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Dec 15, 1999 - Aequorin is one of several photoproteins that emits visible light upon binding to calcium ions. It has been widely used as a Ca2+-indic...
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Bioconjugate Chem. 2000, 11, 65−70

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Bioluminescence and Secondary Structure Properties of Aequorin Mutants Produced for Site-Specific Conjugation and Immobilization J. C. Lewis, J. J. Lo´pez-Moya, and S. Daunert* Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055

Aequorin is one of several photoproteins that emits visible light upon binding to calcium ions. It has been widely used as a Ca2+-indicator and as an alternative highly sensitive bioluminescent label in binding assays. The apoprotein of aequorin binds an imidazopyrazine compound (coelenterazine) and molecular oxygen to form a stable photoprotein complex. Upon addition of calcium, the photoprotein undergoes a conformational change leading to the oxidation of the chromophore with the release of CO2 and blue light. To gain more information of structure-function relationships within the photoprotein that will aid in the design of mutants suitable for site-specific conjugation and immobilization, polymerase chain reaction (PCR)-based site-directed mutagenesis was employed to produce five different aequorin mutants. The five mutants included a cysteine-free mutant and four other mutants with single cysteine residues at selected positions within the protein. The aequorin mutants exhibited different bioluminescence emission characteristics with two mutants showing a decrease in relative light production in comparison to the cysteine-free mutant. Additionally, circular dichroism (CD) spectra revealed that the single amino acid substitutions made for two of the aequorin mutants did alter their secondary structures.

INTRODUCTION

Certain jellyfish within the class Hydrozoa produce proteins termed “photoproteins” that emit light in the visible region. One such photoprotein, aequorin, has become a widely used bioluminescent label in a variety of bioanalytical applications. Aequorin was first isolated from the jellyfish Aequorea victoria found in the Pacific Northwest. The photoprotein consists of an apoprotein, a chromophoric unit (coelenterazine), and molecular oxygen, which when combined form a stable complex. Binding to Ca2+ ions causes the photoprotein to undergo a conformational change, which results in the catalytic oxidation of the noncovalently bound coelenterazine. The coelenterazine is converted to coelenteramide with the release of CO2 and a flash of light (λmax = 469-470 nm) lasting less than 10 s (1). The cDNA sequence for the apoprotein of aequorin (apoaequorin) has been determined (2, 3), and the recombinant protein is readily produced in the laboratory. Amino acid sequencing of apoaequorin revealed a single polypeptide chain consisting of 189 amino acids (21.4 kDa) containing three EF-hand structures (Ca2+-binding sites consisting of a helix-loop-helix structural motif). These sites contained within the apoaequorin structure were shown to be homologous with the EF-hand structures of other Ca2+-binding proteins such as calmodulin and paralbumin, suggesting a common evolutionary origin (4). However, the apoprotein component of aequorin contains an unusually high content of histidine, tryptophan, cysteine, and proline residues in comparison to other Ca2+-binding proteins. It also has an extended carboxy-terminal region, and studies have demonstrated that deletion of the C-terminal proline causes an almost complete loss of luminescence activity. Other site-directed mutagenesis studies have shown that His16, His58, * To whom correspondence should be addressed. Phone: (606) 257-7060. Fax: (606) 323-1069. E-mail: [email protected].

His169, Trp108, and Trp173 are critical to the activity of the photoprotein as well (1). Since 2-mercaptoethanol is required for full regeneration of aequorin, site-directed mutagenesis was also performed on the three cysteine residues present within the protein at positions 145, 152, and 180. Interestingly, a mutant in which all three cysteines were replaced with serine (mutant S) had greater bioluminescence activity in comparison to the wild-type aequorin (5). We have employed aequorin as a highly sensitive label in binding assays for small biomolecules (6, 7). Our studies and those from others have demonstrated that fusion to the N-terminus of the protein with a small peptide (7) or a large protein (8) is possible without a significant decrease in the bioluminescent activity, unlike fusion to the C-terminus (9). We selected gene fusion as one method by which site-specific attachment of our desired analyte to the photoprotein could be achieved in the development of a binding assay for a small biomolecule. To further extend the application of aequorin in bioanalytical methods, we have explored the feasibility of producing a mutant of aequorin (i.e., one containing a single cysteine residue) suited for site-specific conjugation of nonpeptidic molecules and site-specific immobilization of the protein onto a surface. Even though crystallization of the protein has been achieved (10), the threedimensional structure of aequorin has yet to be determined. Thus, to achieve our goal and gain more structureactivity information about the photoprotein, we have produced five mutant aequorins, four of which each contain a unique cysteine residue at selected positions within the apoprotein. PCR-based site-directed mutagenesis was employed to initially replace all three cysteine residues with serine and then introduce a single cysteine residue at positions 5, 53, 71, and 84 in the polypeptide chain. All five mutant aequorins were characterized in terms of their bioluminescence emission properties and secondary structure.

