RESUMEN
G-protein-coupled receptors (GPCRs) are integral proteins of the cell membrane and are directly involved in the regulation of many biological functions and in drug targeting. However, our knowledge of GPCRs' structure and function remains limited. The first bottleneck in GPCR studies is producing sufficient quantities of soluble, functional, and stable receptors. Currently, GPCR production largely depends on the choice of the host system and the type of detergent used to extract the GPCR from the cell membrane and stabilize the protein outside the membrane bilayer. Here, we present three protocols that we employ in our lab to produce and solubilize stable GPCRs: (1) cell-free in vitro translation, (2) HEK cells, and (3) Escherichia coli. Stable receptors can be purified using immunoaffinity chromatography and gel filtration, and can be analyzed with standard biophysical techniques and biochemical assays.
Asunto(s)
Cromatografía de Afinidad , Expresión Génica , Receptores Acoplados a Proteínas G , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Sistema Libre de Células , Escherichia coli , Células HEK293 , Humanos , Receptores Acoplados a Proteínas G/biosíntesis , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
G-protein-coupled receptors (GPCRs) are integral proteins of the cell membrane and are directly involved in the regulation of many biological functions and in drug targeting. However, our knowledge of GPCRs' structure and function remains limited. The first bottleneck in GPCR studies is producing sufficient quantities of soluble, functional, and stable receptors. Currently, GPCR production largely depends on the choice of the overexpression host system and the type of detergent used to extract the GPCR from the cell membrane and stabilize the protein outside the membrane bilayer. Here, we present three protocols that we employ in our lab to produce and solubilize stable GPCRs by cell-free in vitro translation systems, HEK cells, and Escherichia coli. Stable receptors can be purified using immunoaffinity chromatography and gel filtration and can be analyzed with standard biophysical techniques and biochemical assays.
Asunto(s)
Receptores Acoplados a Proteínas G/genética , Cromatografía en Gel , Membrana Dobles de Lípidos , Modelos Moleculares , Conformación Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/aislamiento & purificaciónRESUMEN
Two binder candidates 4-C37L34-B and 3-C15L8-B from a 16-membered set of 42-residue polypeptide conjugates designed to bind human carbonic anhydrase II (HCAII), were shown to bind HCAII with high affinity in a fluorescence-based screening assay. Two carbonic anhydrase isoforms with 60 % homology exist in human blood with HCAI being present in five- to sevenfold excess over HCAII. The ability of the binders to discriminate between HCAI and HCAII was evaluated with regard to what selectivity could be achieved by the conjugation of polypeptides from a 16-membered set to a small organic molecule that binds both isoforms with similar affinities. The polypeptide conjugate 4-C37L34-B bound HCAII with a K(D) of 17 nM and HCAI with a K(D) of 470 nM, that is, with a 30-fold difference in affinity. The corresponding dissociation constants for the complexes formed from 3-C15L8-B and the two carbonic anhydrases were 60 and 390 nM, respectively. This demonstration of selectivity between two very similar proteins is striking in view of the fact that the molecular weight of each one of the conjugate molecules is little more than 5000, the fold is unordered, and the polypeptide sequences were designed de novo and have no prior relationship to carbonic anhydrases. The results suggest that synthetic polypeptide conjugates can be prepared from organic molecules that are considered to be weak binders with low selectivity, yielding conjugates with properties that make them attractive alternatives to biologically generated binders in biotechnology and biomedicine.
Asunto(s)
Anhidrasas Carbónicas/química , Anhidrasas Carbónicas/metabolismo , Cumarinas/química , Péptidos/genética , Sulfonamidas/química , Secuencia de Aminoácidos , Cumarinas/metabolismo , Electroforesis en Gel de Poliacrilamida , Humanos , Espectrometría de Masas , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Isoformas de Proteínas/química , Sulfonamidas/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
The structural heterogeneity and thermal denaturation of a dansyl-labeled four-helix bundle homodimeric peptide was studied with steady-state and time-resolved fluorescence spectroscopy and with circular dichroism (CD). At room temperature the fluorescence decay of the polarity-sensitive dansyl, located in the hydrophobic core region, can be described by a broad distribution of fluorescence lifetimes, reflecting the heterogeneous microenvironment. However, the lifetime distribution is nearly bimodal, which we ascribe to the presence of two major conformational subgroups. Since the fluorescence lifetime reflects the water content of the four-helix bundle conformations, we can use the lifetime analysis to monitor the change in hydration state of the hydrophobic core of the four-helix bundle. Increasing the temperature from 9 degrees C to 23 degrees C leads to an increased population of molten-globule-like conformations with a less ordered helical backbone structure. The fluorescence emission maximum remains constant in this temperature interval, and the hydrophobic core is not strongly affected. Above 30 degrees C the structural dynamics involve transient openings of the four-helix bundle structure, as evidenced by the emergence of a water-quenched component and less negative CD. Above 60 degrees C the homodimer starts to dissociate, as shown by the increasing loss of CD and narrow, short-lived fluorescence lifetime distributions.