Plotting V versus [S] in the M-M equation yields a rectangular hyperbola. Thus, plotting [RL] versus [L] will likewise yield a rectangular hyperbola. Usually, the ligand-binding experiment is setup so that [Rt] is held constant and [R] is monitored as a function of [L]. Note:Although this looks like a M-M equation, K d is a constant. The above derivations are called " binding isotherms " in reference to their ability to solve for Kd under equilibrium conditions and the original equilibrium thermodynamic equation utilized constant temperature.
Plotting data for various values of [L], [R t ], and either [RL] or [R], and fitting to the appropriate function above, allows a more accurate determination of K d although a single experiment will provide an answer.
Due to the similarity of the [RL] versus [L] plot with the M-M plot, it is often the more desirable experimental setup. In particular, a double-reciprocal plot can be used much like the double-reciprocal M-M plot to more accurately determine K d. A semi-permeable membrane can be used in a dialysis-based equilibrium experiment to determined Kd for a ligand-receptor pair.
Figure 6. After equilibrium is reached the ligand concentration on both sides of the semi-permeable membrane is assayed:. Practical relationships between [R], [L], K d and [RL] For the same concentration of added ligand and receptor, more complex formation will occur with a smaller value of K d Example 6. Example 6.
Whispering gallery microresonator WGM WGM is based on wave sensing binding of molecules to the surface of the cavity induces changes of the resonant wavelength changes. The resonant changes of light permit multiple analyses of molecules. WGM is a highly sensitive assay for molecule detection. The possibility to study drug-target interactions under non-equilibrium conditions using this assay is still unclear. The light illuminates the biosensors in microplate at a nominally normal incident angle.
The drug binding of the immobilized receptors results in a shift in the resonant wavelength. RWG assay is effective for affinity screening. This assay is not applicable to study cells due to the large size of cells and limited penetration ability of an evanescent wave.
Biolayer Interferometry Biosensor BIB This assay uses a spectrometer to detect interference patterns formed by light reflected from an optical layer and a biolayer containing proteins of interest. Effective assay to study binding kinetics. This assay has been validated mostly for small molecule detection. Structure-based ligand binding assays Nuclear magnetic resonance NMR NMR analyzes the magnetic characteristics of certain atomic nuclei, which absorb electromagnetic radiation in the magnetic field.
NMR can be applied to analyze the structure of proteins and the molecular details of protein-ligand interactions and to assist structure-based drug design. Besides, NMR can be used for any class of compounds, for almost all soluble proteins, natively unstructured proteins or membrane proteins.
The high cost of the assay, time-consuming, requires a long time to analyze obtained spectra. The resolving power of NMR is lower than X-ray crystallography. X-ray crystallography This assay measures the changes of the diffracted X-ray beams in order to produce a three-dimensional image of electron density within the molecule of interest. Obtained image of electron density is used to determine the structure of the molecules.
The solvent effect can be examined by X-ray crystallography. The whole 3D structure can be obtained by the systematic analysis of a crystallized material. Solutions and behavior of molecules in solution cannot be examined. Direct determination of secondary structures including domain movements cannot be performed.
Thermodynamic binding assays Thermal denaturation assays TDA TDA detect the thermal denaturation of proteins by differential scanning fluorimetry, which applies a prob fluorophore to monitor thermal denaturation process of proteins in the presence and ligands. These methods have found applications in chemical profiling of different protein families, identifying novel ligands, and investigating the stability of proteins in buffers in solution These methods may not be suitable for analysis of very small proteins that do not aggregate upon denaturation.
Isothermal titration calorimetry ITC ITC measures the binding enthalpy variation by sensing the heat caused by the binding reaction This method is useful for detecting highly potent and entropy-driven ligands, compared to others. This method shows low throughput and sensitivity, and requires large sample volumes. Whole cell ligand-binding assays Surface acoustic wave SAW biosensor The surface of the biosensor is excited to oscillate with a high frequency. The phase of the generated acoustic wave is shifted upon mass changes.