10.1021/bc9900800 CCC: $19.00 © 2000 American Chemical Society Published on Web 12/15/1999

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Table 1. Synthetic Oligonucleotides Used for Site-Specific Mutagenesis

a

name

oligonucleotidea

amino acid substitution

C1,2S C3S AQ5 AQ53 AQ71 AQ84

GAA GAT TCC GAG GAA ACA TTC AGA GTG TCC GAT ATT GAT GAA AGT ATG GAC CCT GCT TCC GAA AAG CTC TAC GGT GGA GC AAG CTT ACA TGC GAC TTC GAC TTC GAC AAC CCA AG GGA GCA ACA CCT TGC CAA GCC AAA CGA CAC AAA GAT GGA GCT GGA TGC AAA TAT GGT GTG GAA ACT GAT TGG GCA TAT ATT TGC GGA TGG AAA AAA TTG GCT ACT GAT

Cys145,152 f Ser Cys180 f Ser Ser5 f Cys Glu53 f Cys Met71 f Cys Glu84 f Cys

Lettering highlighted in bold and underlined indicates nucleotide substitutions.

EXPERIMENTAL PROCEDURES

Reagents. All restriction endonucleases, T4 DNA ligase, Luria Bertani (LB) broth, LB agar, DNA mass ladder, ampicillin, and kanamycin were purchased from Gibco-BRL (Gaithersburg, MD). Tris(hydroxymethyl) amino methane (Tris), ethylenediaminetetraacetic acid (EDTA) disodium salt, sodium chloride, dithiothreitol (DTT), and all other reagents were obtained from Sigma (St. Louis, MO). HQH, quaternized polyethyleneimine anion exchanger, was purchased from Perseptive Biosystems (Cambridge, MA). Coelenterazine was obtained from Biosynth International (Naperville, IL). All oligonucleotides (primers) employed for PCR were provided by Operon Technologies (Alameda, CA). The polymerase selected for PCR was Pfu polymerase from Stratagene (La Jolla, CA). All solutions were prepared using deionized (Milli-Q Water Purification system, Millipore, Bedford, MA) distilled water. All chemicals were reagent grade or better and were used as received. Bacterial Strains and Plasmids. The expression vector pSbt and bacterial strain Bacillus subtilis (trpC2, MetB10, npr, apr::cat) was provided by Dr. Philip N. Bryan, University of Maryland. The mammalian expression vector, pMtAEQ, containing the apoaequorin gene was purchased from Molecular Probes (Eugene, OR). The Escherichia coli strain, JM109, employed for initial cloning reactions, was from Gibco-BRL (Gaithersburg, MD). PCR-Based Site-Specific Mutagenesis. The technique of overlap extension PCR was employed to produce the aequorin mutants. Initially, primers C1, 2S, and C3S (see Table 1) were used in order to obtain the apoaequorin gene in which all three cysteine residues were replaced by serine (referred to as mutant S). PCR reactions to amplify DNA fragments used in overlap extension PCR reactions were carried out with 2.5 units of Pfu polymerase, 50 ng of DNA template, 250 µM of each dNTP, and 50 pmol of each primer in a total volume of 100 µL. The overlap extension PCR reactions to amplify the entire mutated apoaequorin gene were performed using 30 ng of each previously amplified fragment, 50 pmol of each outside primer, 250 µM of each dNTP, and 5 units of Pfu polymerase in a total volume of 100 µL. The cycling parameters for the overlap extension PCR reactions were 94 °C for 1 min, 45 °C for 1 min, and 72 °C for 1 min 30 s for the first 10 cycles, followed by 20 cycles with an annealing temperature of 50 °C. An additional overlap extension PCR reaction was performed to attach the sequence for the “pre”-signal peptide of the protein subtilisin, an alkaline serine protease of Bacillus subtilis. This signal peptide facilitates the secretion of a desired protein outside of the bacterial cell and into the culture medium, simplifying the purification process for the protein. All PCR reactions were performed using a Perkin-Elmer GeneAmp PCR System 2400 (Norwalk, CT). The final DNA product containing the cysteine-free mutant apoaequorin (mutant 3) was cloned into the pSbt expression vector as a EcoRI-SalI fragment, to yield the

