Amino acid sequence of the calcium-dependent photoprotein aequorin

The Ca(II)-dependent photoprotein aequorin produces the luminescence of the marine coelenterate Aequorea victoria. The complete amino acid sequence of aequorin has been determined. A complete set of nonoverlapping peptides was produced by cyanogen bromide cleavage. These peptides were aligned by using the amino-terminal sequence of the intact protein and the sequences of selected arginyl and lysyl cleavage products. Although the aequorin preparations employed in these studies were homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the presence of a minimum of 3 isotypes was demonstrated by the location of 17 sites of sequence microheterogeneity. Two amino acid variants were observed at each of 16 positions while 1 position had 3 different replacements. The protein as isolated has 189 amino acids with an unblocked amino terminus. According to the sequence reported here, the molecular weight of the apoprotein is 21 459 while that of the holoprotein is 21 914. The molecule possesses three internally homologous domains which were judged to be EF-hand Ca(II) binding domains by several different criteria. Aequorin is homologous to troponin C and to calmodulin. These findings demonstrate that aequorin is a member of the Ca(II) binding protein superfamily.     

Selective protein modification by the hydroperoxide intermediate in a photoprotein,aequorin

During the course of protein modification program, we employed a recombinant aequorin, the apo-protein reconstituted with coelenterazine, and found out that the photolytic hyperperoxide modified three –S–SCH₂CHOHCHOHCH₂SH groups to –S–SCH₂CHOHCHCH–SO)H or –S–SCH₂CHOHCHCH–S(O)OH of terminal DTT connected to cysteine residues of the C¹⁴⁵, C¹⁵² and C¹⁸⁰, which turned out to locate near the chromophore.     

Bioluminescence of the Ca-binding photoprotein, aequorin, after histidine modification

Modification studies of the 5 histidine residues in aequorin employing site-directed mutagenesis and diethyl pyrocarbonate suggested that His¹⁶⁹ may be the site of binding of molecular oxygen in aequorin. The modification of this residue led to complete loss of activity, whereas modification of the remaining 4 histidine residues yielded mutant aequorins with varying bioluminescence activities.     

EDTA-binding and acylation of the Ca2+-sensitive photoprotein aequorin

The rate of phosphorylation and concomitant inactivation of purified pig heart muscle pyruvate dehydrogenase complex by intrinsic kinase (EC 2.7.1.99) is markedly accelerated by the addition of coenzyme A to the incubation medium, showing a half-maximum effect at 1.8 μM. The pantetheine moiety is the effective part of the coenzyme A molecule. The free thiol group is prerequisite for the stimulatory action, acetyl-CoA, benzoyl-CoA or CoAS-SCoA being ineffectual. The thiol's specificity is evidenced by showing that dithiothreitol, 2-mercaptoethanol or glutathione up to 5 mM failed to replace coenzyme A. The possibility is considered that coenzyme A might act as a physiological modifier of pyruvate dehydrogenase kinase activity.     

Galactose-dependent expression of the recombinant Ca2(+)-binding photoprotein aequorin in yeast

Aequorin is a Ca2(+)-binding protein that emits light upon reacting with Ca2+ and has been used as a probe for monitoring changes in the intracellular free Ca2+ concentration, [Ca2+]i. The protein consists of three components: apoaequorin (apoprotein), molecular oxygen and a chromophore. The present study was designed to conditionally express the apoaequorin cDNA of the jellyfish Aequorea victoria under the control of the GAL1 promoter in the yeast Saccharomyces cerevisiae and to investigate whether apoaequorin can be accumulated in high enough concentration in the cells to detect a Ca2+ signal in vitro. The results showed that the cells accumulated sufficient amounts of recombinant apoaequorin when incubated in the galactose-based medium and that the protein was active and not toxic to the cells, suggesting that the recombinant apoaequorin may be applicable to monitoring changes in [Ca2+]i in intact yeast cells.     

One-step purification and refolding of recombinant photoprotein aequorin by immobilized metal-ion affinity chromatography

A hexahistidine tag was fused to the N-terminus of apoaequorin. A suitable vector encoding the fusion protein was constructed and used for transformation of Escherichia coli JM109 cells. Apoaequorin was overexpressed under the control of tac promoter. It was found, however, that most of the protein existed in the form of inclusion bodies. Inclusion bodies were solubilized with urea, followed by purification and refolding of (His)(6)-apoaequorin in a single chromatographic step by immobilized metal-ion affinity chromatography using Ni(2+)-nitrilotriacetic acid agarose. The purity, as determined by SDS-PAGE analysis, was greater than 80%. The yield was 0.7-1 mg apoaequorin from a 50 ml bacterial culture. The kinetics of light emission of purified aequorin upon addition of Ca(2+) was typical of the commercial aequorin. The luminescence of the purified aequorin was a linear function of its concentration extending over six orders of magnitude. As low as 0.5 attomoles purified aequorin gave a signal-to-noise ratio of 1.8.     

