Expression and spectroscopic analysis of a mutant hepatitis B virus onco-protein HBx without cysteine residues.
Chronic infection of the hepatitis B virus (HBV) is one of the causes leading to liver cancer. The 3.2kb genome of HBV encodes four proteins: core antigen, surface antigen, a DNA polymerase and the X protein (HBx). The biological functions of HBx are not fully understood. It has been shown that HBx is a potent trans-activator, which activates transcription of many cellular and viral promoters indirectly via protein-protein interactions. These transactivating activities of HBx may contribute to the development of hepatocellular carcinoma. In this paper a truncated mini-HBx(-Cys) (18-142) protein, where the cysteines had been either deleted or substituted by serines, was constructed by site-directed mutagenesis and overexpressed as a 6xHis fusion protein in Escherichia coli. The 6xHis-mini-HBx(-Cys) protein was isolated from inclusion bodies, purified by Ni-affinity chromatography under denaturing conditions and refolded by sequential dialysis. The structure of the 6xHis-mini-HBx(-Cys) protein was analyzed by circular dichroism, fluorescence and one-dimensional NMR spectroscopic assays. The data presented here suggest that HBx is unstructured but has a propensity to gain secondary structure under specific experimental conditions. Its conformational flexibility might partially explain its functional complexity, namely its capacity to interact with a wide array of signaling proteins, transcriptional regulators and nucleic acids.
Rui
Edmilson
E
Centro de Biologia Molecular Estrutural, Laboratório Nacional de Luz Síncrotron, Rua Giuseppe Máximo Scolfaro 10.000, CP 6192, Campinas, SP, CEP 13084-971, Brazil.
Moura
Patricia Ribeiro de
PR
Gonçalves
Kaliandra de Almeida
Kde A
Kobarg
Jörg
J
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003545:Cysteine / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006515:Hepatitis B virus / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D054334:Viral Regulatory and Accessory Proteins
D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D003545:Cysteine