Peter E Prevelige

Publications of Peter E Prevelige

• Site-directed coordination chemistry with P22 virus-like particles.

  Authors: Masaki Uchida, David S Morris, Sebyung Kang, Craig C Jolley, Janice Lucon, Lars O Liepold, Ben LaFrance, Peter E Prevelige, Trevor Douglas

  Langmuir : the ACS journal of surfaces and colloids. 12/2011; 28(4):1998-2006.

  Protein cage nanoparticles (PCNs) are attractive platforms for developing functional nanomaterials using biomimetic approaches for functionalization and cargo encapsulation. Many strategies have beenProtein cage nanoparticles (PCNs) are attractive platforms for developing functional nanomaterials using biomimetic approaches for functionalization and cargo encapsulation. Many strategies have been employed to direct the loading of molecular cargos inside a wide range of PCN architectures. Here we demonstrate the exploitation of a metal-ligand coordination bond with respect to the direct packing of guest molecules on the interior interface of a virus-like PCN derived from Salmonella typhimurium bacteriophage P22. The incorporation of these guest species was assessed using mass spectrometry, multiangle laser light scattering, and analytical ultracentrifugation. In addition to small-molecule encapsulation, this approach was also effective for the directed synthesis of a large macromolecular coordination polymer packed inside of the P22 capsid and initiated on the interior surface. A wide range of metals and ligands with different thermodynamic affinities and kinetic stabilities are potentially available for this approach, highlighting the potential for metal-ligand coordination chemistry to direct the site-specific incorporation of cargo molecules for a variety of applications.

• A retroviral chimeric capsid protein reveals the role of the N-terminal β-hairpin in mature core assembly.

  Authors: Juliana R Cortines, Eric B Monroe, Sebyung Kang, Peter E Prevelige

  Journal of molecular biology. 07/2011; 410(4):641-52.

  The human immunodeficiency virus (HIV) is an enveloped virus constituted by two monomeric RNA molecules that encode for 15 proteins. Among these are the structural proteins that are translated as theThe human immunodeficiency virus (HIV) is an enveloped virus constituted by two monomeric RNA molecules that encode for 15 proteins. Among these are the structural proteins that are translated as the gag polyprotein. In order to become infectious, HIV must undergo a maturation process mediated by the proteolytic cleavage of gag to give rise to the isolated structural protein matrix, capsid (CA), nucleocapsid as well as p6 and spacer peptides 1 and 2. Upon maturation, the 13 N-terminal residues from CA fold into a β-hairpin, which is stabilized mainly by a salt bridge between Pro1 and Asp51. Previous reports have shown that non-formation of the salt bridge, which potentially disrupts proper β-hairpin arrangement, generates noninfectious virus or aberrant cores. To date, however, there is no consensus on the role of the β-hairpin. In order to shed light in this subject, we have generated mutations in the hairpin region to examine what features would be crucial for the β-hairpin's role in retroviral mature core formation. These features include the importance of the proline at the N-terminus, the amino acid sequence, and the physical structure of the β-hairpin itself. The presented experiments provide biochemical evidence that β-hairpin formation plays an important role in regard to CA protein conformation required to support proper mature core arrangement. Hydrogen/deuterium exchange and in vitro assembly reactions illustrated the importance of the β-hairpin structure, its dynamics, and its influence on the orientation of helix 1 for the assembly of the mature CA lattice.

• Genetically programmed in vivo packaging of protein cargo and its controlled release from bacteriophage P22.

  Authors: Alison O'Neil, Courtney Reichhardt, Benjamin Johnson, Peter E Prevelige, Trevor Douglas

  Angewandte Chemie (International ed. in English). 06/2011; 50(32):7425-8.

• Templated assembly of organic-inorganic materials using the core shell structure of the P22 bacteriophage.

  Authors: Courtney Reichhardt, Masaki Uchida, Alison O'Neil, Rui Li, Peter E Prevelige, Trevor Douglas

  Chemical communications (Cambridge, England). 06/2011; 47(22):6326-8.

  Biomimetic chemistry offers new approaches to supramolecular materials synthesis and assembly. We have demonstrated that an assembled viral protein cage, comprising an organic core-shell structure,Biomimetic chemistry offers new approaches to supramolecular materials synthesis and assembly. We have demonstrated that an assembled viral protein cage, comprising an organic core-shell structure, can be used as a template for the size constrained synthesis of Fe(2)O(3). Particle nucleation is directed by the inner scaffold protein layer, while the size constraints are determined by the outer capsid layer.

