Folding, stability, and physical properties of the alpha subunit of bacterial luciferase

Bacterial luciferase is a heterodimeric (alphabeta) enzyme composed of homologous subunits. When the Vibrio harveyi luxA gene is expressed in Escherichia coli, the alpha subunit accumulates to high levels. The alpha subunit has a well-defined near-UV circular dichroism spectrum and a higher intrinsic fluorescence than the heterodimer, demonstrating fluorescence quenching in the enzyme which is reduced in the free subunit [Sinclair, J. F., Waddle, J. J., Waddill, W. F., and Baldwin, T. O. (1993) Biochemistry 32, 5036-5044]. Analytical ultracentrifugation of the alpha subunit has revealed a reversible monomer to dimer equilibrium with a dissociation constant of 14.9 +/- 4.0 microM at 18 degrees C in 50 mM phosphate and 100 mM NaCl, pH 7.0. The alpha subunit unfolded and refolded reversibly in urea-containing buffers by a three-state mechanism. The first transition occurred over the range of 0-2 M urea with an associated free-energy change of 2.24 +/- 0.25 kcal/mol at 18 degrees C in 50 mM phosphate buffer, pH 7.0. The second, occurring between 2.5 and 3.5 M urea, comprised a cooperative transition with a free-energy change of 6.50 +/- 0.75 kcal/mol. The intermediate species, populated maximally at ca. 2 M urea, has defined near-UV circular dichroism spectral properties distinct from either the native or the denatured states. The intrinsic fluorescence of the intermediate suggested that, although the quantum yield had decreased, the tryptophanyl residues remained largely buried. The far-UV circular dichroism spectrum of the intermediate indicated that it had lost ca. 40% of its native secondary structure. N-Terminal sequencing of the products of limited proteolysis of the intermediate showed that the C-terminal region of the alpha subunit became protease labile over the urea concentration range at which the intermediate was maximally populated. These observations have led us to propose an unfolding model in which the first transition is the unfolding of a C-terminal subdomain and the second transition represents the unfolding of a more stable N-terminal subdomain. Comparison of the structural properties of the unfolding intermediate using spectroscopic probes and limited proteolysis of the alpha subunit with those of the alphabeta heterodimer suggested that the unfolding pathway of the alpha subunit is the same, whether it is in the form of the free subunit or in the heterodimer.     

Polypeptide folding and dimerization in bacterial luciferase occur by a concerted mechanism in vivo

Bacterial luciferase is a heterodimeric enzyme comprising two nonidentical but homologous subunits, alpha and beta, encoded by adjacent genes, luxA and luxB. The two genes from Vibrio harveyi were separated and expressed from separate plasmids in Escherichia coli. If both plasmids were present within the same E. coli cell, the level of accumulation of active dimeric luciferase was not dramatically less than within cells containing the intact luxAB sequences. Cells carrying the individual plasmids accumulated large amounts of individual subunits, as evidenced by two-dimensional polyacrylamide gel electrophoresis. Mixing of a lysate of cells carrying the luxA gene with a lysate of cells carrying the luxB gene resulted in formation of very low levels of active heterodimeric luciferase. However, denaturation of the mixed lysates with urea followed by renaturation resulted in formation of large amounts of active luciferase. These observations demonstrate that the two subunits, alpha and beta, if allowed to fold independently in vivo, fold into structures that do not interact to form active heterodimeric luciferase. The encounter complex formed between the two subunits must be an intermediate structure on the pathway to formation of active heterodimeric luciferase.     

Functional implications of the unstructured loop in the (beta/alpha)(8) barrel structure of the bacterial luciferase alpha subunit.

Bacterial luciferase catalyzes the conversion of FMNH(2), a long-chain aliphatic aldehyde, and molecular oxygen to FMN, the corresponding carboxylic acid, and H(2)O with the emission of light. The light-emitting species is an enzyme-bound excited state flavin. The enzyme is a heterodimer (alphabeta) of homologous subunits each with an (beta/alpha)(8) barrel structure. A portion of the loop in the alpha subunit that connects beta strand 7 to alpha helix 7 is disordered in the crystal structure. To test the hypothesis that this loop closes over the active site during catalysis and protects the active site from bulk solvent, a mutant was constructed in which the 29 residues that are disordered in the 2.4 A crystal structure were deleted. Deletion of this loop results in a heterodimer with a subunit equilibrium dissociation constant of 1.32 +/- 1.25 microM, whereas the wild-type heterodimer shows no measurable subunit dissociation. This mutant retains its ability to bind substrate flavin and aldehyde with wild-type affinity and can carry out the chemistry of the bioluminescence reaction with nearly wild-type efficiency. However, the bioluminescent quantum yield of the reaction is reduced nearly 2 orders of magnitude from that of the wild-type enzyme.     