Conformational characteristics are indicated by a change in amplitude. Using this assay, the sequential binding of several distinct ligands, targeting different cell surface molecules, to the whole cells can be detected directly in real time. SAW biosensors for analysis of small molecules using living cells have not developed.
RWG biosensor RWG biosensor can transform drug-receptor interactions in cells into cell phenotypic signatures named dynamic mass redistribution. This assay measures the consequence of binding, and the stimulus-response interpretation of the initial interaction of drug and receptor.
Also, RWG can analyze the functional consequences of ligand binding and dissociation. RWG biosensor may not be used to detect ligand binding directly. Figure 1. If the fluorophores are close to each other 1 ].
Figure 2. Surface plasmon resonance SPR determines binding kinetics. A microfluidic device is applied in SPR to detect the association and dissociation of drug compounds binding to the target immobilized on the golden surface. The resonance angle shifts when drug compounds bind to the target. Figure 3. Plasmon-waveguide resonance PWR determines both binding kinetics and conformational changes. A polarized continuous wave laser excites electromagnetic waves in a resonator made of a thin silver film with a layer of SiO2 and a glass prism.
The resonance angle shifts when drug compounds bind to the immobilized target. Figure 4. Microplate-based resonant waveguide grating RWG determines binding affinity screening. Light is coupled into the waveguide via diffraction. The intensity of the reflected light is measured.
The compound binding of the immobilized receptors causes a shift in the resonant wavelength. Figure 5. Biolayer interferometry biosensor BIB. A broadband light resource is used to illuminate the interfaces. Reflected light waves originate from the immobilized receptor surface and the optical layer, interacting with each other and creating interference patterns. Compound binding affects the inference pattern.
Figure 6. The magnetization transfer between two protons is represented by arrows. Nuclear Overhasuer effects NOEs are represented by black dots and white dots represent intermolecular NOEs are represented by white dots. Pharmacol Rev. Fang Y. Ligand-receptor interaction platforms and their applications for drug discovery.
Expert Opin Drug Discov. NMR-based analysis of protein-ligand interactions. Anal Bioanal Chem. Owicki J. Fluorescence polarization and anisotropy in high throughput screening: perspectives and primer. J Biomol Screen. Methods Mol Biol. Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy.
Biophys J. Handl H, Gillies R. Lanthanide-based luminescent assays for ligand-receptor interactions. Life Sci. Rossi A, Taylor C. Analysis of protein-ligand interactions by fluorescence polarization. Nat Protoc. J Vis Exp. Structure and dynamics of the active human parathyroid hormone receptor Labaer J, Ramachandran N. Protein microarrays as tools for functional proteomics. Curr Opin Chem Biol.
Screening kinase inhibitors with a microarray-based fluorescent and resonance light scattering assay. Anal Chem. Fluorescence- and bioluminescence-based approaches to study GPCR ligand binding. Br J Pharmacol. Charest Morin X, Marceau F. Eur J Med Chem. Paracrine signals move by diffusion through the extracellular matrix. These types of signals usually elicit quick responses that last only a short amount of time.
In order to keep the response localized, paracrine ligand molecules are normally quickly degraded by enzymes or removed by neighboring cells. Removing the signals will reestablish the concentration gradient for the signal, allowing them to quickly diffuse through the intracellular space if released again. One example of paracrine signaling is the transfer of signals across synapses between nerve cells. A nerve cell consists of a cell body, several short, branched extensions called dendrites that receive stimuli, and a long extension called an axon, which transmits signals to other nerve cells or muscle cells.
The junction between nerve cells where signal transmission occurs is called a synapse. A synaptic signal is a chemical signal that travels between nerve cells.
Signals within the nerve cells are propagated by fast-moving electrical impulses. When these impulses reach the end of the axon, the signal continues on to a dendrite of the next cell by the release of chemical ligands called neurotransmitters by the presynaptic cell the cell emitting the signal. The neurotransmitters are transported across the very small distances between nerve cells, which are called chemical synapses Figure 2.