Bioluminescent immunoassay using a fusion protein of protein A and the photoprotein aequorin

Aequorin is a photoprotein that emits light in the presence of Ca2+ ions. To develop a bioluminescent immunoassay based on the light emission property of aequorin, we have expressed the apoaequorin fusion protein with S. aureus protein A in E. coli by recombinant DNA techniques. The fusion protein expressed was purified by IgG-Sepharose affinity chromatography, gel filtration and HPLC procedures. The purified protein A-apoaequorin fusion protein has both the luminescent activity of aequorin and the IgG-binding ability of protein A. We compared results obtained using the protein A-aequorin fusion protein with those obtained using a protein A conjugated horseradish peroxidase based immunoassay, and found them to yield similar results.     

Preparation and initial characterization of crystals of the photoprotein aequorin from Aequorea victoria

Crystals of recombinant aequorin, the photoprotein from the jellyfish Aequorea victoria, have been grown from solutions containing sodium phosphate. The crystals grow as thin plates which diffract to beyond 2.2 Å resolution. The crystals are orthorhombic, space group P2₁2₁2 ₁; the axes are a = 89.1(1), b = 88.4(1), and c = 52.7(1) Å. The asymmetric unit contains two molecules. Crystals exposed to calcium ion solutions emit a steady glow and slowly deteriorate, confirming that the crystals consist of a charged, competent photoprotein. This represents the first successful preparation of single crystals of a photoprotein suitable for diffraction analysis. © 1993 Wiley-Liss, Inc.     

Measurement of ionized calcium in blood platelets with the photoprotein aequorin. Comparison with Quin 2

The Ca2+-sensitive photoprotein aequorin (Mr = 20,000) was introduced into human blood platelets by incubation with 10 mM EGTA and 5 mM ATP. Platelet cytoplasmic and granule contents were retained during the loading procedure, and platelet morphology, aggregation, and secretion in response to agonists were normal after aequorin loading. Luminescence indicated an apparent resting cytoplasmic ionized calcium concentration [( Cai2+]) of 2-4 microM in media containing 1 mM Ca2+ and of 0.8-2 microM in 2-4 mM EGTA. The Ca2+ ionophore A23187 and the enzyme thrombin produced dose-related luminescent signals in both Ca2+-containing and EGTA-containing media. Peak [Cai2+] after A23187 or thrombin stimulation of aequorin-loaded platelets was 2-10 microM, while peak [Cai2+] determined using Quin 2 as the [Cai2+] indicator was at least 1 log unit lower. In platelets loaded with both aequorin and Quin 2, the aequorin signal was delayed but not reduced in amplitude. Aequorin loading of Quin 2-loaded cells had no effect on the Quin 2 signal. Ca2+ buffering by Quin 2 (intracellular concentration greater than 1 mM) is also supported by a reciprocal relationship between [Quin 2] and peak [Cai2+] stimulated by A23187 in the presence of EGTA. Parallel experiments with Quin 2 and aequorin may identify inhomogeneous [Cai2+] in platelets and give a more complete picture of platelet Ca2+ homeostasis than either indicator alone.     

Thermostable Bacillus subtilis lipases: in vitro evolution and structural insight

In vitro evolution methods are now being routinely used to identify protein variants with novel and enhanced properties that are difficult to achieve using rational design. However, one of the limitations is in screening for beneficial mutants through several generations due to the occurrence of neutral/negative mutations occurring in the background of positive ones. While evolving a lipase in vitro from mesophilic Bacillus subtilis to generate thermostable variants, we have designed protocols that combine stringent three-tier testing, sequencing and stability assessments on the protein at the end of each generation. This strategy resulted in a total of six stabilizing mutations in just two generations with three mutations per generation. Each of the six mutants when evaluated individually contributed additively to thermostability. A combination of all of them resulted in the best variant that shows a remarkable 15 °C shift in melting temperature and a millionfold decrease in the thermal inactivation rate with only a marginal increase of 3 kcal mol⁻¹ in free energy of stabilization. Notably, in addition to the dramatic shift in optimum temperature by 20 °C, the activity has increased two- to fivefold in the temperature range 25-65 °C. High-resolution crystal structures of three of the mutants, each with 5° increments in melting temperature, reveal the structural basis of these mutations in attaining higher thermostability. The structures highlight the importance of water-mediated ionic networks on the protein surface in imparting thermostability. Saturation mutagenesis at each of the six positions did not result in enhanced thermostability in almost all the cases, confirming the crucial role played by each mutation as revealed through the structural study. Overall, our study presents an efficient strategy that can be employed in directed evolution approaches employed for obtaining improved properties of proteins.     