Identifying the structural boundaries of independent folding domains in the alpha subunit of tryptophan synthase, a beta/alpha barrel protein.

Two equilibrium intermediates have previously been observed in the urea denaturation of the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, an eight-stranded beta/alpha barrel protein. In the current study, a series of amino-terminal fragments were characterized to probe the elementary folding units that may be in part responsible for this complex behavior. Stop-codon mutagenesis was used to produce eight fragments ranging in size from 105-214 residues and containing incremental elements of secondary structure. Equilibrium studies by circular dichroism indicate that all of these fragments are capable of adopting secondary structure. All except for the shortest fragment fold cooperatively. The addition of the fourth, sixth, and eighth beta-strands leads to distinct increases in structure, cooperativity, and/or stability, suggesting that folding involves the modular assembly of betaalphabeta supersecondary structural elements. One-dimensional NMR titrations at high concentrations of urea, probing the environment around His92, were also performed to test for the presence of residual structure in the fragments. All fragments that contained the first four betaalpha units of structure exhibited a cooperative unfolding transition at high concentrations of urea with significant but reduced stability relative to the full-length protein. These results suggest that the residual structure in alphaTS requires the participation of hydrophobic residues in multiple beta-strands that span the entire sequence.     

Mutational analysis of the subunit interface of Vibrio harveyi bacterial luciferase

Bacterial luciferase is a heterodimeric (alphabeta) enzyme which catalyzes a light-producing reaction in Vibrio harveyi. In addition to the alphabeta enzyme, the beta subunit can self-associate to form a stable but inactive homodimer [Sinclair, J. F., Ziegler, M. M., and Baldwin, T. O. (1994) Nat. Struct. Biol. 1, 320-326]. The studies reported here were undertaken to explore the role of the subunit interface in the conformational stability of the enzyme. To this end, we constructed four mutant heterodimers in which residues at the subunit interface were changed in an effort to alter the volume of an apparent solvent accessible channel at the interface or to alter H-bonding groups. Equilibrium unfolding data for the heterodimer have been interpreted in terms of a three-state mechanism [Clark, C. A., Sinclair, J. F., and Baldwin, T. O. (1993) J. Biol. Chem. 268, 10773-10779]. However, we found that unfolding for the wild-type and mutant luciferases is better described by a four-state model. This change in the proposed mechanism of unfolding is based on observation of residual structure in the subunits following dissociation of the heterodimeric intermediate. All of the mutants display modest reductions in activity but, surprisingly, no change in the DeltaG2H2O value for subunit dissociation and no measurable change in the equilibrium dissociation constant relative to that of the wild-type heterodimer. However, the DeltaG1H2O value for the formation of the dimeric intermediate that precedes subunit dissociation is reduced for three of the mutants, indicating that mutations at the interface can alter the stability of a region of the alpha subunit that is distant from the interface. We conclude that the interface region communicates with the distal domains of this subunit, probably through the active center region of the enzyme.     

Identification of two catalytic domains in a luciferase secreted by the copepod Gaussia princeps

Gaussia luciferase secreted by the copepod Gaussia princeps catalyzes the oxidation of coelenterazine to produce blue light. The primary structure of Gaussia luciferase deduced from the cDNA sequence shows two repeat sequences of 71 amino acid residues, suggesting the luciferase consists of two structural domains. Two domains in Gaussia luciferase were expressed independently in Escherichia coli cells, purified and characterized. We found that both domains have luminescence activity with coelenterazine, and the catalytic properties including luminescence spectrum, optimal pH, substrate specificity and luminescence stimulation by halogen ions (Cl⁻, Br⁻ and I⁻) are identical to intact Gaussia luciferase. Thus, Gaussia luciferase has two catalytic domains for the luminescence reaction.     

Spectral properties of Trp182, Trp194, and Trp250 on the alpha subunit of bacterial luciferase.