The small distance between nerve cells allows the signal to travel quickly; this enables an immediate response, such as, Take your hand off the stove! When the neurotransmitter binds the receptor on the surface of the postsynaptic cell, the electrochemical potential of the target cell changes, and the next electrical impulse is launched. The neurotransmitters that are released into the chemical synapse are degraded quickly or get reabsorbed by the presynaptic cell so that the recipient nerve cell can recover quickly and be prepared to respond rapidly to the next synaptic signal.
Signals from distant cells are called endocrine signals , and they originate from endocrine cells. In the body, many endocrine cells are located in endocrine glands, such as the thyroid gland, the hypothalamus, and the pituitary gland. These types of signals usually produce a slower response but have a longer-lasting effect. The ligands released in endocrine signaling are called hormones, signaling molecules that are produced in one part of the body but affect other body regions some distance away.
Hormones travel the large distances between endocrine cells and their target cells via the bloodstream, which is a relatively slow way to move throughout the body. Because of their form of transport, hormones get diluted and are present in low concentrations when they act on their target cells. This is different from paracrine signaling, in which local concentrations of ligands can be very high.
Autocrine signals are produced by signaling cells that can also bind to the ligand that is released. This means the signaling cell and the target cell can be the same or a similar cell the prefix auto- means self, a reminder that the signaling cell sends a signal to itself.
This type of signaling often occurs during the early development of an organism to ensure that cells develop into the correct tissues and take on the proper function. Autocrine signaling also regulates pain sensation and inflammatory responses. Further, if a cell is infected with a virus, the cell can signal itself to undergo programmed cell death, killing the virus in the process.
In some cases, neighboring cells of the same type are also influenced by the released ligand. In embryological development, this process of stimulating a group of neighboring cells may help to direct the differentiation of identical cells into the same cell type, thus ensuring the proper developmental outcome.
Gap junctions in animals and plasmodesmata in plants are connections between the plasma membranes of neighboring cells. These water-filled channels allow small signaling molecules, called intracellular mediators , to diffuse between the two cells. The specificity of the channels ensures that the cells remain independent but can quickly and easily transmit signals. The transfer of signaling molecules communicates the current state of the cell that is directly next to the target cell; this allows a group of cells to coordinate their response to a signal that only one of them may have received.
In plants, plasmodesmata are ubiquitous, making the entire plant into a giant, communication network. Produced by signaling cells and the subsequent binding to receptors in target cells, ligands act as chemical signals that travel to the target cells to coordinate responses. Figure 3. Steroid hormones have similar chemical structures to their precursor, cholesterol. Because these molecules are small and hydrophobic, they can diffuse directly across the plasma membrane into the cell, where they interact with internal receptors.
Small hydrophobic ligands can directly diffuse through the plasma membrane and interact with internal receptors. Important members of this class of ligands are the steroid hormones. Steroids are lipids that have a hydrocarbon skeleton with four fused rings; different steroids have different functional groups attached to the carbon skeleton.
Steroid hormones include the female sex hormone, estradiol, which is a type of estrogen; the male sex hormone, testosterone; and cholesterol, which is an important structural component of biological membranes and a precursor of steroid hormones Figure 3.
Other hydrophobic hormones include thyroid hormones and vitamin D. In order to be soluble in blood, hydrophobic ligands must bind to carrier proteins while they are being transported through the bloodstream. Water-soluble ligands are polar and therefore cannot pass through the plasma membrane unaided; sometimes, they are too large to pass through the membrane at all.
Instead, most water-soluble ligands bind to the extracellular domain of cell-surface receptors. This group of ligands is quite diverse and includes small molecules, peptides, and proteins. Nitric oxide NO is a gas that also acts as a ligand.
It is able to diffuse directly across the plasma membrane, and one of its roles is to interact with receptors in smooth muscle and induce relaxation of the tissue.
NO has a very short half-life and therefore only functions over short distances. Nitroglycerin, a treatment for heart disease, acts by triggering the release of NO, which causes blood vessels to dilate expand , thus restoring blood flow to the heart. NO has become better known recently because the pathway that it affects is targeted by prescription medications for erectile dysfunction, such as Viagra erection involves dilated blood vessels.
Receptors are protein molecules in the target cell or on its surface that bind ligand. There are two types of receptors, internal receptors and cell-surface receptors.
0コメント