Limited Proteolysis of Luciferase as a Reporter in Nanosystem Biology: A Comparative Study

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Photochemistry and Photobiology, 2009, 85: 1162–1167

Limited Proteolysis of Luciferase as a Reporter in Nanosystem Biology: A Comparative Study Farangis Ataei, Saman Hosseinkhani* and Khosro Khajeh Department of Biochemistry, Faculty of Basic Science, Tarbiat Modares University, Tehran, Iran Received 22 February 2009, accepted 9 April 2009, DOI: 10.1111/j.1751-1097.2009.00583.x

ABSTRACT Firefly luciferase is a 62 kDa protein that produces a flash of light on the oxidation of luciferin in the presence of ATP, Oxygen and Mg2+. Luciferase has a broad range of applications for analytical purposes and in vivo imaging for nanosystem biology studies. However, the enzyme is highly susceptible to proteolytic degradation that reduces its half-life. Rate of proteolytic digestion between two members of luciferase family (Photinus pyralis and Lampyris turkestanicus) is compared. Proteolytic sensitivity of L. turkestanicus luciferase was found to be more than P. pyralis luciferase, due to higher rate of hydrolysis under identical conditions. Both luciferases showed more sensitivity to chymotrypsin than trypsin with different digestion pattern. Digestion of P. pyralis by trypsin produced some fragments which were found to be more resistant to further degradation, whereas in L. turkestanicus initial fragments subdigested by trypsin, like chymotrypsin effect on both luciferases. Furthermore, both luciferases become increasingly labile to proteolysis as the temperature increases. The rate of inactivation and the rate of degradation between luciferases were different in a specific time of incubation. Appearance of similar bands for both luciferases confirmed exposure of specific regions, in spite of structural differences.

INTRODUCTION Luciferases are the key enzymes that catalyze the lightemitting reactions in bioluminescence. They catalyze the oxygenation of compounds generally known as luciferin, generating energy-rich peroxidic intermediates, emitting a photon of visible light with high efficiency (1,2). Luciferases make a diverse group of unrelated enzymes acting on chemically different luciferins (3,4). The insects are the richest and most diverse group of bioluminescent organisms and Firefly luciferase is the best characterized bioluminescent protein (5,6). Firefly luciferase (EC is an enzyme that catalyzes the oxidation of luciferin (LH2), in the presence of ATP and Mg2+, giving rise to light (7,8). The bioluminescence reaction involves adenylation of LH2 to form luciferyl-adenylate (LH2AMP) by ATP. LH2-AMP is then oxidized by molecular oxygen and then through a series of intermediates, produces

*Corresponding author email: [email protected] (Saman Hosseinkhani)  2009 The Authors. Journal Compilation. The American Society of Photobiology 0031-8655/09

AMP, inorganic pyrophosphate (PPi), CO2, oxyluciferin and visible light (8). In recent years, luciferases and related genes have become very useful in research and commercial purposes such as: to monitor transcriptional activities (9), the ultra-sensitive detection of ATP (10), study the role of chaperones in protein folding (11), use in DNA sequencing (12) and as a reporter gene in imaging of living animals (13). Many of these techniques are currently used in various industries (14–19). In spite of extensive analytical application of firefly luciferase, however, the enzyme is relatively unstable, thereby decreasing its sensitivity and precision (20). One of distinctive properties of firefly luciferase is pronounced susceptibility to proteolysis in vitro and short half-life in vivo (21). Limited proteolysis has become a powerful tool for probing the higher order structure of proteins which determine the location of particular peptide bonds within the overall fold of the protein (22). This is based on the hypotheses that limited proteolysis exclusively find ‘‘hinges and fringes’’ (23) and conformational parameters such as accessibility and segmental mobility correlate quite well with limited proteolysis sites (22,24,25). This paper describes limited proteolysis of luciferases from firefly Photinus pyralis (P. pyralis) and Glow-worm Lampyris turkestanicus (L. turkestanicus) using trypsin and chymotrypsin as reactive probes. The main goal of this study was identification of protease sensitive regions of firefly luciferases and isolation of digested fragments upon treatment with different proteases. Moreover, in order to correlate the proteolytic sensitivity to differences in primary structures and local flexibility of the enzymes, structural stabilities of luciferases upon proteolysis are compared.

