Misplaced Pages

#816183

37-500: The EF-hand motif contains a helix–loop–helix topology, much like the spread thumb and forefinger of the human hand, in which the Ca ions are coordinated by ligands within the loop. The motif takes its name from traditional nomenclature used in describing the protein parvalbumin , which contains three such motifs and is probably involved in muscle relaxation via its calcium-binding activity. The EF-hand consists of two alpha helices linked by

74-547: A short loop region (usually about 12 amino acids ) that usually binds calcium ions. EF-hands also appear in each structural domain of the signaling protein calmodulin and in the muscle protein troponin-C . The calcium ion is coordinated in a pentagonal bipyramidal configuration. The six residues involved in the binding are in positions 1, 3, 5, 7, 9 and 12; these residues are denoted by X, Y, Z, -Y, -X and -Z. The invariant Glu or Asp at position 12 provides two oxygens for liganding calcium (bidentate ligand). The calcium ion

111-615: A strong preference for oxygen -containing side chains, especially aspartate and glutamate. The sixth residue in the loop is necessarily glycine due to the conformational requirements of the backbone. The remaining residues are typically hydrophobic and form a hydrophobic core that binds and stabilizes the two helices. Upon binding to Ca, this motif may undergo conformational changes that enable Ca-regulated functions as seen in Ca effectors such as calmodulin (CaM) and troponin C (TnC) and Ca buffers such as calreticulin and calbindin D9k . While

148-497: A useful tool in molecular biology for the measurement of intracellular Ca levels. The early successful purification of aequorin led to the first experiments involving the injection of the protein into the tissues of living animals to visualize the physiological release of calcium in the muscle fibers of a barnacle. Since then, the protein has been widely used in many model biological systems , including zebrafish , rats , mice , and cultured cells . Cultured cells expressing

185-400: Is thought to have biological significance. In proteins, a structural motif describes the connectivity between secondary structural elements. An individual motif usually consists of only a few elements, e.g., the 'helix-turn-helix' motif which has just three. Note that, while the spatial sequence of elements may be identical in all instances of a motif, they may be encoded in any order within

222-454: Is "clinically shown" to work. According to the FTC, "the marketers of Prevagen preyed on the fears of older consumers experiencing age-related memory loss ". Quincy said that it would fight the charges. Prior to the suit, a clinical trial run by researchers employed by Quincy Bioscience "found no overall benefit compared to a placebo for its primary endpoints involving memory and cognition", while

259-406: Is a common three-dimensional structure that appears in a variety of different, evolutionarily unrelated molecules. A structural motif does not have to be associated with a sequence motif ; it can be represented by different and completely unrelated sequences in different proteins or RNA. Depending upon the sequence and other conditions, nucleic acids can form a variety of structural motifs which

296-638: Is a member of the superfamily of the calcium-binding proteins, of which there are some 66 subfamilies. The crystal structure revealed that aequorin binds coelenterazine and oxygen in the form of a peroxide , coelenterazine-2-hydroperoxide. The binding site for the first two calcium atoms show a 20 times greater affinity for calcium than the third site. However, earlier claims that only two EF-hands bind calcium were questioned when later structures indicated that all three sites can indeed bind calcium. Thus, titration studies show that all three calcium-binding sites are active but only two ions are needed to trigger

333-433: Is bound by both protein backbone atoms and by amino acid side chains , specifically those of the anionic amino acid residues aspartate and glutamate . These residues are negatively charged and will make a charge-interaction with the positively charged calcium ion. The EF hand motif was among the first structural motifs whose sequence requirements were analyzed in detail. Five of the loop residues bind calcium and thus have

370-466: Is called apoaequorin , which has an approximate molecular weight of 21 kDa , and the prosthetic group coelenterazine , the luciferin. This is to say, apoaequorin is the enzyme produced in the photocytes of the animal, and coelenterazine is the substrate whose oxidation the enzyme catalyzes. When coelenterazine is bound, it is called aequorin. Notably, the protein contains three EF hand motifs that function as binding sites for Ca ions. The protein

407-450: Is made up of two components – the calcium binding component apoaequorin (AQ) and the chemiluminescent molecule coelenterazine . The AQ portion of this protein contains the EF-hand calcium binding domains. Humans proteins containing this domain include: Structural motif#In proteins In a chain-like biological molecule , such as a protein or nucleic acid , a structural motif

SECTION 10

#1732868622817

444-460: Is represented by calbindin D9k and these proteins do not undergo calcium dependent conformational changes. Aequorin is a calcium binding protein (CaBP) isolated from the cnidarian Aequorea victoria . Aequorin belongs to the EF-hand family of CaBPs, with EF-hand loops that are closely related to CaBPs in mammals. In addition, aequorin has been used for years as an indicator of Ca and has been shown to be safe and well tolerated by cells. Aequorin