• Three-dimensional structure of a viral genome-delivery portal vertex.

  Authors: Adam S Olia, Peter E Prevelige, John E Johnson, Gino Cingolani

  Nature structural & molecular biology. 05/2011; 18(5):597-603.

  DNA viruses such as bacteriophages and herpesviruses deliver their genome into and out of the capsid through large proteinaceous assemblies, known as portal proteins. Here, we report two snapshots ofDNA viruses such as bacteriophages and herpesviruses deliver their genome into and out of the capsid through large proteinaceous assemblies, known as portal proteins. Here, we report two snapshots of the dodecameric portal protein of bacteriophage P22. The 3.25-Å-resolution structure of the portal-protein core bound to 12 copies of gene product 4 (gp4) reveals a ~1.1-MDa assembly formed by 24 proteins. Unexpectedly, a lower-resolution structure of the full-length portal protein unveils the unique topology of the C-terminal domain, which forms a ~200-Å-long α-helical barrel. This domain inserts deeply into the virion and is highly conserved in the Podoviridae family. We propose that the barrel domain facilitates genome spooling onto the interior surface of the capsid during genome packaging and, in analogy to a rifle barrel, increases the accuracy of genome ejection into the host cell.

• Fabrication of Ordered Nanostructures of Sulfide Nanocrystal Assemblies over Self-Assembled Genetically Engineered P22 Coat Protein.

  Authors: Liming Shen, Ningzhong Bao, Peter E Prevelige, Arunava Gupta

  Journal of the American Chemical Society. 11/2010;

  Ordered ZnS and CdS nanocrystal assemblies have been synthesized by a facile bioinspired approach consisting of an initial self-assembly of engineered proteins into spherical biotemplates and aOrdered ZnS and CdS nanocrystal assemblies have been synthesized by a facile bioinspired approach consisting of an initial self-assembly of engineered proteins into spherical biotemplates and a subsequent protein-directed nucleation and growth of ZnS and CdS nanocrystals symmetrically distributed over the self-assembled biotemplates.

• Mind the gap: how some viruses infect their hosts.

  Authors: Peter E Prevelige

  Viruses. 11/2010; 2(11):2536-40.

  Cryo-electron microscopy (Cryo-EM) and cryo-electron tomography (Cryo-ET) provide structural insights into complex biological processes. The podoviridae are dsDNA containing phage with short,Cryo-electron microscopy (Cryo-EM) and cryo-electron tomography (Cryo-ET) provide structural insights into complex biological processes. The podoviridae are dsDNA containing phage with short, non-contractile tails which nevertheless translocate their DNA into the cytoplasm of their host cells. Liu et al. [1] used a combination of cryo-EM and cryo-ET to study the structural changes accompanying infection of P. marinus by the phage P-SSP7 and thereby provide unique molecular insight into the process by which the DNA transits from phage to host during infection.

• Hydrogen/deuterium exchange analysis of HIV-1 capsid assembly and maturation.

  Authors: Eric B Monroe, Sebyung Kang, Sampson K Kyere, Rui Li, Peter E Prevelige

  Structure (London, England : 1993). 11/2010; 18(11):1483-91.

  Following budding, HIV-1 virions undergo a maturation process where the Gag polyprotein in the immature virus is cleaved by the viral protease and rearranges to form the mature infectious virion.Following budding, HIV-1 virions undergo a maturation process where the Gag polyprotein in the immature virus is cleaved by the viral protease and rearranges to form the mature infectious virion. Despite the wealth of structures of isolated capsid domains and an in vitro-assembled mature lattice, models of the immature lattice do not provide an unambiguous model of capsid-molecule orientation and no structural information is available for the capsid maturation pathway. Here we have applied hydrogen/deuterium exchange mass spectrometry to immature, mature, and mutant Gag particles (CA5) blocked at the final Gag cleavage event to examine the molecular basis of capsid assembly and maturation. Capsid packing arrangements were very similar for all virions, whereas immature and CA5 virions contained an additional intermolecular interaction at the hexameric, 3-fold axis. Additionally, the N-terminal β-hairpin was observed to form as a result of capsid-SP1 cleavage rather than driving maturation as previously postulated.

• Implementation of p22 viral capsids as nanoplatforms.

  Authors: Sebyung Kang, Masaki Uchida, Alison O'Neil, Rui Li, Peter E Prevelige, Trevor Douglas

  Biomacromolecules. 10/2010; 11(10):2804-9.