MATERIALS AND METHODS Expression of recombinant luciferase. In order to purify luciferases, E. coli harboring the expression plasmid was grown at 37C in 5 mL of LB medium, containing 50 lg mL)1 of ampicillin (for P. pyralis) and kanamycin (for L. turkestanicus), as reported earlier (26). After overnight culture, the medium was transferred to 500 mL of fresh LB medium. The growth continued at 37C with vigorous shaking until the cell density reached an absorbance of 0.6–0.8 at 600 nm (A600), the culture was then induced with lactose (4 mM) and the incubation was continued for an additional 12 h at 22C. After centrifuging the culture medium at 2500 g for 30 min at 4C, the bacterial pellet was resuspended in lysis buffer (50 mM Tris-NaOH, 300 mM NaCl, 10 mM imidazole, pH 7.8) containing 1 mM phenylmethilsulfonyl fluoride (PMSF) and sonicated to disrupt the bacterial cells.


Photochemistry and Photobiology, 2009, 85 Luciferase purification by using affinity chromatography. The cell lysate was centrifuged at 8000 g for 20 min at 4C. The supernatant was applied to nickel–nitrilotriacetic acid (Ni-NTA) Sepharose column (Qiagen) and then washed with an imidazole step gradient (20, 60 and 100 mM imidazole in 50 mM Tris-NaOH, 300 mM NaCl, pH 7.8) in order to weakly bound and contaminating nonspecific proteins were removed. The polyhistidine-tagged luciferase was finally eluted from the column by increasing the imidazole concentration gradient to 250.0 mM and then glycerol was added to a final concentration of 10% (vol ⁄ vol), followed by storage at )20C. Determination of protein purity and concentration. The purity of the fractions containing purified luciferase was determined by SDS-PAGE by using 12.5% slab gels. Proteins were visualized by coomassie blue staining. The concentration of purified protein was determined by ‘‘Bradford’’ assay at 595 nm. Limited proteolysis. Highly purified firefly luciferase (0.5 mg mL)1) was suspended in assay buffer (50 mM HEPES, pH 7.8, 10 mM MgSO4) and then incubated at 23C or 37C with 5 lg mL)1 trypsin (50 mM Tris-NaOH, 10 mM CaCl2, pH 7.8) or 1 lg mL)1 chymotrypsin (50 mM K2HPO4, pH 7.8) for various lengths of time. At the end of the incubation time, PMSF (1 mM) was added to stop the reaction. The reaction mixtures were denatured by boiling in SDS-b-mercaptoethanol, and samples were then run on SDS-PAGE. Luciferase assay throughout limited proteolysis. The luciferase activity was determined by light emission measurements using a Sirius Single Tube Luminometer (Berthold Detection Systems, Pforzheim, Germany), as reported earlier (27,28). Assays were initiated by injecting samples which were incubated with trypsin ([E ⁄ S] ratio 1:100) and chymotrypsin ([E ⁄ S] ratio 1:500) in enzyme dilution buffer [1:500] (50 mM Tris-NaOH, 1 mM EDTA, 1 mM b-mercaptoethanol, 2% vol ⁄ vol glycerol, pH 7.8). Then 5 lL of the diluted sample was added to 5 lL [1:1] of the substrate solution (50 mM Tris-NaOH, 4 mM ATP, 2 mM D-luciferin, 10 mM MgSO4, and pH 7.8). The enzymatic activity was measured in aliquots taken at suitable time intervals.


