Analysis of the multimeric state of proteins by matrix assisted laser desorption/ionization mass spectrometry after cross-linking with glutaraldehyde

Helin, Jari; Caldentey, Javier; Kalkkinen, Nisse; Bamford, Dennis H.

doi:10.1002/(sici)1097-0231(19990215)13:3<185::aid-rcm481>3.0.co;2-o

Cited by 17 publications

(11 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…(35) with phage 6 structural proteins (10 to 85 kDa) and the phage PRD1 protein P3 trimer (120 kDa) as standards. Glutaraldehyde-cross-linked protein multimers were also identified by Western blotting with either polyclonal anti-P1 serum (1:20,000 dilution) or polyclonal anti-P2 serum (1:100,000 dilution) and anti-rabbit immunoglobulin G antibodies (Dako) as secondary antibodies detected by enhanced chemiluminescence reagents (Biological Industries).…”

Section: Methodsmentioning

confidence: 99%

Bacteriophage PM2 Has a Protein Capsid Surrounding a Spherical Proteinaceous Lipid Core

Kivelä

Kalkkinen

Bamford

2002

J Virol

Self Cite

View full text Add to dashboard Cite

The marine double-stranded DNA (dsDNA) bacteriophage PM2, studied since 1968, is the type organism of the family Corticoviridae, infecting two gram-negative Pseudoalteromonas species. The virion contains a membrane underneath an icosahedral protein capsid composed of two structural proteins. The purified major capsid protein, P2, appears as a trimer, and the receptor binding protein, P1, appears as a monomer. The C-terminal part of P1 is distal and is responsible for receptor binding activity. The rest of the structural proteins are associated with the internal phospholipid membrane enclosing the viral genome. This internal particle is designated the lipid core. The overall structural organization of phage PM2 resembles that of dsDNA bacteriophage PRD1, the type organism of the family Tectiviridae.PM2, the only isolate in the family Corticoviridae, is the first bacteriophage for which the presence of lipids in the virion was firmly demonstrated (15,24,26). It is a lytic virus, infecting two Pseudoalteromonas species: marine gram-negative Pseudoalteromonas espejiana BAL-31 (originally Pseudomonas sp. strain BAL-31), isolated along with the virus (25, 32), and Pseudoalteromonas sp. strain ER72M2, obtained from the East River, New York, by Leonard Mindich (38). Originally, considerable interest was directed toward this virus system (about 200 references), but since 1983, only a few studies have been published. Recently, we decided to examine this virus system (38, 39), as our laboratory is focused on lipid-containing bacterial viruses.The mass of the virion (ϳ4.5 ϫ 10 7 Da) is distributed among nucleic acid (14%), lipid (14%), and protein (72%) constituents (15,16,17). The average diameter of the icosahedral particle is ϳ60 nm (24,34,49), and the lipids are located internally (15,34). The sedimentation coefficient (s 20 , w ) of the particle is 293S, and buoyant densities in sucrose and cesium chloride are 1.26 and 1.28 g/cm 3 , respectively (16, 38). The stability of the virion is strongly dependent on sodium and especially on calcium ions, and the virion equilibrated in sucrose is inactive (38). Calcium ions are also essential in the final assembly process during PM2 infection (50).The PM2 genome is a highly supercoiled circular doublestranded DNA (dsDNA) molecule (27) with the highest number of negative supercoils (Ϫ51) observed in a natural molecule (33). The organization of the genes and the transcriptional regulation of operons in the genome have been determined (39; R. H. Männistö, A. M. Grahn, D. H. Bamford, and J. K. H. Bamford, submitted for publication). The 10,079-bp genome replicates via a rolling-circle mechanism (18, 28, 39) while associated with the host cytoplasmic membrane (11).The viral lipids are derived from the host cell membrane during the assembly process (26), and the composition is ϳ64% phosphatidylglycerol, ϳ27% phosphatidylethanolamine, ϳ8% neutral lipids, and small amounts of asylphosphatidylglycerol (15, 53). The proportion of the major phospholipids in the virion is nearly the inverse...

show abstract

Section: Methodsmentioning

confidence: 99%

Bacteriophage PM2 Has a Protein Capsid Surrounding a Spherical Proteinaceous Lipid Core

