock protein Hsp27 are linked to its molecular chaperone function and influenced by post-translational modifications, including phosphorylation. Phosphorylation of Hsp27 promotes oligomer dissociation and can enhance chaperone activity. This study explored the impact of phosphorylation on the quaternary structure and dynamics of Hsp27. Using mutations that mimic phosphorylation, and ion mobility mass spectrometry, we show that successive substitutions result in an increase in the conformational heterogeneity of Hsp27 dimers. In contrast, we did not detect any changes in the structure of an Hsp27 12-mer, representative of larger Hsp27 oligomers. Our data suggest that oligomer dissociation and increased flexibility of the dimer contribute to the enhanced chaperone activity of phosphorylated Hsp27. Thus, post-translational modifications such as phosphorylation play a crucial role in modulating both the tertiary and quaternary structure of Hsp27, which is pivotal to its function as a key component of the proteostasis network in cells. Our data demonstrate the utility of ion mobility mass spectrometry for probing the structure and dynamics of heterogeneous proteins.Blagojce Jovcevski , Megan A. Kelly, J. Andrew Aquilina, Justin L. P. Benesch and Heath Ecroy
Mass spectrometry: Come of age for structural and dynamical biology
No full textOver the past two decades, mass spectrometry (MS) has emerged as a bone fide approach for structural biology. MS can inform on all levels of protein organization, and enables quantitative assessments of their intrinsic dynamics. The key advantages of MS are that it is a sensitive, high-resolution separation technique with wide applicability, and thereby allows the interrogation of transient protein assemblies in the context of complex mixtures. Here we describe how molecular-level information is derived from MS experiments, and how it can be combined with spatial and dynamical restraints obtained from other structural biology approaches to allow hybrid studies of protein architecture and movements. © 2011 Elsevier Ltd
Dynamical structure of αB-crystallin.
No full textThe human small heat-shock protein αB-crystallin is an extremely difficult molecule to study, with its inherent structural dynamics posing unique challenges to all biophysical and structural biology techniques. Here we highlight how the polydispersity and quaternary dynamics of αB-crystallin are intrinsically inter-twined, and how this can impact on measurements of the oligomeric distribution. We show that, in spite of these difficulties, considerable understanding of the varied fluctuations αB-crystallin undergoes at equilibrium has emerged in the last few years. By reporting on data obtained from a variety of biophysical techniques, we demonstrate how the αB-crystallin solution ensemble is governed by molecular motions of varying amplitude and time-scales spanning several orders of magnitude. We describe how these diverse measurements are being used to construct an integrated view of the dynamical structure of αB-crystallin, and highlight areas that require further interrogation. With its study motivating the refinement of experimental techniques, and the development of new approaches to combine the hybrid datasets, we conclude that αB-crystallin continues to represent a paradigm for dynamical biology
Torture Laid Bare
No full textTorture, while internationally sanctioned, is not well-defined. This paper sets out a Minimal English definition of the crime of 'torture' in international law. The four elements of torture are: (1) infliction of severe pain and suffering (2) acting with intent (3) for a purpose (4) by the state. The connection between intention and outcome is considered in the light of presumptions. I then briefly consider the concept of 'lawful sanctions' and the UN Standard Minimum Rules that apply to the treatment of prisoners to establish a baseline against which allegations of torture can be measured. Finally, I argue that current regimes of British benefit sanctions, whereby social welfare payments are stopped, may in some cases constitute torture. This argument considers the effects of sanctions and the discourses and ideologies attached to social welfare claimants.© 2017. This is an author produced version of a paper published in the Journal of Language and Politics, uploaded in accordance with the publisher’s self- archiving policy. The final published version (version of record) is available online at http://www.jbe-platform.com/content/journals/10.1075/jlp.15040.moo. Some minor differences between this version and the final published version may remain. We suggest you refer to the final published version should you wish to cite from it
EM∩IM: software for relating ion mobility mass spectrometry and electron microscopy data
No full textWe present EM∩IM, software that allows the calculation of collision cross-sections from electron density maps obtained for example by means of transmission electron microscopy. This allows the assessment of structures other than those described by atomic coordinates with ion mobility mass spectrometry data, and provides a new means for contouring and validating electron density maps. EM∩IM thereby facilitates the use of data obtained in the gas phase within structural biology studies employing diverse experimental methodologies
Structural and functional aspects of the interaction partners of the small heat-shock protein in Synechocystis