Figure 1. SDS-PAGE of the proteolytic digestion of luciferase from P. pyralis ( and L. turkestanicus (L.tur) by trypsin (Try). Proteolysis was conducted at 23C (protease to substrate ratio [E ⁄ S]; 1:100 by weight). Samples were taken from the reaction mixtures at indicated times and stopped by PMSF. Luciferase and proteolytic fragments were detected by staining with coomassie blue. The molecular weights of the fragments corresponding to digestion of enzyme and marker (M) are indicated.

RESULTS Structural comparison of luciferases from P. pyralis with L. turkestanicus indicated high structural similarity (29) and 85.3% sequence similarity (30). Differences in primary structure of L. turkestanicus compared to P. pyralis luciferase have changed the protein conformation (29). In order to compare proteolytic sensitivity, a series of preliminary proteolysis experiments were carried out with two proteases. Proteolytic digestion of luciferases from Photinus pyralis and Lampyris turkestanicus Limited proteolysis experiments have been successfully used to probe conformational properties of globular proteins (22,23). In this study, trypsin and chymotrypsin were used to compare the flexibility of luciferases from P. pyralis and L. turkestanicus. The intact purified proteins were incubated individually with trypsin and chymotrypsin under various experimental conditions of protease to luciferase ratio, temperature and duration of incubation, and the extent of the enzymatic digestion was estimated by SDS-PAGE. The SDS-PAGE analysis (under reducing conditions) of the proteolyzed mixture at 23C reveals that the digestion of L. turkestanicus luciferase was more than P. pyralis luciferase (Figs. 1 and 2). The extensive degradation of the L. turkestanicus luciferase was quite distinct from the digestion pattern of P. pyralis luciferase under identical conditions. The occurrence of low amount fragments in P. pyralis digest indicates clearly that, under identical conditions, most of the protein remained intact. These results showed that P. pyralis

Figure 2. SDS-PAGE of the proteolytic digestion of luciferase from P. pyralis ( and L. turkestanicus (L.tur) by chymotrypsin (Chy). Proteolysis was conducted at 23C (protease to substrate ratio [E ⁄ S]; 1:500 by weight). Samples were taken from the reaction mixtures at the indicated times and stopped by PMSF. Luciferase and proteolytic fragments were detected by staining with coomassie blue. The molecular weights of the fragments corresponding to digestion of enzyme and marker (M) are indicated.

luciferase is relatively more stable against proteolysis and is not degraded at the same rate as L. turkestanicus luciferase. Cleavage of L. turkestanicus with trypsin or chymotrypsin yields similar product patterns. The initial products are three clear bands about 45, 35 and 30 kDa followed by a number of smaller fragments (Fig. 3). The proteolyzed fragments are unstable, which subsequently further digested by trypsin and chymotrypsin. In fact, with increasing digestion time, the primary fragments become more susceptible to cleavage, leading to formation of smaller fragments at longer incubation. Trypsin is classified as a protease with narrow specificity which hydrolyses peptide bonds at C-terminal of basic amino acids, lysine and arginine (22,23,31), and chymotrypsin considered as a broader-specificity protease that cleaves many peptide bonds but has a strong preference for hydrolysis of aromatic amino acids (23,31–33). The digestion pattern of L. turkestanicus luciferase by trypsin and chymotrypsin can be explained by its more flexibility. Limited proteolysis of P. pyralis luciferase with trypsin and chymotrypsin, surprisingly, reveals different digestion pattern (Fig. 4). The luciferase from P. pyralis, like that from

1164 Farangis Ataei et al. that the rate of proteolysis by chymotrypsin is much faster than the rate of digestion with trypsin in both cases. The increased susceptibility of these firefly luciferases to chymotrypsin could be explained by the presence of hydrophobic patches on the protein surface that was confirmed earlier (34). Proteolysis at thermal inactivation temperature

Figure 3. Limited proteolysis of L. turkestanicus (L.tur) luciferase with chymotrypsin (Chy) and trypsin (Try) at 23C. Native luciferase was digested at a protease to substrate ratio of 1:100 (by mass) for trypsin and 1:500 for chymotrypsin. After proteolysis (at indicated times), PMSF was added to stop the reaction, and an aliquot of the proteolysis mixture was analyzed by SDS-PAGE. The molecular weights of the fragments corresponding to digestion of enzyme and marker (M, Lane 1) are indicated.