  Viral capsids are dynamic macromolecular machines which self-assemble and undergo concerted conformational changes during their life cycle. We have taken advantage of the inherent structuralViral capsids are dynamic macromolecular machines which self-assemble and undergo concerted conformational changes during their life cycle. We have taken advantage of the inherent structural flexibility of viral capsids and generated two morphologically different types of viral nanoplatforms from the bacteriophage P22 capsids. Their interior surfaces were genetically manipulated for site-specific attachment of a biotin linker. The extent of internal modifications in each capsid form was characterized by high-resolution mass spectrometry and the analyses revealed that the reactivity of the genetically introduced residues located on the internal surface changes according to the structural transformation of the capsid. Internally modified capsids having 10 nm diameter pores at the 12 icosahedral vertices, so-called wiffle-balls (WB), exhibited the capability to entrap the large tetrameric protein complex streptavidin via the biotin linker anchored onto the interior surface of the WB.

• A docking model based on mass spectrometric and biochemical data describes phage packaging motor incorporation.

  Authors: Chi-yu Fu, Charlotte Uetrecht, Sebyung Kang, Marc C Morais, Albert J R Heck, Mark R Walter, Peter E Prevelige

  Molecular & cellular proteomics : MCP. 08/2010; 9(8):1764-73.

  The molecular mechanism of scaffolding protein-mediated incorporation of one and only one DNA packaging motor/connector dodecamer at a unique vertex during lambdoid phage assembly has remainedThe molecular mechanism of scaffolding protein-mediated incorporation of one and only one DNA packaging motor/connector dodecamer at a unique vertex during lambdoid phage assembly has remained elusive because of the lack of structural information on how the connector and scaffolding proteins interact. We assembled and characterized a phi29 connector-scaffolding complex, which can be incorporated into procapsids during in vitro assembly. Native mass spectrometry revealed that the connector binds at most 12 scaffolding molecules, likely organized as six dimers. A data-driven docking model, using input from chemical cross-linking and mutagenesis data, suggested an interaction between the scaffolding protein and the exterior of the wide domain of the connector dodecamer. The connector binding region of the scaffolding protein lies upstream of the capsid binding region located at the C terminus. This arrangement allows the C terminus of scaffolding protein within the complex to both recruit capsid subunits and mediate the incorporation of the single connector vertex.

• In vitro incorporation of the phage Phi29 connector complex.

  Authors: Chi-Yu Fu, Peter E Prevelige

  Virology. 10/2009;

  The incorporation of the DNA packaging connector complex during lambdoid phage assembly in vivo is strictly controlled-one and only one of the twelve identical icosahedral vertices is differentiatedThe incorporation of the DNA packaging connector complex during lambdoid phage assembly in vivo is strictly controlled-one and only one of the twelve identical icosahedral vertices is differentiated by the inclusion of a portal or connector dodecamer. Proposed control mechanisms include obligate nucleation from a connector containing complex, addition of the connector as the final step during assembly, and a connector-mediated increase in the growth rate. The inability to recapitulate connector incorporation in vitro has made it difficult to obtain direct biochemical evidence in support of one model over another. Here we report the development an in vitro assembly system for the well characterized dsDNA phage Phi29 which results in the co-assembly of connector with capsid and scaffolding proteins to form procapsid-like particles (PLPs). Immuno-electron microscopy demonstrates the specific incorporation of connector vertex in PLPs. The connector protein increases both the yield and the rate of capsid assembly suggesting that the incorporation of the connector in Phi29 likely promotes nucleation of assembly.

• Characterization of a Mumps Virus Nucleocapsid-like Particle.

  Authors: Robert Cox, Todd J Green, Shihong Qiu, Jungsoon Kang, Jun Tsao, Peter E. Prevelige, Biao He, Ming Luo

  Journal of virology. 09/2009;

  The nucleocapsid protein (NP) of mumps virus (MuV), a paramyxovirus, was co-expressed with the phosphoprotein (P) in E. coli. The NP and P proteins form a soluble complex containing RNA. Under aThe nucleocapsid protein (NP) of mumps virus (MuV), a paramyxovirus, was co-expressed with the phosphoprotein (P) in E. coli. The NP and P proteins form a soluble complex containing RNA. Under a transmission electron microscope, the NP-RNA complex appears as a nucleocapsid-like ring that has a diameter of approximately 20nm with thirteen subunits. There is a piece of single strand RNA with a length of 78 nucleotides in the NP-RNA ring. Shorter RNA pieces are also observed. The MuV NP protein may provide weaker protection of the RNA, reflecting the degree of NP protein association.

