RESUMEN

Protein folding is a fundamental process of life with important implications throughout biology. Indeed, tens of thousands of mutations have been associated with diseases, and most of these mutations are believed to affect protein folding rather than function. Correct folding is also a key element of design. These factors have motivated decades of research on protein folding. Unfortunately, knowledge of membrane protein folding lags that of soluble proteins. This gap is partly caused by the greater technical challenges associated with membrane protein studies, but also because of additional complexities. While soluble proteins fold in a homogenous water environment, membrane proteins fold in a setting that ranges from bulk water to highly charged to apolar. Thus, the forces that drive folding vary in different regions of the protein, and this complexity needs to be incorporated into our understanding of the folding process. Here, we review our understanding of membrane protein folding biophysics. Despite the greater challenge, better model systems and new experimental techniques are starting to unravel the forces and pathways in membrane protein folding.

Asunto(s)

Proteínas de la Membrana , Pliegue de Proteína , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo

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 & purificación

RESUMEN

G protein-coupled receptors (GPCRs) are vital for diverse biological functions, including vision, smell, and aging. They are involved in a wide range of diseases, and are among the most important targets of medicinal drugs. Tools that facilitate GPCR studies or GPCR-based technologies or therapies are thus critical to develop. Here we report using our QTY (glutamine, threonine, tyrosine) code to systematically replace 29 membrane-facing leucine, isoleucine, valine, and phenylalanine residues in the transmembrane α-helices of the GPCR CXCR4. This variant, CXCR4QTY29, became more hydrophilic, while retaining the ability to bind its ligand CXCL12. When transfected into HEK293 cells, it inserted into the cell membrane, and initiated cellular signaling. This QTY code has the potential to improve GPCR and membrane protein studies by making it possible to design functional hydrophilic receptors. This tool can be applied to diverse α-helical membrane proteins, and may aid in the development of other applications, including clinical therapies.

Asunto(s)

Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Receptores CXCR4/química , Receptores CXCR4/metabolismo , Quimiocina CXCL12/química , Quimiocina CXCL12/metabolismo , Cromatografía de Afinidad , Dicroismo Circular , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía Confocal , Unión Proteica , Estructura Secundaria de Proteína , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiología

RESUMEN

The question of how proteins manage to organize into a unique three-dimensional structure has been a major field of study since the first protein structures were determined. For membrane proteins, the question is made more complex because, unlike water-soluble proteins, the solvent is not homogenous or even unique. Each cell and organelle has a distinct lipid composition that can change in response to environmental stimuli. Thus, the study of membrane protein folding requires not only understanding how the unfolded chain navigates its way to the folded state, but also how changes in bilayer properties can affect that search. Here we review what we know so far about the impact of lipid composition on bilayer physical properties and how those properties can affect folding. A better understanding of the lipid bilayer and its effects on membrane protein folding is not only important for a theoretical understanding of the folding process, but can also have a practical impact on our ability to work with and design membrane proteins.

Asunto(s)

Membrana Celular , Membrana Dobles de Lípidos , Proteínas de la Membrana , Modelos Moleculares , Pliegue de Proteína , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Estructura Secundaria de Proteína

RESUMEN

Structure and function studies of membrane proteins, particularly G protein-coupled receptors and multipass transmembrane proteins, require detergents. We have devised a simple tool, the QTY code (glutamine, threonine, and tyrosine), for designing hydrophobic domains to become water soluble without detergents. Here we report using the QTY code to systematically replace the hydrophobic amino acids leucine, valine, isoleucine, and phenylalanine in the seven transmembrane α-helices of CCR5, CXCR4, CCR10, and CXCR7. We show that QTY code-designed chemokine receptor variants retain their thermostabilities, α-helical structures, and ligand-binding activities in buffer and 50% human serum. CCR5QTY, CXCR4QTY, and CXCR7QTY also bind to HIV coat protein gp41-120. Despite substantial transmembrane domain changes, the detergent-free QTY variants maintain stable structures and retain their ligand-binding activities. We believe the QTY code will be useful for designing water-soluble variants of membrane proteins and other water-insoluble aggregated proteins.