Kivelä

Kalkkinen

Bamford

2002

J Virol

Self Cite

View full text Add to dashboard Cite

show abstract

“…Two hundred forty copies of these trimers are organized on an icosahedral lattice with pseudo T ϭ 25 triangulation (2). The vertices of the icosahedral capsid lattice are occupied by the minor capsid protein P31 (14 kDa), which forms pentamers in solution (3,4). This organization is similar to that of adenovirus, in which the hexameric and pentameric capsid proteins are called hexons and pentons, respectively.…”

mentioning

confidence: 99%

“…The majority of vertices, designated as binding vertices, were shown to contain proteins P2 and P5. Protein P2 is a monomeric receptor-binding protein (4,17,18). P5, designated as the spike protein, is released as a soluble trimer from the virion (19).…”

mentioning

confidence: 99%

Tale of two spikes in bacteriophage PRD1

Huiskonen

Manole

Butcher

2007

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

View full text Add to dashboard Cite

Structural comparisons between bacteriophage PRD1 and adenovirus have revealed an evolutionary relationship that has contributed significantly to current ideas on virus phylogeny. However, the structural organization of the receptor-binding spike complex and how the different symmetry mismatches are mediated between the spike-complex proteins are not clear. We determined the architecture of the PRD1 spike complex by using electron microscopy and three-dimensional image reconstruction of a series of PRD1 mutants. We constructed an atomic model for the fulllength P5 spike protein by using comparative modeling. P5 was shown to be bound directly to the penton base protein P31. P5 and the receptor-binding protein P2 form two separate spikes, interacting with each other near the capsid shell. P5, with a tumor necrosis factor-like head domain, may have been responsible for host recognition before capture of the current receptor-binding protein P2.electron cryomicroscopy ͉ symmetry mismatch ͉ vertex ͉ virus

show abstract

“…Chemical cross-linking of noncovalent protein complexes and analysis of the cross-linked species by SDS-PAGE has long been employed in biochemistry laboratories to determine the stoichiometry of the constituent monomers~Davies & Stark, 1970;Jung & Moroi, 1983!. Cross-linking followed by MALDI-MS analysis has more recently been utilized to determine the stoichiometry of noncovalent protein complexes~Farmer & Caprioli, 1998;Helin et al, 1999!, and some studies have extended this to proteolytic digestion of the crosslinked species and subsequent identification of the linked peptides~Artemyev et al., 1993;Ngai et al, 1994;Haniu et al, 1995;Rossi et al, 1995;Vater et al, 1996;Yang et al, 1996;Yu et al, 1997;Hegyi et al, 1998;Mills et al, 1998;Scaloni et al, 1998!. One group has utilized photoactivatible thiol-cleavable cross-linkers to covalently link a protein and an interacting peptide, followed by MALDI-MS analysis of the proteolytic digest of the cross-linked species~Kaufmann et Machold et al, 1995!. To date, however, there is no report in the literature that utilizes a thiol-cleavable cross-linker to covalently link protein subunits or interacting molecules, and differential MALDI-MS peptide mapping~6 thiol reagent! to determine the contact sites in the noncovalent complex.…”

mentioning

confidence: 99%

Chemical cross‐linking with thiol‐cleavable reagents combined with differential mass spectrometric peptide mapping—A novel approach to assess intermolecular protein contacts

et al. 2000

View full text Add to dashboard Cite

The intermolecular contact regions between monomers of the homodimeric DNA binding protein ParR and the interaction between the glycoproteins CD28 and CD80 were investigated using a strategy that combined chemical crosslinking with differential MALDI-MS analyses. ParR dimers were modified in vitro with the thiol-cleavable cross-linker 3,39-dithio-bis~succinimidylproprionate!~DTSSP!, proteolytically digested with trypsin and analyzed by MALDI-MS peptide mapping. Comparison of the peptide maps obtained from digested cross-linked ParR dimers in the presence and absence of a thiol reagent strongly supported a "head-to-tail" arrangement of the monomers in the dimeric complex. Glycoprotein fusion constructs CD28-IgG and CD80-F ab were cross-linked in vitro by DTSSP, characterized by nonreducing SDS-PAGE, digested in situ with trypsin and analyzed by MALDI-MS peptide mapping~6 thiol reagent!. The data revealed the presence of an intermolecular cross-link between the receptor regions of the glycoprotein constructs, as well as a number of unexpected but nonetheless specific interactions between the fusion domains of CD28-IgG and the receptor domain of CD80-F ab . The strategy of chemical cross-linking combined with differential MALDI-MS peptide mapping~6 thiol reagent! enabled localization of the interface region~s! of the complexes studied and clearly demonstrates the utility of such an approach to obtain structural information on interacting noncovalent complexes.

show abstract

Analysis of the multimeric state of proteins by matrix assisted laser desorption/ionization mass spectrometry after cross-linking with glutaraldehyde

Cited by 17 publications

References 22 publications

Bacteriophage PM2 Has a Protein Capsid Surrounding a Spherical Proteinaceous Lipid Core

Bacteriophage PM2 Has a Protein Capsid Surrounding a Spherical Proteinaceous Lipid Core

Tale of two spikes in bacteriophage PRD1

Chemical cross‐linking with thiol‐cleavable reagents combined with differential mass spectrometric peptide mapping—A novel approach to assess intermolecular protein contacts

Contact Info

Product

Resources

About