481-411: Is unclear if the cDNA variants can account for all of the isoforms of the protein. Early studies of the bioluminescence of Aequorea by E. Newton Harvey had noted that the bioluminescence appears as a ring around the bell, and occurs even in the absence of air. This was remarkable because most bioluminescence reactions require oxygen , and led to the idea that the animals somehow store oxygen. It

518-484: The calmodulin -based sensor cameleon , developed by Roger Tsien and the troponin -based sensor, TN-XXL , developed by Oliver Griesbeck. Apoaequorin is an ingredient in "Prevagen", which is marketed by Quincy Bioscience as a memory supplement. In 2017, the US Federal Trade Commission (FTC) charged the maker with falsely advertising that the product improves memory, provides cognitive benefits, and

555-496: The plasma membrane of higher eukaryotes, making aequorin suitable as a Ca reporter in plants, fungi, and mammalian cells. Aequorin has a number of advantages over other Ca indicators. Because the protein is large, it has a low leakage rate from cells compared to lipophilic dyes such as DiI . It lacks phenomena of intracellular compartmentalization or sequestration as is often seen for Voltage-sensitive dyes , and does not disrupt cell functions or embryo development. Moreover,

592-459: The Quincy-sponsored study (known as "Madison Memory Study") was negative, but that the company utilized p-hacking to find favorable results. She wrote that their cited safety studies were all rat studies and their claim that apoaequorin crosses the blood–brain barrier was based solely on a dog study. The American Pharmacists Association warns that Apoaequorin "is unlikely to be absorbed to

629-474: The absence of oxygen. Later, Osamu Shimomura began work into the bioluminescence of Aequorea in 1961. This involved tedious harvesting of tens of thousands of jellyfish from the docks in Friday Harbor, Washington . It was determined that light could be produced from extracts with seawater, and more specifically, with calcium . It was also noted during the extraction the animal creates green light due to

666-435: The aequorin gene can effectively synthesize apoaequorin; however, recombinant expression yields only the apoprotein . Therefore it is necessary to add coelenterazine into the culture medium of the cells to obtain a functional protein and thus use its blue light emission to measure Ca concentration. Coelenterazine is a hydrophobic molecule, and therefore is easily taken up across plant and fungal cell walls , as well as

703-442: The binding site, as well as the ion solvation properties. Pattern (motif signature) search is one of the most straightforward ways to predict continuous EF-hand Ca-binding sites in proteins. Based on the sequence alignment results of canonical EF-hand motifs, especially the conserved side chains directly involved in Ca binding, a pattern PS50222 has been generated to predict canonical EF-hand sites. Prediction servers may be found in

740-559: The class of consumers who purchased Prevagen over the previous four years. The trial in the case was set for October 2020. As of September 21, 2020 , Quincy Bioscience agreed to settle the claims that it misrepresented its Prevagen products as supporting brain health and helping with memory loss. Under the terms of the settlement, eligible purchasers applying by October 26, 2020, for purchases made from 2007 through July 31, 2020, could recover refunds of up to $ 70. Dr. Harriet Hall , writing for Science-Based Medicine , noted that

777-513: The company's advertising misleadingly cited a few contested subgroup analyses that showed slight improvements. The suit ( Spath, et al. v. Quincy Bioscience Holding Company, Inc., et al. , Case No. 18-cv-12416, D. NJ.) was dismissed in the District court, but an appeal seeking to overturn the dismissal was filed. The suit was consolidated with another against Quincy Pharmaceuticals, Vanderwerff v. Quincy Bioscience (Case No. 17-cv-784, D. NJ), which

SECTION 20

#1732868622817

814-555: The entire 211 people who completed the study there was no statistically significant difference". The court vigorously dismissed allegations by the company lawyers that the FTC pursued its action for political reasons. On March 23, 2020, a federal magistrate judge in the United States District Court for the Southern District of Florida entered a report and recommendations certifying a nationwide class action for

851-430: The enzymatic reaction. Other studies have shown the presence of an internal cysteine bond that maintains the structure of aequorin. This has also explained the need for a thiol reagent like beta mercaptoethanol in the regeneration of the protein since such reagents weaken the sulfhydryl bonds between cysteine residues, expediting the regeneration of the aequorin. Chemical characterization of aequorin indicates

888-460: The external links section. Since the delineation of the EF-hand motif in 1973, the family of EF-hand proteins has expanded to include at least 66 subfamilies thus far. EF-hand motifs are divided into two major structural groups: Additional points: Among all the structures reported to date, the majority of EF-hand motifs are paired either between two canonical or one pseudo and one canonical motifs. For proteins with odd numbers of EF-hands, such as