Limited proteolysis with trypsin and chymotrypsin was also carried out at 37C. The SDS-PAGE gels reveal that both firefly luciferases exhibit the same digestion pattern at 37C as 23C. However, the rates of proteolysis at 37C are much faster than 23C. Much time was required for luciferase digestion at 23C, as at this temperature, luciferase maintains a native-like structure (27). However, at elevated temperatures (like 37C), local unfolding of some segments may be induced (23); therefore the proteins adopt a more flexible state and thus become more prone to proteolytic attack (33). In reality, when proteins are partially denatured by heating, the inherent susceptibility of the proteins to proteolytic attack is increased (23). In addition, at 37C, two major fragments of tryptic digestion of P. pyralis luciferase appeared to be quite resistant to further proteolysis as 23C, because their electrophoretic bands remain in the stained gel after several hours of reaction (Fig. 5). Rates of inactivation and of cleavage

Figure 4. Limited proteolysis of P. pyralis ( luciferase with chymotrypsin (Chy) and trypsin (Try) at 23C. Native luciferase was digested at a protease to substrate ratio of 1:100 (by mass) for trypsin and 1:500 for chymotrypsin. After proteolysis, PMSF was added to stop the reaction, and an aliquot of the proteolysis mixture was analyzed by SDS-PAGE. The molecular weights of the fragments corresponding to digestion of enzyme and marker (M, Lane 1) are indicated.

L. turkestanicus, is extensively degraded by chymotrypsin; primary fragments were produced and then followed by complete proteolysis. However, a detail investigation of these results revealed the occurrence of subtle differences. As shown in Fig. 4, tryptic digestion indicates that the P. pyralis luciferase is cut in a limited manner and the nicked protein is stable and remains resistant to further degradation. Digestion of the P. pyralis luciferase using trypsin yields two major fragments with molecular masses of 40 and 30 kDa and some other peptides in low amounts. Two major peptides are rapidly produced and are persistent during proteolysis. Therefore, the results of this experiment suggest that these fragments conserve their conformation upon limited proteolysis.

The possibility that peptide bond cleavage might cause inactivation after a preliminary conformational change, rather than by a direct effect upon the active center, required comparison of the rate of inactivation with the rate of hydrolysis. The remaining activity was measured in the presence of both proteases at 23C and 37C. A comparative analysis of the time-course of proteolysis and inactivation reveals that the rate of inactivation of the L. turkestanicus enzyme by trypsin and chymotrypsin is faster than that for the P. pyralis luciferase, as well as the rate of degradation (Fig. 6). Furthermore, according to kinetic data, it is clear that luciferases appeared to be relatively more sensitive in the presence of chymotrypsin than trypsin under different conditions and times (Fig. 6). This observation revealed that the rate of enzyme inactivation with chymotrypsin is faster.

Comparison of lability to trypsin and chymotrypsin One of the most important points in this study is comparison of luciferase lability to different proteases. Incubation of luciferases from P. pyralis and L. turkestanicus with chymotrypsin showed significant differences compared to the results obtained by trypsin. Figures 1 and 2 show that both luciferases are more sensitive to chymotrypsin than trypsin. The figures clearly show

Figure 5. Proteolytic stability of P. pyralis ( luciferase and its tryptic fragments was incubated at 37C and 23C for the indicated times with 1:100 ratio of trypsin:luciferase (by mass), followed by SDSPAGE analysis. The molecular weights of the fragments corresponding to digestion of enzyme and marker (M) are indicated.

Photochemistry and Photobiology, 2009, 85


Figure 6. Loss of luciferase activity during incubation with the proteases. Approximately 0.5 mg mL)1 native luciferase ( and L.tur) incubated with (A) 5 lg mL)1 trypsin (Try) and (B) 1 lg mL)1 chymotrypsin (Chy) for various durations (minutes) at 23C. Reactions were stopped at the specified times with addition of 1 mM PMSF and Luciferase activity was recorded immediately. The error bars show the variation between various reactions.