Figure 1. Schematic representation of the plasmids pSD110114 containing the oligonucleotide sequences of the “pre”-signal peptide and the mutated apoaequorin genes, fused in frame.

pSD110 vector shown in Figure 1. Bacteria (E. coli strain JM109) were then transformed with the pSD110 vector. Clones carrying the mutant plasmid were identified through restriction analysis using the restriction enzymes EcoRI/SalI and a unique restriction enzyme site introduced as a silent mutation in the apoaequorin gene during the first set of PCR reactions. The apoaequorin mutants containing unique cysteine residues at positions 5, 53, 71, and 84 in the polypeptide chain were prepared in the same manner (see Table 1) as described above using the gene encoding for apoaequorin mutant 3 as a template in the PCR reactions. The site-specific mutations were confirmed through DNA sequencing performed at the Macromolecular Center (University of Kentucky). All molecular biology procedures were conducted using standard protocols (11). Expression and Purification of Mutant Apoaequorins. Plasmids containing the mutant apoaequorin genes were transformed into competent B. subtilis cells, and cultured to express the mutant proteins. Specifically, bacteria were grown in 100 mL of LB broth supplemented with 30 µg/mL of kanamycin for 16 h at 37 °C with shaking (250 rpm). The culture was centrifuged at 5930g at 10 °C for 40 min to pellet the cells. The culture medium containing the secreted protein was removed and filtered with a 0.2 µM cellulose acetate syringe filter to ensure the complete removal of any remaining cells. The pH of the medium ranged 7.5-7.8 and was decreased to 4.2 with concentrated acetic acid to precipitate the protein. The mixture was incubated at 4 °C for 2 h before centrifugation at 12100g at 4 °C for 40 min. The precipitate was dissolved in 40 mM Tris-HCl, pH 8.0, buffer containing 5 mM EDTA and 5 mM DTT. The mixture was incubated at 37 °C for 15 min before it was purified by perfusion chromatography using an HQH anion-exchange column (4.6 mm × 100 mm). The column was preequilibrated with a 40 mM Tris-HCl, pH 7.0,

Aequorin Mutants for Site-Specific Conjugation

buffer containing 5 mM EDTA and 5 mM DTT. DTT was not added during the purification process for mutant S. A salt gradient from 0.0 to 4.0 M NaCl was employed to elute the protein. All the apoaequorin mutant proteins eluted between 0.15 and 0.20 M NaCl. The fractions containing the mutant protein were combined and concentrated using a Centriplus 3000 device from Millipore (Bedford, MA). The desired buffer exchange was achieved employing a polyacrylamide 6000 desalting column from Pierce (Rockford, IL). The purity of the mutant proteins was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using an electrophoresis PhastSystem from Pharmacia Biotech (Uppsala, Sweden). Polyacrylamide (12.5%) gels were developed by the method of silver staining. The protein concentrations were determined by measuring absorbance at 280 nm using the formula E1%,1cm ) 18.0. Conversion of Apoaequorin Mutants to Photoproteins. Generation of the aequorin mutants from the apoproteins was achieved by mixing a set amount of the purified proteins in a glass test tube with a molar excess of coelenterazine (0.2 µg) in Tris-HCl, pH 7.6, buffer containing 2 mM EDTA with or without 5 mM DTT as indicated. The mixture was briefly vortexed and then placed on ice for the required time. Assay for Mutant Aequorin Activity. The luminescence activity of the mutant aequorins was measured by placing 10 µL of the regeneration mixture described above in a glass test tube, which was then placed in a Optocomp I luminometer from GEM Biomedical (Carrborro, NC). A volume of 100 µL of a 100 mM CaCl2 and 100 mM Tris-HCl, pH 7.6, buffer (luminescence triggering buffer) was injected into the sample solution in order to trigger light emission. The luminescence signal was collected at 0.1 s intervals over a 5 s time period. CD Spectra of Apoaequorin Mutants. CD spectra were obtained using a Jasco model J-710 (Tokyo) spectropolarimeter with a 0.1 cm path-length cell. The mutant protein concentrations were 0.1-0.2 mg/mL in 10 mM Tris-HCl, pH 7.6, buffer containing 2 mM EDTA. Protein samples were stored on ice, and then placed in a RT bath for 60 min before taking the CD spectra. The CD spectra shown are an average of 16 scans per sample. Bioluminescence Emission Spectra of Apoaequorin Mutants. Emission spectra were obtained using a Fluorolog-τ2 spectrofluorometer from Spex Industries (Edison, NJ) without turning on the lamp or chopper. Luminescence was triggered by careful addition of 1.5 mL of a 2.5 × 10-6 M solution of mutant aequorin using a constant-rate syringe pump into 0.5 mL of luminescence triggering buffer. The spectra were not corrected for changes in the sensitivity of the photomultiplier tube to the luminescence at different wavelengths. RESULTS

