High-resolution mass spectrometry of viral assemblies: Molecular composition and stability of dimorphic hepatitis B virus capsids

Uetrecht, Charlotte; Versluis, Cees; Watts, Norman R.; Roos, Wouter H.; Wuite, Gijs J. L.; Wingfield, Paul T.; Steven, Alasdair C.; Heck, Albert J. R.

doi:10.1073/pnas.0800406105

Cited by 200 publications

(224 citation statements)

References 43 publications

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“…This process can occur repeatedly, limited by the absolute number of charges and activation energy available [49]. Indeed, in favorable cases as many as seven successive dissociation steps have been observed [50].…”

Section: Collision-induced Dissociationmentioning

confidence: 99%

Collisional activation of protein complexes: Picking up the pieces

Benesch

2009

J. Am. Soc. Mass Spectrom.

187

250

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Mass spectrometry is fast becoming a vital approach not only for the identification and quantification of proteins, but also for the study of the noncovalent assemblies they form. Approaches for ionizing, transmitting, and detecting protein complexes intact in the mass spectrometer are now well established. The challenge has therefore shifted to developing and applying mass spectrometry approaches to elucidate the structure of such species. A crucial aspect to this goal is inducing their disassembly in the gas phase to mine information as to their composition and organization. Here the consequences of collisionally activating protein complexes are illustrated through ion mobility mass spectrometry measurements and discussed in the context of the current literature. Although a consensus view of the mechanism of dissociation is starting to emerge, it is also clear that a number of aspects remain unresolved. These outstanding questions and frontier challenges must be addressed if gas-phase dissociative approaches are to reach their full potential in the study of protein assemblies. . Since that time technological and methodological developments have continued apace [2][3][4][5], such that the MS of such large species is no longer merely a technical curiosity, but rather a bona fide approach for structural biologists [6]. The many advantages that MS possesses, including speed and sensitivity of analysis [7], have made it integral to the fields of proteomics and systems biology [8,9]. The long-term challenge now is to extend the technology and methodology such that all higher levels of protein structure, from the secondary to the quinary [10], might be characterized rapidly and effectively by means of MS.Such a revolution will require the addition to, and adaptation of, the current conventional MS-based proteomic strategy. A cornerstone of this is tandem MS, wherein ions of interest are subjected to gas-phase dissociation and the fragments analyzed to provide protein sequence, and thus identity, information [11]. Recently much effort has been made to perform analogous experiments on protein complexes, whereby they are dissociated in the mass spectrometer to provide mass information on their constituents [12]. Furthermore, some evidence suggests that, aside from just providing the identity of subunits, the gas-phase dissociation process may even reveal details as to how these subunits are organized within the oligomer [13]. As such, the possibility has arisen that gas-phase dissociation coupled to MS might eventually allow the reverse engineering of protein assemblies.Over the last few years a considerable body of literature has emerged regarding the mechanism of the gas-phase dissociation of protein assemblies. A variety of activation techniques have been used [14 -18], but the most popular approach for the gas-phase dissociation of protein assemblies is currently collision-induced dissociation (CID). It is likely this is primarily attributable to its ease of implementation and its incorporation into the quadrupole t...

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Section: Collision-induced Dissociationmentioning

confidence: 99%

Collisional activation of protein complexes: Picking up the pieces

Benesch

2009

J. Am. Soc. Mass Spectrom.

187

250

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“…Furthermore, it was shown that capsid mutations can alter the mechanical strength of cowpea chlorotic mottle virus and minute virus of mice (25,26). Nanoindentation techniques have also been used to analyze the morphology of different capsids of one virus (e.g., the T ϭ 3 and T ϭ 4 hepatitis B capsids), which were shown to have mechanically similar shells (27).…”

mentioning

confidence: 99%

Scaffold expulsion and genome packaging trigger stabilization of herpes simplex virus capsids

Roos

Radtke

Kniesmeijer

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Herpes simplex virus type 1 (HSV1) capsids undergo extensive structural changes during maturation and DNA packaging. As a result, they become more stable and competent for nuclear egress. To further elucidate this stabilization process, we used biochemical and nanoindentation approaches to analyze the structural and mechanical properties of scaffold-containing (B), empty (A), and DNA-containing (C) nuclear capsids. Atomic force microscopy experiments revealed that A and C capsids were mechanically indistinguishable, indicating that the presence of DNA does not account for changes in mechanical properties during capsid maturation. Despite having the same rigidity, the scaffold-containing B capsids broke at significantly lower forces than A and C capsids. An extraction of pentons with guanidine hydrochloride (GuHCl) increased the flexibility of all capsids. Surprisingly, the breaking forces of the modified A and C capsids dropped to similar values as those of the GuHCl-treated B capsids, indicating that mechanical reinforcement occurs at the vertices. Nonetheless, it also showed that HSV1 capsids possess a remarkable structural integrity that was preserved after removal of pentons. We suggest that HSV1 capsids are stabilized after removal of the scaffold proteins, and that this stabilization is triggered by the packaging of DNA, but independent of the actual presence of DNA.atomic force microscopy ͉ nanoindentation ͉ penton ͉ viral structure ͉ virus mechanics H erpes simplex virus type 1 (HSV1) virions encapsidate their 152 kbp double-stranded DNA genome in an icosahedral capsid that is surrounded by an amorphous protein layer, called the tegument, and a lipid-containing envelope. Assembly of herpesviruses is initiated in the nucleus where procapsids selfassemble around a protein scaffold and subsequently mature (1-4). This transformation is characterized by massive conformational changes of the Ϸ200 MDa shell, resulting in stable, mature capsids, and is in many aspects analogous to maturation processes in bacteriophages (5, 6). The scaffold is proteolytically cleaved and removed, whereas the outer shell transforms from a spherical into an icosahedral shape (7, 8). The mature capsid has an outer diameter of 125 nm with an overall shell thickness of Ϸ15 nm (9). Capsomeres converge at their proximal ends, forming a contiguous shell of Ϸ4 nm thickness that is only interrupted by channels passing through all of the 162 capsomeres (10, 11). The capsomeres are hexamers (hexons) and pentamers (pentons) of the major capsid protein VP5 that form a shell with a triangulation number of T ϭ 16. In total, 320 triplexes, each formed by a heterotrimer of 1 VP19c and 2 VP23 molecules, connect these capsomeres (10). One of the 12 capsid vertices is occupied by the pUL6 portal (12). Three particle types can be isolated from the nuclei of infected cells due to their different sedimentation behavior: B capsids, which still have the scaffold inside; the lighter A capsids, which are empty; and the denser C capsids, which contain the DN...

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“…Analysis of large MDa complexes such as the ribosome 19 and highly ordered virus capsids [20][21][22] , in defining substrate binding to molecular machines [23][24][25] , or the characterization of subunit interaction networks 26,17,16,17,27,28 serve as but a few examples of the value of this approach.…”

Section: Discussionmentioning

confidence: 99%

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Kirshenbaum

Michaelevski

Sharon

2010

JoVE

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Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes 1 . To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multiprotein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS) [2][3][4][5] . This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture.Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

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High-resolution mass spectrometry of viral assemblies: Molecular composition and stability of dimorphic hepatitis B virus capsids

Cited by 200 publications

References 43 publications

Collisional activation of protein complexes: Picking up the pieces

Collisional activation of protein complexes: Picking up the pieces

Scaffold expulsion and genome packaging trigger stabilization of herpes simplex virus capsids

Analyzing Large Protein Complexes by Structural Mass Spectrometry

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