Moreover, the remaining activity of both luciferases during proteolysis at 23C and 37C were compared and results are shown in Fig. 7. As can be seen in this figure, at 37C, both enzymes are easily inactivated by the proteases, and after a

short time of reaction, they lost almost all of their activity (Fig. 7A). However when the temperature decreased near the optimum temperature (23C), the rate of luminescence inactivation is rather slow (Fig. 7B). In fact, the proteins lose their activity in about 15 min at 37C, whereas they are somewhat more resistant to proteolysis and inactivation at 23C, because the active protein is still visible after a 3 h reaction in SDSPAGE. The results of enzyme inactivation experiments are in agreement with limited proteolysis data.


Figure 7. Remaining activity of luciferase in the presence of proteases at different interval times at 37C (A) and 23C (B). Approximately 0.5 mg mL)1 native luciferase ( and L.tur) incubated with 5 lg mL)1 trypsin (Try) and 1 lg mL)1 chymotrypsin (Chy) for various times (minutes). Reactions were stopped at the specified times with addition of 1 mM PMSF and luciferase activity was recorded immediately. The error bars show the variation between various reactions.

The firefly emits brilliant yellow–green light by an enzymatic reaction which is detected easily (35). During recent years, the firefly luciferases have been extensively used for sensitive detection of metabolites and research applications. However, the use of luciferase in the analytical purposes suffers from losses in sensitivity and precision as a result of the susceptibility to proteolytic degradation and decrease in its half-life (36). Due to the protease contamination in most in vivo or in vitro systems, it was of interest to study the effect of proteolytic enzymes on this protein. The main goal of this study was comparison of peptic digestion between two members of the firefly luciferase family. The main finding of this study was that P. pyralis luciferase is more stable than L. turkestanicus luciferase against proteolysis. As shown in Figs. 1 and 2, L. turkestanicus luciferase is fully digested in few minutes, while P. pyralis luciferase requires up to hours for its complete digestion, in particular in the presence of trypsin. The conformational properties of P. pyralis and L. turkestanicus luciferase have been compared by a variety of biochemical and biophysical techniques, such as circular dichroism (CD), fluorescence emission, and binding of hydrophobic dyes, which shows subtle differences (29). Therefore, the relatively lower resistance of L. turkestanicus luciferase to proteolysis derives from the fact that the decline in the protein structure and enhanced segmental mobility of the luciferase structural building blocks is the key feature to acceleration of proteolytic attack. That is to say, as the sequences of the two luciferases are slightly different and even slight variation in sequence can lead to different building blocks and variation in conformation, thereby changing the sensitivity against proteases (37). Moreover, some building