Measurement of thromboxane A2-induced elevation of ionized calcium in collagen-stimulated platelets with the photoprotein, aequorin

Using aequorin-loaded rat platelets stimulated with collagen, we found two phases of Ca2+ mobilization, one coinciding with a shape change and the other with aggregation, which have not yet been detected in quin2-loaded platelets. U46619, a stable analogue of prostaglandin H2, induced only a shape change and a concomitant rapid rise in the cytoplasmic ionized calcium concentration ([Cai2+]). However, upon addition of U46619 to platelets previously stimulated with collagen in the presence of indomethacin, a rapid increase in [Cai2+] and a shape change occurred, and, after about 1 min, second increase in [Cai2+] and aggregation occurred. The actions of U46619 were inhibited by an antagonist for the thromboxane A2 (TXA2) receptor. These results suggest that the collagen-induced shape change is initiated by TXA2-induced Ca2+ mobilization, and aggregation is induced by the secondary Ca2+ mobilization induced by TXA2 and the occupation of the receptor by collagen.     

Interchange of aequorin and obelin bioluminescence color is determined by substitution of one active site residue of each photoprotein

The bioluminescence spectra from the Ca2+-regulated photoproteins aequorin (lambdamax=469 nm) and obelin (lambdamax=482 nm) differ because aequorin has an H-bond from its Tyr82 to the bound coelenteramide, not present in obelin at the corresponding Phe88. Substitutions of this Phe88 by Tyr, Trp, or His shifted the obelin bioluminescence to shorter wavelength with F88Y having lambdamax=453 nm. Removal of the H-bond by the substitution of Y82F in aequorin shifted its bioluminescence to lambdamax=501 nm. All mutants were stable with good activity and were expressible in mammalian cells, thereby demonstrating potential for monitoring multiple events in cells using multi-color detection.     

Blue fluorescent protein from the calcium-sensitive photoprotein aequorin: catalytic properties for the oxidation of coelenterazine as an oxygenase

Blue fluorescent protein from the calcium-binding photoprotein aequorin (BFP-aq) is a complex of Ca2+ -bound apoaequorin and coelenteramide, and shows luminescence activity like a luciferase, catalyzing the oxidation of coelenterazine with molecular oxygen. To understand the catalytic properties of BFP-aq, various fluorescent proteins (FP-aq) have been prepared from semi-synthetic aequorin and characterized in comparison with BFP-aq. FP-aq has luciferase activity and could be regenerated into native aequorin by incubation with coelenterazine. The results from substrate specificity studies of FP-aq using various coelenterazine analogues have suggested that the oxidation of coelenterazine by BFP-aq in the luciferase reaction and the regeneration process to aequorin might involve the same catalytic site of BFP-aq.     

NMR analysis of the Mg2+-binding properties of aequorin, a Ca2+-binding photoprotein

Aequorin, which is a calcium-sensitive photoprotein and a member of the EF-hand superfamily, binds to Mg2+ under physiological conditions, which modulates its light emission. The Mg2+ binding site and its stabilizing influence were examined by NMR spectroscopy. The binding of Mg2+ to aequorin prevented the molecule from aggregating and stabilized it in the monomeric form. To determine the structural differences between Mg2+-bound and free aequorin, we have performed backbone NMR assignments of aequorin in the Mg2+-free state. Mg2+ binding induces conformational changes that are localized in the EF-hand loops. The chemical shift difference data indicated that there are two Mg2+-binding sites, EF-hands I and III. The Mg2+ titration experiment revealed that EF-hand III binds to Mg2+ with higher affinity than EF-hand I, and that only EF-hand III seems to be occupied by Mg2+ under physiological conditions.     

A C-terminal proline is required for bioluminescence of the Ca(2+)-binding photoprotein, aequorin.

The requirement for a proline residue at the C-terminus of the Ca(2+)-binding photoprotein, aequorin, was investigated by measuring luminescence activities of a series of C-terminal deletion mutants, substitution mutants and an addition mutant. CD spectral measurements of apoaequorin with the C-terminal proline deleted showed a small change in secondary structure. In all cases studied, the C-terminal proline was required for bioluminescence activity.     

Renilla luciferin as the substrate for calcium induced photoprotein bioluminescence. Assignment of luciferin tautomers in aequorin and mnemiopsin.

A study was made of the effects of pH and protic and aprotic solvents on the spectral properties of Renilla (sea pansy) luciferin and a number of its analogs. The results have made possible the assignment of two tautomeric forms of Renilla luciferin, one which absorbs maximally at 435 nm and another which exhibits an absorption maximum at 454 nm. Furthermore the results provide an explanation for the visible absorption characteristics of the photoproteins aequorin (lambda-max 454 nm) and mnemiopsin (lambda-max 435 nm). In addition a Renilla-like luciferin can be extracted from both of these photoproteins. This luciferin produces light with Renilla luciferase, at a rate dependent upon the concentration of dissolved oxygen, and in other respects is indistinguishable from Renilla luciferin in this bioluminescent reaction. The results suggest that the native chromophore in both photoproteins is Renilla luciferin (or a nearly identical derivative). The results also suggest that a hydroperoxide intermediate probably exists in photoproteins, on energetic grounds, and to account for the oxygen concentration independency of the rate of photoprotein reactions. This hydroperoxide may be attached initially to an amino-acid side chain (possibly indolyl-OOH, imidazoyl-OOH, or -SOOH) rather than to the luciferin chromophore.