• The P22 tail machine at subnanometer resolution reveals the architecture of an infection conduit.

  Authors: Gabriel C Lander, Reza Khayat, Rui Li, Peter E Prevelige, Clinton S Potter, Bridget Carragher, John E Johnson

  Structure (London, England : 1993). 07/2009; 17(6):789-99.

  The portal channel is a key component in the life cycle of bacteriophages and herpesviruses. The bacteriophage P22 portal is a 1 megadalton dodecameric oligomer of gp1 that plays key roles in capsidThe portal channel is a key component in the life cycle of bacteriophages and herpesviruses. The bacteriophage P22 portal is a 1 megadalton dodecameric oligomer of gp1 that plays key roles in capsid assembly, DNA packaging, assembly of the infection machinery, and DNA ejection. The portal is the nucleation site for the assembly of 39 additional subunits generated from multiple copies of four gene products (gp4, gp10, gp9, and gp26), which together form the multifunctional tail machine. These components are organized with a combination of 12-fold (gp1, gp4), 6-fold (gp10, trimers of gp9), and 3-fold (gp26, gp9) symmetry. Here we present the 3-dimensional structures of the P22 assembly-naive portal formed from expressed subunits (gp1) and the intact tail machine purified from infectious virions. The assembly-naive portal structure exhibits a striking structural similarity to the structures of the portal proteins of SPP1 and phi29 derived from X-ray crystallography.

• Synthesis of biotin-tagged chemical cross-linkers and their applications for mass spectrometry.

  Authors: Sebyung Kang, Liyuan Mou, Jason Lanman, Sadanandan Velu, Wayne J Brouillette, Peter E Prevelige

  Rapid communications in mass spectrometry : RCM. 07/2009; 23(11):1719-26.

  Chemical cross-linking combined with mass spectrometry (MS) has been used to elucidate protein structures and protein-protein interactions. However, heterogeneity of the samples and the relativelyChemical cross-linking combined with mass spectrometry (MS) has been used to elucidate protein structures and protein-protein interactions. However, heterogeneity of the samples and the relatively low abundance of cross-linked peptides make this approach challenging. As an effort to overcome this hurdle, we have synthesized lysine-reactive homobifunctional cross-linkers with the biotin in the middle of the linker and used them to enrich cross-linked peptides. The reaction of biotin-tagged cross-linkers with purified HIV-1 CA resulted in the formation of hanging and intramolecular cross-links. The peptides modified with biotinylated cross-linkers were effectively enriched and recovered using a streptavidin-coated plate and MS-friendly buffers. The enrichment of modified peptides and removal of the dominantly unmodified peptides simplify mass spectra and their analyses. The combination of the high mass accuracy of Fourier transform ion cyclotron resonance (FT-ICR) MS and the tandem mass spectrometric (MS/MS) capability of the linear ion trap allows us to unambiguously identify the cross-linking sites and additional modification, such as oxidation. Copyright (c) 2009 John Wiley & Sons, Ltd.

• Controlled assembly of bifunctional chimeric protein cages and composition analysis using noncovalent mass spectrometry.

  Authors: Sebyung Kang, Luke M Oltrogge, Chris C Broomell, Lars O Liepold, Peter E Prevelige, Mark Young, Trevor Douglas

  Journal of the American Chemical Society. 01/2009; 130(49):16527-9.

• Send for reinforcements! Conserved binding of capsid decoration proteins.

  Authors: Peter E Prevelige

  Structure (London, England : 1993). 10/2008; 16(9):1292-3.

  In this issue of Structure, Lander et al. (2008) highlight the confluence of three exciting developments in structural virology: an appreciation of the evolutionary relatedness of viruses, theIn this issue of Structure, Lander et al. (2008) highlight the confluence of three exciting developments in structural virology: an appreciation of the evolutionary relatedness of viruses, the advances in cryo-EM technology, and the renewed interest of biophysicists in understanding the forces involved in DNA packaging and capsid stability.

• Hydrogen/deuterium exchange on protein solutions containing nucleic acids: utility of protamine sulfate.

  Authors: Anton Poliakov, Paul Jardine, Peter E Prevelige

  Rapid communications in mass spectrometry : RCM. 08/2008; 22(16):2423-8.