No full textThe canonical function of small heat-shock proteins (sHSPs) is to interact with proteins destabilized under conditions of cellular stress. While the breadth of interactions made by many sHSPs is well-known, there is currently little knowledge about what structural features of the interactors form the basis for their recognition. Here, we have identified 83 in vivo interactors of the sole sHSP in the cyanobacterium Synechocystis sp. PCC 6803, HSP16.6, reflective of stable associations with soluble proteins made under heat-shock conditions. By performing bioinformatic analyses on these interactors, we identify primary and secondary structural elements that are enriched relative to expectations from the cyanobacterial genome. In addition, by examining the Synechocystis interactors and comparing them with those identified to bind sHSPs in other prokaryotes, we show that sHSPs associate with specific proteins and biological processes. Our data are therefore consistent with a picture of sHSPs being broadly specific molecular chaperones that act to protect multiple cellular pathways
Combining tandem mass spectrometry with ion mobility separation to determine the architecture of polydisperse proteins
No full textPolydispersity presents a considerable challenge for the detailed molecular characterisation of many proteins. This is because in most biophysical and structural biology approaches the molecules in solution are ensemble-averaged, obscuring differences between individual proteins or conformational states. Mass spectrometry is however inherently dispersive, allowing the specific interrogation of molecules with distinct mass-to-charge ratios. Here, we exploit this intrinsic benefit to develop a means for determining directly the stoichiometries and sizes of oligomers comprising a polydisperse protein ensemble. Our method exploits the quadrupole-(ion-mobility)-(time-of-flight) geometry by submitting selected mass-to-charge ranges for ion mobility separation followed by collision-induced dissociation. In this sequential experiment the ion mobility information of the precursors is reported by the arrival times of the fragments, which are highly separated in mass-to-charge by virtue of the dissociation process. We observe small differences in the measured arrival time between fragments arising due to ion transit conditions after the ion mobility cell. To accommodate these systematic deviations, we develop a mass-to-charge dependent correction, leading to a reduction in the error of the collision cross-section measurement to around 0.5%. We characterise our method using HSP16.9, a small heat-shock protein that undergoes a mono- to polydisperse transition upon lowering pH, and reveal that the oligomers it forms have collisional cross-sections consistent with the polyhedral and double-ring architectures exhibited by other members of the protein family
Structural and functional consequences of age-related isomerization in α-crystallins
No full textLong-lived proteins are subject to spontaneous degradation and may accumulate a range of modifications over time, including subtle alterations such as side-chain isomerization. Recently, tandem MS has enabled identification and characterization of such peptide isomers, including those differing only in chirality. However, the structural and functional consequences of these perturbations remain largely unexplored. Here, we examined the impact of isomerization of aspartic acid or epimerization of serine at four sites mapping to crucial oligomeric interfaces in human αA- and αB-crystallin, the most abundant chaperone proteins in the eye lens. To characterize the effect of isomerization on quaternary assembly, we utilized synthetic peptide mimics, enzyme assays, molecular dynamics calculations, and native MS experiments. The oligomerization of recombinant forms of αA- and αB-crystallin that mimic isomerized residues deviated from native behavior in all cases. Isomerization also perturbs recognition of peptide substrates, either enhancing or inhibiting kinase activity. Specifically, epimerization of serine (αASer-162) dramatically weakened inter-subunit binding. Furthermore, phosphorylation of αBSer-59, known to play an important regulatory role in oligomerization, was severely inhibited by serine epimerization and altered by isomerization of nearby αBAsp-62. Similarly, isomerization of αBAsp-109 disrupted a vital salt bridge with αBArg-120, a contact that when broken has previously been shown to yield aberrant oligomerization and aggregation in several disease-associated variants. Our results illustrate how isomerization of amino acid residues, which may seem to be only a minor structural perturbation, can disrupt native structural interactions with profound consequences for protein assembly and activity
Analysis of αB-crystallin polydispersity in solution through native microfluidic electrophoresis
No full textIn recent years, significant advancements have been made in the understanding of the population distributions and dynamic oligomeric states of the molecular chaperone αB-crystallin and its core domain variants. In this work, we provide solution-phase evidence of the polydispersity of αB-crystallin using microfluidic methods, used for separating the oligomeric species present in solution according to their different electrophoretic mobilities on-chip in a matter of seconds. We in particular demonstrate that microfluidic high-field electrophoresis and diffusion can detect the oligomerisation of these highly dynamic molecular chaperones and characterise the dominant oligomeric species present. We thereby provide a robust microfluidic method for characterising the individual species within complex protein mixtures of biological relevance
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