Asunto(s)

Glutamina/metabolismo , Receptores de Quimiocina/metabolismo , Treonina/metabolismo , Tirosina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Detergentes/química , Glutamina/química , Glutamina/genética , Calor , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Unión Proteica , Estabilidad Proteica , Estructura Secundaria de Proteína , Receptores de Quimiocina/química , Receptores de Quimiocina/genética , Solubilidad , Treonina/química , Treonina/genética , Tirosina/química , Tirosina/genética , Agua/química

RESUMEN

Protein folding is a fundamental life process with many implications throughout biology and medicine. Consequently, there have been enormous efforts to understand how proteins fold. Almost all of this effort has focused on water-soluble proteins, however, leaving membrane proteins largely wandering in the wilderness. The neglect has occurred not because membrane proteins are unimportant but rather because they present many theoretical and technical complications. Indeed, quantitative membrane protein folding studies are generally restricted to a handful of well-behaved proteins. Single-molecule methods may greatly alter this picture, however, because the ability to work at or near infinite dilution removes aggregation problems, one of the main technical challenges of membrane protein folding studies.

Asunto(s)

Transferencia Resonante de Energía de Fluorescencia/métodos , Espectrometría de Masas/métodos , Proteínas de la Membrana/química , Microscopía de Fuerza Atómica/métodos , Pliegue de Proteína , Imagen Individual de Molécula/métodos , Animales , Humanos , Lípidos de la Membrana/química , Proteínas de la Membrana/aislamiento & purificación

RESUMEN

Insects transmit numerous devastating diseases, including malaria, dengue fever, and sleeping sickness. Olfactory cues guide insects to their hosts, and are thus responsible for disease transmission. Understanding the molecular basis of insect olfaction could facilitate the development of interventions. The first step is to heterologously overexpress and purify insect olfactory receptors (ORs). This is challenging, as ORs are membrane proteins. Here, we show that insect ORs and their co-receptor can be expressed in an E. coli cell-free system. After immunoaffinity chromatography, the ORs are ~95% pure, and up to 1 mg/10 ml reaction is obtained. Circular dichroism together with microscale thermophoresis indicate that each receptor is properly folded, and can bind its respective ligand. This is the first time insect ORs have been expressed in an E. coli system. The methods described here could facilitate future structure-function studies, which may aid in developments to alleviate the suffering of millions caused by insect-transmitted diseases.

Asunto(s)

Proteínas de Drosophila/aislamiento & purificación , Drosophila melanogaster/genética , Neuronas Receptoras Olfatorias/metabolismo , Receptores de Superficie Celular/aislamiento & purificación , Receptores Odorantes/aislamiento & purificación , Animales , Sistema Libre de Células , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiología , Escherichia coli/genética , Expresión Génica/genética , Ligandos , Unión Proteica , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/genética , Receptores Odorantes/biosíntesis , Receptores Odorantes/genética

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 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 & purificación

RESUMEN

The first bottleneck in olfactory receptor (OR) studies is producing sufficient quantities of soluble, -functional, and stable receptors. Commercial cell-free in vitro translation systems can be used to produce milligrams of soluble and functional receptors within several hours directly from plasmid DNA. The receptors can be purified using immunoaffinity chromatography and gel filtration, and can be analyzed using gel electrophoresis and with other standard techniques.

Asunto(s)

Escherichia coli/genética , Ingeniería Genética/métodos , Receptores Odorantes/biosíntesis , Anticuerpos Monoclonales/química , Cromatografía en Gel , Escherichia coli/citología , Células HEK293 , Humanos , Microesferas , Receptores Odorantes/química , Receptores Odorantes/aislamiento & purificación , Receptores Odorantes/metabolismo , Sefarosa/química , Factores de Tiempo

RESUMEN

The detergents used to solubilize GPCRs can make crystal growth the rate-limiting step in determining their structure. The Kobilka laboratory showed that insertion of T4-lysozyme (T4L) in the 3rd intracellular loop is a promising strategy towards increasing the solvent-exposed receptor area, and hence the number of possible lattice-forming contacts. The potential to use T4L with the olfactory-related receptors hOR17-4 and hVN1R1 was thus tested. The structure and function of native and T4L-variants were compared. Both receptors localized to the cell membrane, and could initiate ligand-activated signaling. Purified receptors not only had the predicted alpha-helical structures, but also bound their ligands canthoxal (M(W) = 178.23) and myrtenal (M(W) = 150.22). Interestingly, the T4L variants had higher percentages of soluble monomers compared to protein aggregates, effectively increasing the protein yield that could be used for structural and function studies. They also bound their ligands for longer times, suggesting higher receptor stability. Our results indicate that a T4L insertion may be a general method for obtaining GPCRs suitable for structural studies.