The phosphorescence and fluorescence properties of bacterial luciferase (alphabeta) mutants from Xenorhabdus luminescens were investigated. All tryptophans in the alpha and beta subunits were replaced with tyrosines except for one or two tryptophans in the alpha subunit. Because one luciferase mutant (W250) retained only a single tryptophan in the alpha subunit while two other mutants (W182/250 and W194/250) each contained two tryptophans in the alpha subunit, it was possible to deduce the spectral properties of these specific tryptophans (Trp182, Trp194, Trp250). Analyses of the phosphorescence properties were particularly revealing as only a single phosphorescence emission peak at 411-414 nm was observed for the W250 and W194/250 mutants while peaks at 409 and 414 nm could be clearly observed for the W182/250 mutant. Coupled with intrinsic fluorescence quenching experiments, these results show that alphaTrp182 is in a distinctly polar environment while alphaTrp250 is in a hydrophobic region and illustrate the advantages of using phosphorescence to recognize different microenvironments for tryptophan residues.     

Nucleotide sequence of the luxA gene of Vibrio harveyi and the complete amino acid sequence of the alpha subunit of bacterial luciferase

The nucleotide sequence of the 1.85-kilobase EcoRI fragment from Vibrio harveyi that was cloned using a mixed-sequence synthetic oligonucleotide probe (Cohn, D. H., Ogden, R. C., Abelson, J. N., Baldwin, T. O., Nealson, K. H., Simon, M. I., and Mileham, A. J. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 120-123) has been determined. The alpha subunit-coding region (luxA) was found to begin at base number 707 and end at base number 1771. The alpha subunit has a calculated molecular weight of 40,108 and comprises a total of 355 amino acid residues. There are 34 base pairs separating the start of the alpha subunit structural gene and a 669-base open reading frame extending from the proximal EcoRI site. At the 3' end of the luxA coding region there are 26 bases between the end of the structural gene and the start of the luxB structural gene. Approximately two-thirds of the alpha subunit was sequenced by protein chemical techniques. The amino acid sequence implied by the DNA sequence, with few exceptions, confirmed the chemically determined sequence. Regions of the alpha subunit thought to comprise the active center were found to reside in two discrete and relatively basic regions, one from around residues 100-115 and the second from around residues 280-295.     

Dynamic stabilization of nuclear receptor ligand binding domains by hormone or corepressor binding

We have developed a novel assembly assay to examine structural changes in the ligand binding domain (LBD) of the thyroid hormone receptor (TR). Fragments including the first helix of the TR LBD interact only weakly with the remainder of the LBD in the absence of hormone, but this interaction is strongly enhanced by the addition of either hormone or the corepressor NCoR. Since neither the ligand nor the corepressor shows direct interaction with this helix, we propose that both exert their effects by stabilizing the overall structure of the LBD. Current models of activation of nuclear hormone receptors focus on a ligand-induced allosteric shift in the position of the C-terminal helix 12 that generates the coactivator binding site. Our results suggest that ligand binding also has more global effects that dynamically alter the structure of the receptor LBD.     

Thermodynamic analysis of ligand binding and ligand binding-induced tertiary structure formation by the thiamine pyrophosphate riboswitch

The thi-box riboswitch regulates gene expression in response to the intracellular concentration of thiamine pyrophosphate (TPP) in archaea, bacteria, and eukarya. To complement previous biochemical, genetic, and structural studies of this phylogenetically widespread RNA domain, we have characterized its interaction with TPP by isothermal titration calorimetry. This shows that TPP binding is highly dependent on Mg²⁺ concentration. The dissociation constant decreases from ∼200 nM at 0.5 mM Mg²⁺ concentration to ∼9 nM at 2.5 mM Mg²⁺ concentration. Binding is enthalpically driven, but the unfavorable entropy of binding decreases as Mg²⁺ concentration rises, suggesting that divalent cations serve to pre-organize the RNA. Mutagenesis, biochemical analysis, and a new crystal structure of the riboswitch suggest that a critical element that participates in organizing the riboswitch structure is the tertiary interaction formed between the P3 and L5 regions. This tertiary contact is distant from the TPP binding site, but calorimetric analysis reveals that even subtle mutations in L5 can have readily detectable effects on TPP binding. The thermodynamic signatures of these mutations, namely decreased favorable enthalpy of binding and small effects on entropy of binding, are consistent with the P3–L5 association contributing allosterically to TPP-induced compaction of the RNA.     

