RESUMO
Virus-like particles (VLPs) have many potentially useful applications. The core proteins of human hepatitis B virus self-assemble into icosahedral VLPs. As previously reported, core protein dimers (CPDs), produced by connecting two core proteins via a peptide linker, can also assemble into VLPs. CPDs in which heterologous proteins were connected to the C-terminus (CPD1) were found to rearrange into symmetrical octahedra during crystallization. In this study, a heterologous protein was inserted into the peptide linker of the CPD (CPD2). CPD2 was expressed in Escherichia coli, assembled into VLPs, purified and crystallized. A single crystal diffracted to 2.8 Å resolution and belonged to the cubic space group F432, with unit-cell parameters a = b = c = 218.6 Å. Single-crystal analysis showed that CPD1 and CPD2 rearranged into the same octahedral organization in a crystallization solution.
Assuntos
Regulação Viral da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/química , Mutagênese Insercional , Fragmentos de Peptídeos/genética , Multimerização Proteica , Proteínas do Core Viral/química , Cristalização , Cristalografia por Raios X , Antígenos do Núcleo do Vírus da Hepatite B/química , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Mutagênese Insercional/genética , Mutagênese Insercional/imunologia , Fragmentos de Peptídeos/química , Multimerização Proteica/fisiologia , Proteínas do Core Viral/genética , Proteínas do Core Viral/isolamento & purificaçãoRESUMO
Recombinant hepatitis B virus core proteins dimerize to form building blocks that are capable of self-assembly into a capsid. A core capsid protein dimer (CPD) linked to a green fluorescent protein variant, EGFP, at the C-terminus has been designed. The recombinant fusion CPD was expressed in Escherichia coli, assembled into virus-like particles (VLPs), purified and crystallized. The single crystal diffracted to 2.15 Å resolution and belonged to the cubic space group F432, with unit-cell parameters a = b = c = 219.7 Å. The fusion proteins assembled into icosahedral VLPs in aqueous solution, but were rearranged into octahedral symmetry through the crystal-packing process under the crystallization conditions.
Assuntos
Vírus da Hepatite B/metabolismo , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/isolamento & purificação , Capsídeo/química , Cristalização , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Vírus da Hepatite B/ultraestrutura , Microscopia de Fluorescência , Multimerização Proteica , Ultracentrifugação , Vírion/ultraestruturaRESUMO
GABA(B) receptor is a G protein-coupled receptor for GABA and drug target for neurological and psychiatric disorders. From the analysis of GTPgammaS binding assay, we found that a synthesized peptide (GABAb: ETKSVSTEKINDHR) corresponding to the intracellular third loop region of metabotropic GABA(B) receptor could activate G(i) protein alpha subunit directly. The three dimensional molecular structure of the peptide in SDS-d(25) micelles was determined by 2D (1)H-NMR spectroscopy. GABAb peptide formed an alpha helical structure and a positive charge cluster at the C-terminal site. These structural features were also found in several other G protein activating peptides. From the comparison among these peptides, we found that peptides with high helical content show the high activity.
Assuntos
Modelos Químicos , Modelos Moleculares , Receptores de GABA-B/química , Receptores de GABA-B/ultraestrutura , Simulação por Computador , Humanos , Espectroscopia de Ressonância Magnética , Conformação ProteicaRESUMO
Human APG1 gene is homologous to Drosophila methuselah gene associated with extended life span. A peptide (APG1: RNGKRSNRTLREE) corresponding to a predicted region of the intracellular third loop of G protein-coupled receptor coded in human APG1 gene could activate Gi protein alpha subunit directly. The three-dimensional molecular structure of the peptide in SDS-d25 micelles was determined by 2D 1H NMR spectroscopy. APG1 formed an alpha-helical structure at the C-terminal site and a positive charge cluster at the N-terminal site. The cluster was also found in several other Gi protein-coupled receptor peptides. Therefore, the positive charge cluster on the helical structure might be engaged in G protein activation.
Assuntos
Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/genética , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/genéticaRESUMO
We found that a peptide (EP3a: TIKALVSRCRAKAAV) corresponding to the N-terminal site of the intracellular third loop of human prostaglandin EP3alpha receptor could activate G protein alpha-subunit directly. The activity was almost same as Mastoparan-X, a G protein activating peptide from wasp venom. The three-dimensional molecular structure of the peptide in SDS-d(25) micelles was determined by 2D (1)H NMR spectroscopy. The structure of EP3a consists of a positive charge cluster on the C-terminal helical site. The cluster was also found in several corresponding receptor peptides. Therefore, the positive charge cluster on the helical structure might play a crucial role in activation of G protein.
