title: Disulfide linkage and structure of highly stable yeast-derived virus-like particles of murine polyomavirus.
abstract: VP1 is the major coat protein of murine polyomavirus and forms virus-like particles (VLPs) in vitro. VLPs consist of 72 pentameric VP1 subunits held together by a terminal clamp structure that is further stabilized by disulfide bonds and chelation of calcium ions. Yeast-derived VLPs (yVLPs) assemble intracellularly in vivo during recombinant protein production. These in vivo assembled yVLPs differ in several properties from VLPs assembled in vitro from bacterially produced pentamers. We found several intermolecular disulfide linkages in yVLPs involving 5 of the 6 cysteines of VP1 (Cys(115)-Cys(20), Cys(12)-Cys(20), Cys(16)-Cys(16), Cys(12)/ Cys(16)-Cys(115), and Cys(274)-Cys(274)), indicating a highly coordinated disulfide network within the in vivo assembled particles involving the N-terminal region of VP1. Cryoelectron microscopy revealed structured termini not resolved in the published crystal structure of the bacterially expressed VLP that appear to clamp the pentameric subunits together. These structural features are probably the reason for the observed higher stability of in vivo assembled yVLPs compared with in vitro assembled bacterially expressed VLPs as monitored by increased thermal stability, higher resistance to trypsin cleavage, and a higher activation enthalpy of the disassembly reaction. This high stability is decreased following disassembly of yVLPs and subsequent in vitro reassembly, suggesting a role for cellular components in optimal assembly.
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes Strasse 03, 06120 Halle, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Simon
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
authors.forename: Thomas
authors.initials: T
authors.lastname: Klose
authors.affiliation: Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, Wolfgang-Langenbeck Strasse 04, 06120 Halle, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Herbst
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720.
authors.forename: Bong Gyoon
authors.initials: BG
authors.lastname: Han
authors.affiliation: Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, Wolfgang-Langenbeck Strasse 04, 06120 Halle, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Sinz
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Glaeser
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes Strasse 03, 06120 Halle, Germany.
authors.forename: Milton T
authors.initials: MT
authors.lastname: Stubbs
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes Strasse 03, 06120 Halle, Germany. Electronic address: hauke.lilie@biochemtech.uni-halle.de.
authors.forename: Hauke
authors.initials: H
authors.lastname: Lilie
mesh_terms: D000595:Amino Acid Sequence; D002213:Capsid / Q000737:chemistry; D036022:Capsid Proteins / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry; D020285:Cryoelectron Microscopy; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D006358:Hot Temperature; D007700:Kinetics; D007716:Kluyveromyces / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011120:Polyomavirus / Q000737:chemistry* / Q000648:ultrastructure; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry; D014461:Ultracentrifugation; D014771:Virion / Q000737:chemistry; D019065:Virus Assembly
chemical_list: D036022:Capsid Proteins; D003432:Cross-Linking Reagents; D004220:Disulfides; D010455:Peptides; D011994:Recombinant Proteins; C062481:VP1 protein, polyomavirus; D012259:Ribonuclease, Pancreatic; D014357:Trypsin; D003545:Cysteine
keywords: Protein Self-assembly; Protein Stability; Viral Protein; Virus Assembly; Yeast