1166 Farangis Ataei et al. blocks have high degree of similarity and structural conservation. These elements are critically important for the protein function and coupling folding (38). Two proteases with different specificities are used in this study to create such conditions where the sensitivity of the cleavage is not, or limited by, the specificity of the enzyme. The grouping of the preferential cleavage sites within the same region of the protein regardless of the proteases used is strongly indicative of the consistency of the data and suggestive of the exposure of that particular region, in both luciferases (Figs. 1–4). It is worthwhile to underline the differences to hydrolytic attack by trypsin in these two luciferase species. In the case of P. pyralis luciferase, some fragments were found to be more resistant to further degradation, whereas in L. turkestanicus case, fragments released from the intact protein were subdigested by trypsin, giving rise to smaller peptides, like chymotrypsin effect on both luciferases. The building blocks are contiguous sequence fragments of variable sizes, and their stability derives from local interaction. Some building blocks are highly stable; whereas others may be only marginally stable (39). Apparently, the results obtained in this investigation may explain that in P. pyralis luciferase, the building blocks that were observed in the native state are those that would manifest as peptide fragments in solution containing trypsin, but this does not occur for L. turkestanicus luciferase. This suggestion derived from the fact that limited proteolysis occurs at flexible sites along the protein chain and that stable, autonomous folded fragments are more resistant to further proteolytic degradation than relatively unstructured fragments (22,40–43). Folding funnels of protein is another important conception, which can be considered to interpret the differences in proteolysis of luciferases. The shape of the folding funnel of the monomer enables making reasonable guesses regarding the shape of the corresponding binding funnel (44). Different sequences imply different folding funnels and preferred conformations which are very important for biological activity. On the other hand, since proteases prefer cutting in flexible regions, and these are normally in surface loops, a slight change in primary structure and preferred conformation will affect the precise location of these and their populations. Energy landscapes of proteins have been depicted as a hill, corresponding to a high energy conformation and valleys with more favorable conformations (45). Therefore, it may be assumed that the ensemble in solution will contain similar conformations since these are homologous proteins; the relative populations of certain conformers in the ensemble due to energy landscape of protein are varied. Alteration in protein sequence like mutations can stabilize or destabilize folding intermediates. Hence, in effect, mutations or sequence differences are able to change the energy landscape in a manner similar to environmental conditions such as pH, ionic strength, temperature, the presence of denaturant or pressure changes (46). Therefore, it may be concluded differences in primary structure of P. pyralis and L. turkestanicus luciferases are responsible for changes in building blocks and folding funnel of both luciferases, and can thereby shift the conformational ensemble of each luciferase in solution and proteolytic sensitivity. Moreover, two pathways for degradation of the compact, globular structure of folded proteins and its relation to protein stability have been proposed (47). Accordingly, it seems, in

P. pyralis luciferase the initial large fragments (building blocks) are stable and remained resistant to sub digestion, whereas L. turkestanicus luciferase is looked more labile to proteolysis and further degradation. Another part of our data supports the higher rate of hydrolysis by chymotrypsin (Figs. 3 and 4). Also, initial fragments which were generated at early times lose their stability and general degradation takes place. The present results from digestion experiments can be explained by the fact that hydrophobic regions of firefly luciferases are more accessible to chymotrypsin, compared to eligible site for trypsin hydrolysis. Moreover, bioinformatics comparison of the putative peptide cleavage sites of both luciferases using peptide cutter software, confirmed more chymotrypsin cleavage sites (hydrophobic residues) compared to trypsin cleavage sites (data not shown). Proteolysis of luciferase by trypsin and chymotrypsin has been conducted at 23C and 37C which show faster cleavage at 37C (Fig. 5). It may be suggested at 23C, the equilibrium between native and unfolded states dictates the rate of proteolysis, and thus, the proteolytic probe can discriminate between the native form (more resistant to proteolysis) and the unfolded species which is easily digested. On the other hand, at 37C the population of the unfolded state is much enhanced and becomes the suitable substrate for limited proteolysis. In fact, higher temperatures similar to mutation change the energy landscape and make conformation more prone to proteolysis (46). In agreement with these results, a comparison between kinetics of proteolysis clearly interprets cleavage observations (Figs. 6 and 7). The rate of loss of activity and the rate of degradation were similar, in a specific period of time. In spite of these apparent differences between the luciferases, it is clear that both luciferases have protease-lability in the same regions. This point is important as flexibility of substrate protein is the main determinant factor in proteolytic cleavage (40–42,48). The importance of the current study is due to application of firefly luciferase in bioluminescence imaging and a central component of recent studies in nanosystems biology (28,49). System biology is an approach in which the digital information of the genome, acted upon by environmental cues, generates the many molecular signatures of gene and protein expression, as well as other, more phenomenological experimental observations. In conclusion, the result presented in this manuscript indicates, in spite of variation in primary structure of firefly luciferases and differences in proteolysis rates, the general structural feature of firefly luciferase have been conserved. Acknowledgement—Financial support of this work was provided by Research Council of Tarbiat Modares University.

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