RESUMO
Determination of the oligomeric state of integral membrane proteins in detergent solutions is a challenging task because the amount of detergent associated with the protein is typically unknown and unpredictable. Methods that estimate the molecular weight of proteins from their hydrodynamic properties in solution are not suitable for detergent-solubilized membrane proteins. However, size-exclusion chromatography (SEC) performed in combination with analyses of static light scattering (SLS), ultraviolet absorbance (UV), and refractive index (RI) provides a universal method for determination of the molar masses of biopolymers and protein-detergent complexes. The light scattered by a protein is directly proportional to its molecular mass, irrespective of shape, and any additional contributions due to bound detergent molecules can be quantitatively accounted for by the additional combined analysis of ultraviolet absorbance and refractive index information. The primary intention of this unit is to describe how to apply the combination of high-performance liquid chromatography SEC and SLS-UV-RI to evaluate molecular mass and the physicochemical heterogeneity of purified membrane protein-detergent complexes.
Assuntos
Cromatografia em Gel/métodos , Cromatografia Líquida de Alta Pressão/métodos , Detergentes/química , Proteínas de Membrana/química , Animais , Bovinos , Fracionamento Químico , Padrões de Referência , Soroalbumina Bovina/metabolismoRESUMO
The ectodomain of HIV-1 gp41 mediates the fusion of viral and host cellular membranes. The peptide-based drug Enfuvirtide(1) is precedent that antagonists of this fusion activity may act as anti HIV-agents. Here, NMR screening was used to discover non-peptide leads against this target and resulted in the discovery of a new benzamide 1 series. This series is non-peptide, low molecular weight, and analogs have activity in a cell fusion assay with EC50 values ranging 3-41microM. Structural work on the gp41/benzamide 1 complex was determined by NMR spectroscopy using a designed model peptide system that mimics an open pocket of the fusogenic form of the protein.
Assuntos
Fármacos Anti-HIV/química , Benzamidas/química , Proteína gp41 do Envelope de HIV/química , Inibidores da Fusão de HIV/química , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Benzamidas/síntese química , Benzamidas/farmacologia , Cristalografia por Raios X , Enfuvirtida , Proteína gp41 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/farmacologia , Inibidores da Fusão de HIV/síntese química , Inibidores da Fusão de HIV/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
TRPV1 is a ligand-gated cation channel that is involved in acute thermal nociception and neurogenic inflammation. By using the GP67 signal peptide, high levels of full-length human TRPV1 was expressed in High Five insect cells using the baculovirus expression system. The functional activity of the expressed TRPV1 was confirmed by whole-cell ligand-gated ion flux recordings in the presence of capsaicin and low pH and via specific ligand binding to the isolated cellular membranes. Efficient solubilization and purification protocols have resulted in milligram amounts of detergent-solubilized channel at 80-90% purity after Ni2+ IMAC chromatography and size exclusion chromatography. Western blot analysis of amino and carboxyl terminal domains and MS of tryptic digestions of purified protein confirmed the presence of the full-length human TRPV1. Specific ligand binding experiments confirmed the protein integrity of the purified human TRPV1.
Assuntos
Baculoviridae , Expressão Gênica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Canais de Cátion TRPV/biossíntese , Canais de Cátion TRPV/isolamento & purificação , Animais , Linhagem Celular , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Spodoptera , Canais de Cátion TRPV/química , Canais de Cátion TRPV/genéticaRESUMO
Calsenilin is a member of the recoverin branch of the EF-hand superfamily that is reported to interact with presenilins, regulate prodynorphin gene expression, modulate voltage-gated Kv4 potassium channel function, and bind to neurotoxins. Calsenilin is a Ca+2-binding protein and plays an important role in calcium signaling. Despite its importance in numerous neurological functions, the structure of this protein has not been reported. In the absence of Ca+2, the protein has limited spectral resolution that increases upon the addition of Ca+2. Here, we describe the three-dimensional solution structure of EF-hands 3 and 4 of calsenilin in the Ca+2-bound form. The Ca+2-bound structure consists of five alpha-helices and one two-stranded antiparallel beta-sheet. The long loop that connects EF hands 3 and 4 is highly disordered in solution. In addition to its structural effects, Ca+2 binding also increases the protein's propensity to dimerize. These changes in structure and oligomerization state induced upon Ca+2 binding may play important roles in molecular recognition during calcium signaling.
Assuntos
Cálcio/química , Proteínas Interatuantes com Canais de Kv/química , Sequência de Aminoácidos , Cálcio/metabolismo , Dicroísmo Circular , Dimerização , Escherichia coli/metabolismo , Humanos , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Canais de Potássio/química , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Two new proteins of approximately 70 amino acids in length, corresponding to an unnaturally-linked N- and C-helix of the ectodomain of the gp41 protein from the human immunodeficiency virus (HIV) type 1, were designed and characterized. A designed tripeptide links the C-terminus of the C-helix with the N-terminus of the N-helix in a circular permutation so that the C-helix precedes the N-helix in sequence. In addition to the artificial peptide linkage, the C-helix is truncated at its N-terminus to expose a region of the N-helix known as the "Trp-Trp-Ile" binding pocket. Sedimentation, crystallographic, and nuclear magnetic resonance studies confirmed that the protein had the desired trimeric structure with an unoccupied binding site. Spectroscopic and centrifugation studies demonstrated that the engineered protein had ligand binding characteristics similar to previously reported constructs. Unlike previous constructs which expose additional, shallow, non-conserved, and undesired binding pockets, only the single deep and conserved Trp-Trp-Ile pocket is exposed in the proteins of this study. This engineered version of gp41 protein will be potentially useful in research programs aimed at discovery of new drugs for therapy of HIV-infection in humans.