To produce the aequorin mutants containing unique cysteine residues, PCR-based site-directed mutagenesis was employed to construct expression vectors pSD110114 (Figure 1) using the oligonucleotides shown in Table 1. Restriction analysis was performed to verify the presence of the mutated apoaequorin genes in the expression vectors followed by DNA sequencing to confirm the desired mutations. Following expression of the apoaequorin mutants in B. subtilis, the mutant proteins were successfully secreted into the culture medium by the “pre”-signal peptide of subtilisin. The proteins were purified using perfusion chromatography with a HQH anion-exchange column. All five mutant apoaequorins

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Figure 2. Flash-type bioluminescence emission obtained from each aequorin mutant [mutant S (9), mutant 5 (2), mutant 53 (b), mutant 71 ([), mutant 84 (+)]. The bioluminescence signal was measured as light intensity in relative units, and then converted to percent using the maximum signal obtained for each aequorin mutant. Table 2. Characterization of Aequorin Mutants in Terms of Luminescence Activity and Secondary Structure

Aequorin mutant

luminescence activity (%)a

half-life (s)

λmax

R-helix content (%)

mutant S (no Cys) mutant 5 (Ser5Cys) mutant 53 (Glu53Cys) mutant 71 (Met71Cys) mutant 84 (Glu84Cys)

100 102 98 13 14

1.00 0.99 1.02 1.10 1.13

465 463 467 465 469

27.2 27.4 26.9 19.6 31.0

a Composition of regeneration mixtures were 1 µg of purified mutant protein in 350 µL of 30 mM Tris-HCl, 2 mM EDTA, 5 mM DTT, pH 7.6, buffer containing 0.2 µg of coelenterazine (0.1 µg/ µL). All measurements were performed in triplicates with 10 µL aliquots of the regeneration mixture. Relative activities are reported as the percentage of maximum activity obtained for mutant S (100%).

were eluted from the column at salt concentrations between 0.15 and 0.20 M. The purity of the eluted fractions was determined by SDS-PAGE using the method of silver staining, which indicated a purity of greater than 95% (11). The yields for the protein purification ranged 22-43 mg of protein/L of culture. The purified apoaequorin mutants were converted to the corresponding photoproteins (regeneration of the photoprotein) by mixing the apoproteins with a molar excess of coelenterazine followed by incubation of these mixtures on ice or at 4 °C. A bioluminescence emission study was performed to determine if the mutant proteins had retained the same flash-type emission characteristics of native aequorin and to compare the mutants containing a specifically placed cysteine residue with mutant S (Figure 2). All five mutants did emit approximately 95% of their total bioluminescence signal within 3 s. The halflives for the bioluminescence light emission of the mutants are shown in Table 2. The half-lives for the mutants are longer in comparison to the native aequorin with a value of 0.60 s. Also, the values calculated for mutants 71 and 84 are slightly larger than those for mutants 3, 5, and 53. These difference could be attributed to the presence of the mutations within the apoprotein structures. To compare the luminescence activity of the individual mutants to mutant S, as well as determine the time