Asunto(s)

Bacteriófago T4/genética , Muramidasa/genética , Ingeniería de Proteínas/métodos , Receptores Odorantes/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Virales/genética , Monoterpenos Bicíclicos , Factores Quimiotácticos/química , Factores Quimiotácticos/genética , Factores Quimiotácticos/metabolismo , Dicroismo Circular , Detergentes/química , Células HEK293 , Humanos , Inmunohistoquímica , Ligandos , Muramidasa/química , Muramidasa/metabolismo , Unión Proteica , Conformación Proteica , Receptores Odorantes/química , Receptores Odorantes/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Plasma Seminal/química , Proteínas de Plasma Seminal/genética , Proteínas de Plasma Seminal/metabolismo , Solubilidad , Terpenos/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo

RESUMEN

A crucial bottleneck in membrane protein studies, particularly G-protein coupled receptors, is the notorious difficulty of finding an optimal detergent that can solubilize them and maintain their stability and function. Here we report rapid production of 12 unique mammalian olfactory receptors using short designer lipid-like peptides as detergents. The peptides were able to solubilize and stabilize each receptor. Circular dichroism showed that the purified olfactory receptors had alpha-helical secondary structures. Microscale thermophoresis suggested that the receptors were functional and bound their odorants. Blot intensity measurements indicated that milligram quantities of each olfactory receptor could be produced with at least one peptide detergent. The peptide detergents' capability was comparable to that of the detergent Brij-35. The ability of 10 peptide detergents to functionally solubilize 12 olfactory receptors demonstrates their usefulness as a new class of detergents for olfactory receptors, and possibly other G-protein coupled receptors and membrane proteins.

Asunto(s)

Detergentes/química , Lípidos/química , Péptidos/química , Receptores Odorantes/metabolismo , Animales , Sistema Libre de Células , Dicroismo Circular , Humanos , Concentración de Iones de Hidrógeno , Ligandos , Ratones , Modelos Moleculares , Polietilenglicoles/química , Estructura Secundaria de Proteína , Receptores Odorantes/química , Receptores Odorantes/aislamiento & purificación , Tinción con Nitrato de Plata , Solubilidad , Temperatura

RESUMEN

Membrane proteins, particularly G-protein coupled receptors (GPCRs), are notoriously difficult to express. Using commercial E. coli cell-free systems with the detergent Brij-35, we could rapidly produce milligram quantities of 13 unique GPCRs. Immunoaffinity purification yielded receptors at >90% purity. Secondary structure analysis using circular dichroism indicated that the purified receptors were properly folded. Microscale thermophoresis, a novel label-free and surface-free detection technique that uses thermal gradients, showed that these receptors bound their ligands. The secondary structure and ligand-binding results from cell-free produced proteins were comparable to those expressed and purified from HEK293 cells. Our study demonstrates that cell-free protein production using commercially available kits and optimal detergents is a robust technology that can be used to produce sufficient GPCRs for biochemical, structural, and functional analyses. This robust and simple method may further stimulate others to study the structure and function of membrane proteins.

Asunto(s)

Receptores Acoplados a Proteínas G/metabolismo , Línea Celular , Electroforesis en Gel de Poliacrilamida , Humanos , Ligandos , Conformación Proteica , Estructura Secundaria de Proteína , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/fisiología , Solubilidad

RESUMEN

Two major bottlenecks in elucidating the structure and function of membrane proteins are the difficulty of producing large quantities of functional receptors, and stabilizing them for a sufficient period of time. Selecting the right surfactant is thus crucial. Here we report using peptide surfactants in commercial Escherichia coli cell-free systems to rapidly produce milligram quantities of soluble G protein-coupled receptors (GPCRs). These include the human formyl peptide receptor, human trace amine-associated receptor, and two olfactory receptors. The GPCRs expressed in the presence of the peptide surfactants were soluble and had α-helical secondary structures, suggesting that they were properly folded. Microscale thermophoresis measurements showed that one olfactory receptor expressed using peptide surfactants bound its known ligand heptanal (molecular weight 114.18). These short and simple peptide surfactants may be able to facilitate the rapid production of GPCRs, or even other membrane proteins, for structure and function studies.