  Obtaining global hydrogen/deuterium (H/D) exchange data on proteins is an important first step in amide proton exchange experiments. Important information such as the mode of exchange, theObtaining global hydrogen/deuterium (H/D) exchange data on proteins is an important first step in amide proton exchange experiments. Important information such as the mode of exchange, the cooperativity of folding/unfolding reactions, and the effects of ligand binding can be readily obtained in global exchange experiments. Many interesting biological systems are complexes containing both proteins and nucleic acids. The low pH conditions required to quench H/D exchange reactions result in the formation of stable protein/nucleic acid precipitates which interfere with the liquid chromatography step of the experiment and preclude obtaining mass spectrometric data. In this work we show that the precipitation of proteins and nucleic acids is electrostatic in nature and can be prevented by high ionic strength and by removing nucleic acids by protamine sulfate. Using protamine sulfate in quenching solution, we were able to obtain global H/D data with protein samples containing large amounts of DNA or RNA.

• Detection of Intermediates and Kinetic Control during Assembly of Bacteriophage P22 Procapsid.

  Authors: Roman Tuma, Hiro Tsuruta, Kenneth H French, Peter E Prevelige

  Journal of molecular biology. 07/2008;

  Bacteriophage P22 serves as a model for the assembly and maturation of other icosahedral double-stranded DNA viruses. P22 coat and scaffolding proteins assemble in vitro into an icosahedralBacteriophage P22 serves as a model for the assembly and maturation of other icosahedral double-stranded DNA viruses. P22 coat and scaffolding proteins assemble in vitro into an icosahedral procapsid, which then expands during DNA packaging (maturation). Efficient in vitro assembly makes this system suitable for design and production of monodisperse spherical nanoparticles (diameter approximately 50 nm). In this work, we explore the possibility of controlling the outcome of assembly by scaffolding protein engineering. The scaffolding protein exists in monomer-dimer-tetramer equilibrium. We address the role of monomers and dimers in assembly by using three different scaffolding proteins with altered monomer-dimer equilibrium (weak dimer, covalent dimer, monomer). The progress and outcome of assembly was monitored by time-resolved X-ray scattering, which allowed us to distinguish between closed shells and incomplete assembly intermediates. Binding of scaffolding monomer activates the coat protein for assembly. Excess dimeric scaffolding protein resulted in rapid nucleation and kinetic trapping yielding incomplete shells. Addition of monomeric wild-type scaffold with excess coat protein completed these metastable shells. Thus, the monomeric scaffolding protein plays an essential role in the elongation phase by activating the coat and effectively lowering its critical concentration for assembly.

• Development of bacteriophage p22 as a platform for molecular display: genetic and chemical modifications of the procapsid exterior surface.

  Authors: Sebyung Kang, Gabriel C Lander, John E Johnson, Peter E Prevelige

  Chembiochem : a European journal of chemical biology. 04/2008; 9(4):514-8.

• Probing Conserved Helical Modules of Portal Complexes by Mass Spectrometry-based Hydrogen/Deuterium Exchange.

  Authors: Sebyung Kang, Anton Poliakov, Jennifer Sexton, Matthew B Renfrow, Peter E Prevelige

  Journal of molecular biology. 04/2008;

  The Double-stranded DNA bacteriophage P22 has a ring-shaped dodecameric complex composed of the 84 kDa portal protein subunit that forms the central channel of the phage DNA packaging motor. TheThe Double-stranded DNA bacteriophage P22 has a ring-shaped dodecameric complex composed of the 84 kDa portal protein subunit that forms the central channel of the phage DNA packaging motor. The overall morphology of the P22 portal complex is similar to that of the portal complexes of Phi29, SPP1, T3, T7 phages and herpes simplex virus. Secondary structure prediction of P22 portal protein and its threading onto the crystal structure of the Phi29 portal complexes suggested that the P22 portal protein complex shares conserved helical modules that were found in the dodecameric interfaces of the Phi29 portal complex. To identify the amino acids involved in intersubunit contacts in the P22 portal ring complexes and validate the threading model, we performed comparative hydrogen/deuterium exchange analysis of monomeric and in vitro assembled portal proteins of P22 and the dodecameric Phi29 portal. Hydrogen/deuterium exchange experiments provided evidence of intersubunit interactions in the P22 portal complex similar to those in the Phi29 portal that map to the regions predicted to be conserved helical modules.