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465 nm (13). Therefore, we decided to generate bioluminescence emission spectra for each of the five mutants employed here, as shown in Figure 5. The curves were obtained by following a modification of the method by Shimomura (14). Specifically, the aequorin mutants were added to the luminescence triggering buffer using a constant-rate syringe pump. The mutant aequorin solution is assumed to be completely saturated with calcium ions as soon as it is added to the quartz cell, thus, producing a constant luminescence signal over the required time period of 3-4 min. As seen from the figure, certain mutants (S, 53, and 5) appear to have a shoulder in their spectrum between 420 and 440 nm. However, mutants 71 and 84 exhibit no such shoulder. Emission maxima for each of the individual mutants were similar with values ranging 463-469 nm (Table 2). DISCUSSION Figure 3. Time course study for conversion of apoaequorin mutants [mutant S (9), mutant 5 (2), mutant 53 (b), mutant 71 ([), mutant 84 (+)] to fully regenerated photoproteins. Bioluminescence measurements were taken at various times for up to 36 h using protein solutions containing 1 µg of apoprotein and 0.2 µg of coelenterazine in 30 mM Tris-HCl, 2 mM EDTA, 5 mM DTT, pH 7.6, buffer.

required for full regeneration of the photoproteins, a regeneration time course study was performed using each of the mutant apoaequorins. Due to the possible formation of dimers for the mutants containing a single cysteine residue, the study was conducted using a TrisHCl/EDTA buffer with 5 mM DTT, the presence of which has been shown not to affect the luminescence activity of mutant S (5). Figure 3 shows that mutants S, 5, and 53 have similar regeneration characteristics with each reaching >90% of its maximum signal after 9 h. However, mutants 71 and 84 required 18 h to reach the same point. The luminescence activities of each of the four mutants with unique cysteine residues at positions 5, 53, 71, and 84 in comparison to mutant S with no cysteine residues are shown in Table 2. While mutants 5 and 53 demonstrate no decrease in bioluminescence activity, mutants 71 and 84 have less than 15% of the bioluminescence activity of mutant S. To further study the affects of the mutagenesis on the photoprotein structures, circular dichroism (CD) spectra were taken with each individual mutant using mutant 3 as a baseline for comparison of secondary structure. The acquired CD spectra are shown in Figure 4 and exhibit typical protein secondary structures of R-helix and β-sheet. As seen from the figure, mutant 84 shows the largest change in secondary structure, while mutant 53 appears almost identical to mutant S. The R-helix content for each apoaequorin mutant (Table 2) was determined by analysis of the data using the program by Yang et al. (12). Whereas mutants 5 and 53 have similar values for their R-helix content in comparison to mutant S, both mutants 71 and 84 have either a significant decrease or an increase in the amount of R-helix contained within the apoprotein structure. The bioluminescence activity was measured before and after taking each CD spectra to ensure no denaturation of the proteins had taken place. Interestingly, it would appear that an alteration in the R-helix content of protein, whether positive or negative, could possibly lead to a siginifcant loss in activity. Previously, it was demonstrated that a single amino acid substitution (Trp86Phe) produced a mutant aequorin with two emission maxima at 400 and 465 nm, unlike the native protein with a single emission maximum at

The photoprotein, aequorin, produces a highly sensitive bioluminescent signal allowing for detection of the protein down to subattomol levels (15). Therefore, the photoprotein has found application as a label in a variety of bioanalytical methods. Since there is no available crystal structure for the protein, site-directed mutagenesis studies have been performed in order to elucidate information on structure-function relationships within the protein. The protein contains three calcium-binding sites (EFhand structures) that consist of 12 amino acid residues arranged sequentially in a loop. When a key glycine residue was replaced at position 6 within the EF-hand structures, it was demonstrated that only Ca2+-binding sites I and II are required for full bioluminescence activity (16). Another important consideration for the bioluminescence activity of the protein is the O2-binding site and the hydrophobic regions in which the chromophore becomes noncovalently bound. The hydrophobic regions of the protein are between residue positions 4050, 55-65, and 100-110 within the polypeptide chain (16). The unusually high content of histidine residues within the protein suggested the possibility that one of these residues was part of the O2-binding site. Modification of the five histidine residues present within the protein (positions 16, 18, 27, 58, and 169) to alanine produced four mutants with decreased bioluminescent activity, and one mutant (169) with a complete loss of activity. Thus, His169, has been suggested as the O2binding site (17). The six tryptophan residues present within the protein were altered as well; each was replaced with the close analogue phenylalanine. Five of the six mutant aequorins showed a significant decrease in luminescence activity. CD spectra of these mutants demonstrated that complex changes had occurred within the secondary structure of the proteins as a result of the single amino acid substitutions. Only one mutant (Trp79Phe) had retained full activity, and this mutant showed only a small change in secondary structure in comparison to the native aequorin (16). Critical to the work proposed here were site-directed mutagenesis studies on the three cysteine residues present within apoaequorin at positions 145, 152, and 180 (6, 16). The requirement for 2-mercaptoethanol in the regeneration of the photoprotein indicated that these residues played an important role in the bioluminescence activity of aequorin (16). Replacement of the individual cysteine residues with serine produced three mutants, all of which had significantly lower activity in comparison to native aequorin. Interestingly, however, was a mutant produced in a later study in which all three cysteine residues were simultaneously replaced with serine (5).