925-413: The ground state, blue light ( wavelength of 465 nm) is emitted. Before coelenteramide is exchanged out, the entire protein is still fluorescent blue. because of the connection between bioluminescence and fluorescence , this property was ultimately important in the discovery of the luciferin coelenterazine . Since the emitted light can be easily detected with a luminometer , aequorin has become

962-423: The light emitted by the oxidation of coelenterazine does not depend on any optical excitation, so problems with auto-fluorescence are eliminated. The primary limitation of aequorin is that the prosthetic group coelenterazine is irreversibly consumed to produce light, and requires continuous addition of coelenterazine into the media. Such issues led to developments of other genetically encoded calcium sensors including

999-508: The majority of the known EF-hand calcium-binding proteins (CaBPs) contain paired EF-hand motifs, CaBPs with single EF hands have also been discovered in both bacteria and eukaryotes. In addition, "EF-hand-like motifs" have been found in a number of bacteria. Although the coordination properties remain similar with the canonical 29-residue helix–loop–helix EF-hand motif, the EF-hand-like motifs differ from EF-hands in that they contain deviations in

1036-429: The penta-EF-hand calpain, EF-hand motifs were coupled through homo- or hetero-dimerization. The recently-identified EF-hand containing ER Ca sensor protein, stromal interaction molecule 1 and 2 (STIM1, STIM2), has been shown to contain a Ca-binding canonical EF-hand motif that pairs with an immediate, downstream atypical "hidden" non-Ca-binding EF-hand. Single EF-hand motifs can serve as protein-docking modules: for example,

1073-507: The presence of the green fluorescent protein , which changes the native blue light of aequorin to green. While the main focus of his work was on the bioluminescence, Shimomura and two others, Martin Chalfie and Roger Tsien , were awarded the Nobel Prize in 2008 for their work on green fluorescent proteins . Aequorin is a holoprotein composed of two distinct units, the apoprotein that

1110-421: The protein is somewhat resilient to harsh treatments. Aequorin is heat resistant. Held at 95 °C for 2 minutes the protein lost only 25% activity. Denaturants such as 6-M urea or 4-M guanidine hydrochloride did not destroy the protein. Aequorin is presumably encoded in the genome of Aequorea. At least four copies of the gene were recovered as cDNA from the animal. Because the genome has not been sequenced, it

1147-493: The same animal. Apoaequorin, the protein portion of aequorin, is an ingredient in the dietary supplement Prevagen. The US Federal Trade Commission (FTC) has charged the maker with false advertising for its memory improvement claims. Work on aequorin began with E. Newton Harvey in 1921. Though Harvey was unable to demonstrate a classical luciferase - luciferin reaction, he showed that water could produce light from dried photocytes and that light could be produced even in

EF hand - Misplaced Pages Continue

1184-477: The secondary structure of the flanking sequences and/or variation in the length of the Ca-coordinating loop. EF hands have very high selectivity for calcium. For example, the dissociation constant of alpha parvalbumin for Ca is ~1000 times lower than that for the similar ion Mg. This high selectivity is due to the relatively rigid coordination geometry, the presence of multiple charged amino acid side chains in

1221-572: The single EF hand in the NKD1 and NKD2 proteins binds the Dishevelled (DVL1, DVL2, DVL3) proteins. Functionally, the EF-hands can be divided into two classes: The first group is the largest and includes the most well-known members of the family such as calmodulin, troponin C and S100B. These proteins typically undergo a calcium-dependent conformational change which opens a target binding site. The latter group

1258-449: The underlying gene . In addition to secondary structural elements, protein structural motifs often include loops of variable length and unspecified structure. Structural motifs may also appear as tandem repeats . Aequorin Aequorin is a calcium-activated photoprotein isolated from the hydrozoan Aequorea victoria . Its bioluminescence was studied decades before the protein

1295-438: Was isolated from the animal by Osamu Shimomura in 1962. In the animal, the protein occurs together with the green fluorescent protein to produce green light by resonant energy transfer , while aequorin by itself generates blue light. Discussions of "jellyfish DNA" that can make "glowing" animals often refer to transgenic animals that express the green fluorescent protein, not aequorin, although both originally derive from

1332-410: Was later discovered that the apoprotein can stably bind coelenterazine-2-hydroperoxide, and oxygen is required for the regeneration to this active form of aequorin. However, in the presence of calcium ions, the protein undergoes a conformational change and converts its prosthetic group, coelenterazine-2-hydroperoxide, into excited coelenteramide and CO 2 . As the excited coelenteramide relaxes to

1369-563: Was the lead case. On February 21, 2019, the United States Court of Appeals for the Second Circuit ruled that the FTC and the state of New York could proceed with their lawsuit against Quincy Bioscience for its claims that Prevagen can improve memory. The order came less than two weeks after the parties argued the case before a three-judge panel of the circuit, where company lawyers admitted they did not "dispute that if you look across

#816183