Asunto(s)

Péptidos/química , Receptores Acoplados a Proteínas G/química , Tensoactivos/química , Aminas/química , Sistema Libre de Células , Dicroismo Circular , Escherichia coli/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Ligandos , Conformación Molecular , Neuronas Receptoras Olfatorias/metabolismo , Biosíntesis de Proteínas , Estructura Secundaria de Proteína , Solubilidad

RESUMEN

Here we report the study of two bioengineered variants of human trace amine-associated receptor 5 (hTAAR5) that were expressed in stable tetracycline-inducible HEK293S cell lines. A systematic detergent screen showed that fos-choline-14 was the optimal detergent to solubilize and subsequently purify the receptors. Milligram quantities of both hTAAR5 variants were purified to near homogeneity using immunoaffinity chromatography followed by gel filtration. Circular dichroism showed that the purified receptors had helical secondary structures, indicating that they were properly folded. The purified receptors are not only suitable for functional analyses, but also for subsequent crystallization trials. To our knowledge, this is the first mammalian TAAR that has been heterologously expressed and purified. Our study will likely stimulate in the development of therapeutic drug targets for TAAR-associated diseases, as well as fabrication of TAAR-based sensing devices.

Asunto(s)

Isoformas de Proteínas/fisiología , Receptores Acoplados a Proteínas G/fisiología , Cromatografía de Afinidad , Cromatografía en Gel , Dicroismo Circular , Células HEK293 , Humanos , Isoformas de Proteínas/química , Estructura Secundaria de Proteína , Receptores Acoplados a Proteínas G/química

RESUMEN

The vomeronasal system is one of several fine-tuned scent-detecting signaling systems in mammals. However, despite significant efforts, how these receptors detect scent remains an enigma. One reason is the lack of sufficient purified receptors to perform detailed biochemical, biophysical and structural analyses. Here we report the ability to express and purify milligrams of purified, functional human vomeronasal receptor hVN1R1. Circular dichroism showed that purified hVN1R1 had an alpha-helical structure, similar to that of other GPCRs. Microscale thermophoresis showed that hVN1R1 bound its known ligand myrtenal with an EC(50) approximately 1 µM. This expression system can enable structural and functional analyses towards understanding how mammalian scent detection works.

Asunto(s)

Factores Quimiotácticos/química , Factores Quimiotácticos/fisiología , Regulación de la Expresión Génica , Olfato , Dicroismo Circular , Detergentes/farmacología , Células HEK293 , Humanos , Ligandos , Unión Proteica , Estructura Terciaria de Proteína , Transducción de Señal

RESUMEN

The contractile behavior of cells is relevant in understanding wound healing and scar formation. In tissue engineering, inhibition of the cell contractile response is critical for the regeneration of physiologically normal tissue rather than scar tissue. Previous studies have measured the contractile response of cells in a variety of conditions (e.g. on two-dimensional solid substrates, on free-floating tissue engineering scaffolds and on scaffolds under some constraint in a cell force monitor). Tissue engineering scaffolds behave mechanically like open-cell elastomeric foams: between strains of about 10 and 90%, cells progressively buckle struts in the scaffold. The contractile force required for an individual cell to buckle a strut within a scaffold has been estimated based on the strut dimensions (radius, r, and length, l) and the strut modulus, E(s). Since the buckling force varies, according to Euler's law, with r(4)/l(2), and the relative density of the scaffold varies as (r/l)(2), the cell contractile force associated with strut buckling is expected to vary with the square of the pore size for scaffolds of constant relative density. As the cell density increases, the force per cell to achieve a given strain in the scaffold is expected to decrease. Here we model the contractile response of fibroblasts by analyzing the response of a single tetrakaidecahedron to forces applied to individual struts (simulating cell contractile forces) using finite element analysis. We model tetrakaidecahedra of different strut lengths, corresponding to different scaffold pore sizes, and of varying numbers of loaded struts, corresponding to varying cell densities. We compare our numerical model with the results of free-floating contraction experiments of normal human dermal fibroblasts (NHDF) in collagen-GAG scaffolds of varying pore size and with varying cell densities.

Asunto(s)

Colágeno/química , Fibroblastos/citología , Andamios del Tejido/química , Recuento de Células , Proliferación Celular , Supervivencia Celular , Dermis/citología , Humanos , Modelos Biológicos , Porosidad , Ingeniería de Tejidos