Aequorin Mutants for Site-Specific Conjugation

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Figure 4. CD spectra for apoaequorin mutants. Each panel shows the spectra for mutant S (dashed lines) in comparison to spectra obtained for one of the apoaequorin with a unique cysteine residue (solid lines).

Figure 5. Bioluminescence emission spectra for aequorin mutants [mutant S (9), mutant 5 (2), mutant 53 (b), mutant 71 ([), mutant 84 (+)] using spectrofluorometer with lamp and chopper turned off.

This cysteine-free mutant had an increase (124%) in bioluminescence activity in comparison to the wild-type photoprotein without the additional requirement of a reducing agent. These studies suggested that introduction of a unique cysteine residue within apoaequorin for the purpose of site-specific chemistries on the protein, was possible while still retaining the complete lightproducing properties of photoprotein. However, without an available three-dimensional structure for the protein, selection of an appropriate site for a unique cysteine residue within the apoprotein required additional studies on structure-function relationships present within aequorin. On the basis of information obtained from the

previous studies discussed above, we generated a cysteine-free mutant of aequorin (mutant S), and used this mutant to produce four additional mutants with unique cysteine residues at various positions (5, 53, 71, and 84) within the photoprotein. The positions selected were not part of any of the amino acid sequences previously identified within apoaequorin as being critical to its bioluminescence activity including the three EF-hand structures (Ca2+-binding sites), hydrophobic regions of the protein, and the C-terminal region. Since the apoprotein contains only a single serine residue (position 5 in the polypeptide chain) that is not part of a critical region within the protein, replacement of this serine with cysteine was the only conservative mutation possible. Also, this residue was at the N-terminus of the protein, and studies have demonstrated that N-terminal fusions are possible without significant losses in the luminescence activity of aequorin (7, 8), thus, indicating that amino acid substitution at this position was unlikely to produce an aequorin mutant with significantly decreased luminescence activity. The incorporation of a unique cysteine residue within the protein structure allows for the site-specific attachment of a desired biomolecule or the site-specific immobilization of the photoprotein onto a surface. The apoaequorin mutant produced in our laboratory containing all three cysteine residues present within the native protein replaced with serines was successfully converted to an active photoprotein as previously reported. This mutant functioned as a baseline for comparison between the four additional mutants generated containing specifically placed unique cysteine residues. The four aequorin mutants containing single cysteine residues were different in terms of their light emission properties and secondary structure features. In particular, two of the mutants (5 and 53) showed similar

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secondary structure characteristics and bioluminescence emission properties to mutant S. However, the other two mutants produced (71 and 84) exhibited a large decrease in their light-generating capabilities in comparison to mutant S. CD spectra revealed that the mutagenesis had produced significant changes in the secondary structure of the proteins in comparison to mutant S with either increasing or decreasing R-helix content. These two proteins also required a much longer regeneration time of 18 h versus 9 h for the other mutants. On the other hand, two of the mutants obtained (5 and 53) demonstrated no loss in bioluminescence activity and exhibited similar secondary structures in comparison to mutant S, as well as regeneration time and half-lives. Due to the dramatic decrease in bioluminescence signal observed for mutants 71 and 84, it could be postulated that these amino acid residues are in close proximity to one of the active sites within the photoprotein structure, most presumably the hydrophobic region in which coelenterazine binds. Further studies should reveal whether the decrease in activity is a result of decreased stability, quantum yield, or binding of the chromophore. The properties of the four different mutants produced indicate that mutants 5 and 53 are more suitable for site-specific immobilization of the photoprotein because these mutants retained the same degree of bioluminescence activity as compared to the cysteine-free aequorin mutant. However, while mutants 5 and 53 would also be excellent candidates for site-specific conjugation of desired analytes, mutants 71 and 84 are viable as well. Since these mutants have different bioluminescence and secondary structure characteristics, it is conceivable that they might demonstrate a change in their luminescence signal upon binding to an appropriate binder. Thus, mutants 71 and 84 could function as bioluminescence labels in homogeneous binding assays for desired biomolecules. ACKNOWLEDGMENT