Assuntos
Desenho de Fármacos , Proteína gp41 do Envelope de HIV/química , HIV-1/química , Engenharia de Proteínas , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Proteína gp41 do Envelope de HIV/genética , HIV-1/genética , Humanos , Dados de Sequência Molecular , Conformação ProteicaRESUMO
The human immunodeficiency virus type 1 (HIV-1) gp41 plays an important role in mediating the fusion of HIV with host cells. During the fusion process, three N-terminal helices and three C-terminal helices pack in an anti-parallel direction to form a six-helix bundle. X-ray crystallographic analysis of the gp41 core demonstrated that within each coiled-coil interface, there is a deep and large pocket, formed by a cluster of residues in the N-helix coiled-coil. In this report, we systematically analyzed the role of seven conserved residues that are either lining or packing this pocket on the infectivity and interhelical interaction using novel approaches. Our results show that residues L568, V570, W571, and K574 of the N-helix that are lining the side chain and right wall of the pocket are important for establishing a productive infection. Mutations V570A and W571A completely abolished replication, while replication of the L568A and K574A mutants was significantly attenuated relative to wild type. Similarly, residues W628, W631, and I635 of the C-helix that insert into the pocket are essential for infectivity. The impaired infectivity of these seven mutants is in part attributed to the loss in binding affinity of the interhelical interaction. Molecular modeling of the crystal structure of the coiled-coil further shows that alanine substitution of those residues disrupts the hydrophobic interaction between the N- and C-helix. These results suggest that the conserved residues in the coiled-coil domain play a key role in HIV infection and this coiled-coil pocket is a good target for development of inhibitors against HIV. In addition, our data indicate that the novel fluorescence polarization assay described in this study could be valuable in screening for inhibitors that block the interhelical interaction and HIV entry.
Assuntos
Proteína gp41 do Envelope de HIV/genética , HIV-1/genética , Replicação Viral/genética , Sequência de Aminoácidos , Biomarcadores Tumorais , Peptídeo C/química , Peptídeo C/genética , Linhagem Celular Transformada , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Polarização de Fluorescência/métodos , Proteína gp41 do Envelope de HIV/química , HIV-1/patogenicidade , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fosfopiruvato Hidratase/química , Fosfopiruvato Hidratase/genética , Mutação Puntual , Relação Estrutura-Atividade , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genéticaRESUMO
Fluorescence methods are commonly used in pharmaceutical drug discovery to assay the binding of drug-like compounds to signaling proteins and other bio-particles. For binding studies of non-fluorescent compounds, a competitive format may be used in which the binding of the compound results in displacement of another fluorescently labeled ligand. Highly-sensitive measurements within nano-liter sized open probe volumes can be accomplished using a confocal epi-illumination geometry and thus key tools for such drug-binding studies include fluorescence correlation spectroscopy (FCS) and its related techniques. This paper reviews the general protocol for application of FCS to biomolecular compound-binding assays and it focuses on methods for the reduction of experimental photon count data to obtain the normalized autocorrelation function (ACF), on theoretical models of the ACF, and on statistical and systematic errors in the experimental ACF. Results from a detailed Monte Carlo simulation of FCS, which are useful for testing theoretical models and validating short-duration assay capabilities, are discussed. An illustrative example is presented on the use of FCS to assay binding of Alexa-488-labeled Bak peptide with Bcl-x(L), which is an intracellular protein that acts to protect against programmed cell death.
Assuntos
Algoritmos , Biopolímeros/química , Avaliação Pré-Clínica de Medicamentos/métodos , Preparações Farmacêuticas/química , Proteínas/química , Espectrometria de Fluorescência/métodos , Estatística como Assunto , Desenho de Fármacos , Substâncias Macromoleculares , Proteínas de Membrana/química , Preparações Farmacêuticas/análise , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Proteína Killer-Antagonista Homóloga a bcl-2 , Proteína bcl-XRESUMO
Fluorescence correlation spectroscopy (FCS) enables direct observation of the translational diffusion of single fluorescent molecules in solution. When fluorescent hapten binds to antibody, analysis of FCS data yields the fractional amounts of free and bound hapten, allowing determination of the equilibrium binding constant. Equilibrium dissociation constants of anti-digoxin antibodies and corresponding fluorescein-labeled digoxigenin obtained by FCS and fluorescence polarization measurements are identical. It is also possible to follow a competitive displacement of the tracer from the antibody by unlabeled hapten using FCS in an immunoassay format. The fluorescence polarization immunoassay for vancomycin detection was used to test the FCS approach. Fitting of the FCS data for the molar fractions of free and bound fluorescein-labeled vancomycin yielded a calibration curve which could serve for determination of the vancomycin concentration in biological samples.