title: Localization of the N terminus of hepatitis B virus capsid protein by peptide-based difference mapping from cryoelectron microscopy.
abstract: Recently, cryoelectron microscopy of isolated macromolecular complexes has advanced to resolutions below 10 A, enabling direct visualization of alpha-helical secondary structure. To help correlate such density maps with the amino acid sequences of the component proteins, we advocate peptide-based difference mapping, i. e., insertion of peptides, approximately 10 residues long, at targeted points in the sequence and visualization of these peptides as bulk labels in cryoelectron microscopy-derived difference maps. As proof of principle, we have appended an extraneous octapeptide at the N terminus of hepatitis B virus capsid protein and determined its location on the capsid surface by difference imaging at 11 A resolution. Hepatitis B virus capsids are icosahedral particles, approximately 300 A in diameter, made up of T-shaped dimers (subunit Mr, 16-21 kDa, depending on construct). The stems of the Ts protrude outward as spikes, whereas the crosspieces pack to form the contiguous shell. The two N termini per dimer reside on either side of the spike-stem, at the level at which it enters the shell. This location is consistent with formation of the known intramolecular disulfide bond between the cysteines at positions 61 and -7 (in the residual propeptide) in the "e-antigen" form of the capsid protein and has implications for why this clinically important antigen remains unassembled in vivo.
authors.affiliation: Laboratory of Structural Biology Research, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: J F
authors.initials: JF
authors.lastname: Conway
authors.forename: N
authors.initials: N
authors.lastname: Cheng
authors.forename: A
authors.initials: A
authors.lastname: Zlotnick
authors.forename: S J
authors.initials: SJ
authors.lastname: Stahl
authors.forename: P T
authors.initials: PT
authors.lastname: Wingfield
authors.forename: A C
authors.initials: AC
authors.lastname: Steven
mesh_terms: D000595:Amino Acid Sequence; D002213:Capsid / Q000737:chemistry* / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy; D006515:Hepatitis B virus / Q000737:chemistry* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D017510:Protein Folding