We would like to thank the Department of Energy (DEFG05-95ER62010) and the National Institutes of Health (GM47915) for support of this work. S. Daunert is a Cottrell Scholar and a Lilly Faculty Awardee. J. C. Lewis acknowledges support from the National Science Foundation for an IGERT Predoctoral Fellowship. We would also like to thank Dr. Paul Bummer of the Department of Pharmaceutical Sciences of the University of Kentucky for his assistance in aquiring the CD spectra of the apoaequorin mutants. LITERATURE CITED (1) Ohmiya, Y., and Hirano, T. (1998) Shining the light: mechanism of the bioluminescence reaction of calciumbinding photoproteins. Chem. Biol. 3, 337.

Lewis et al. (2) Prasher, D., McCann, R. O., and Cormier, M. J. (1985) Cloning and expression of the cDNA coding for aequorin, a bioluminescent calcium-binding protein. Biochem. Biophys. Res. Commun. 126, 1259. (3) Inouye, S., Noguchi, M., Sakaki, Y., Takagi, Y., Miyata, T., Iwanaga, S., and Tsuji, F. I. (1985) Cloning and sequence analysis for the cDNA for the luminescent protein aequorin. Proc. Natl. Acad. Sci. U.S.A. 82, 3154. (4) Charbonneau, H., Walsh, K. A., McCann, R. O., Prendergast, F. G., Cormier, M. J., and Vanaman T. C. (1985) Amino acid sequence of calcium-dependent photoprotein aequorin. Biochemistry 24, 6762. (5) Kurose, K., Inouye, S., Sakaki, Y., and Tsuji, F. I. (1989) Bioluminescence of the Ca2+-binding photoprotein aequorin after cysteine modification. Proc. Natl. Acad. Sci. U.S.A. 86, 80. (6) Witkowski, A., Ramanathan, S., and Daunert, S. (1994) Bioluminescence binding assay for biotin with attomol detection based on recombinant aequorin. Anal. Chem. 66, 1837. (7) Ramanthan, S., Lewis, J. C., Kindy, M. S., and Daunert, S. (1998) Heterogeneous bioluminescence binding assay for an octapeptide using recombinant aequorin. Anal. Chim. Acta 369, 181. (8) Zenno, S., and Inouye, S. (1990) Bioluminescent immunoassay using a fusion protein of protein A and the photoprotein aequorin. Biochem. Biophys. Res. Commun. 171, 169. (9) Nomura, M., Inouye, S., Ohmiya, Y., and Tsuji, F. I. (1991) A C-terminal proline is required for bioluminescence of the Ca2+-binding protein, aequorin. FEBS Lett. 295, 63. (10) Hannick, L. I., Prasher, D. C., Schultz, L. W., Deschamps, J. R., and Ward, K. B. (1993) Preparation and initial characterization of crystals of the photoprotein aequorin from Aequorea victoria. Proteins 15, 103. (11) Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY. (12) Yang, J. T., Wu, C. C., and Martinez, H. M. (1986) Calculation of protein conformation from circular dichroism. Methods Enzymol. 130, 208. (13) Ohmiya, Y., Ohashi, M., and Tsuji, F. I. (1992) Two excited states in aequorin bioluminescence induced by tryptophan modification. FEBS Lett. 301, 197. (14) Shimomura, O. (1986) Isolation and properties of various molecular forms of aequorin. Biochem. J. 234, 271. (15) Actor, J. K., Kuffner, T., Dezzutti, C. S., Hunter, R. L., and McNichol, J. M. (1998) A flash-type bioluminescent immunoassay that is more sensitive than radioimaging: quantitative detection of cytokine cDNA in activated and resting human cells. J. Immunol. Methods 211, 65. (16) Tsuji, F. I., Inouye, S., Goto, T., and Sakaki, Y. (1986) Sitespecific mutagenesis of the calcium-binding photoprotein aequorin. Proc. Natl. Acad. Sci. U.S.A. 83, 8107. (17) Ohmiya, Y., and Tsuji, F. (1993) Bioluminescence of the Ca2+-binding photoprotein, aequorin, after histidine modification. FEBS Lett. 320, 267.

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