Synergism in folding of a double mutant of the alpha subunit of tryptophan synthase

The urea-induced unfolding of the inactive single mutants Tyr-175----Cys and Gly-211----Glu and the active double mutant Cys-175/Glu-211 of the alpha subunit of tryptophan synthase from Escherichia coli was examined by using ultraviolet difference spectroscopy. Equilibrium techniques were used to determine the equilibrium free energies of unfolding for the mutant proteins to permit comparison with the wild-type protein. The sum of the changes in stability for the single mutants is not equal to the change seen in the double mutant. This inequality is evidence for a structural interaction between these two residues. Kinetic studies show that this synergism, which destabilizes the native form by 1.5-2.0 kcal/mol at pH 7.8, 25 degrees C, occurs only after the final rate-limiting step of domain association.     

Characterization of a slow folding reaction for the alpha subunit of tryptophan synthase

The equilibria and kinetics of urea-induced unfolding and refolding of the alpha subunit of tryptophan synthase of E. coli have been examined for their dependences on viscosity, pH, and temperature in order to investigate the properties of one of the rate-limiting steps, domain association. A viscosity enhancer, 0.58 M sucrose, was found to slow unfolding and accelerate refolding. This apparently anomalous result was shown to be due to the stabilizing effect of sucrose on the folding reaction. After accounting for this stabilization effect by using linear free-energy plots, the unfolding and refolding kinetics were found to have a viscosity dependence. A decrease in pH was found to stabilize the domain association reaction by increasing the refolding rate and decreasing the unfolding rate. This effect was accounted for by protonation of a single residue with a pK value of 8.8 in the native state and 7.1 in the intermediate, in which the two domains are not yet associated. The activation energy of unfolding is 4.8 kcal/mol, close to the diffusion limit. The negative activation entropy of unfolding, -47 cal/deg-mol, which controls this reaction, may result from ordering of solvent about the newly exposed domain interface of the transition state. These results may provide information on the types of noncovalent interactions involved in domain association and improve the ability to interpret the folding of mutants with single amino-acid substitutions at the interface.     

Characterization of an early intermediate in the folding of the alpha subunit of tryptophan synthase by hydrogen exchange measurement

The development of the hydrogen bonding network in the early stages of the folding of the alpha subunit of tryptophan synthase was monitored with a hydrogen exchange technique. The orders of magnitude difference between the rapid conversions of the unfolded forms to two stable intermediates (milliseconds) and the subsequent slow conversions of the intermediates to the native form (greater than 100 s) was used to selectively label with tritium the hydrogen bonds that form in the first 30 s of folding at 0 degree C. Rapid removal of the tritiated solvent by gel filtration ensured that hydrogen bonds formed in subsequent folding reactions would be unlabeled. Limited proteolysis and separation of peptides by high-pressure liquid chromatography permitted the determination of the amount of label retained in individual peptides by scintillation counting. Peptides 1-70 and 71-188, which when covalently linked comprise the stable amino domain in the native conformation, retain 91% and 93%, respectively, of the label retained when the protein is allowed to completely refold in tritiated solvent. Peptide 189-268, the marginally stable carboxyl domain, only retains 43% of the label. The striking difference in retention of label confirms the independent folding of these two domains and shows that the kinetic intermediates that appear in the folding of alpha subunit correspond to structural domains in the native conformation. The near-equality of the labeling of the two peptides comprising the amino domain shows that this domain folds as a single entity and that subdomain folding is unlikely.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermal stability of the hemagglutinin-neuraminidase from Sendai virus evidences two folding domains

The domain structure of hemagglutinin-neuraminidase from Sendai virus (cHN) was investigated by studying the thermal stability in the 20-100 degrees C range. Differential scanning calorimetry evidences two conformational transitions. The first transition is apparently a reversible two-state process, with Tm 48.3 degrees C, and is shifted to 50.1 degrees C in the presence of the substrate analogue 2,3-dehydro-2-deoxy-N-acetyl neuraminic acid, meaning that the substrate binding domain is involved in the transition. The second transition, with apparent Tm 53.2 degrees C, is accompanied by irreversible loss of enzymatic activity of the protein, and the presence of the substrate analogue does not affect the Tm. The data indicate that cHN is composed of two independent folding domains, and that only one domain is involved in the binding of the substrate. Our results suggest that the paramyxovirus neuraminidases have the folding properties of a two-domain protein.     

Role of diffusion in the folding of the alpha subunit of tryptophan synthase from Escherichia coli

The rate-limiting step in the folding of the alpha subunit of tryptophan synthase has been proposed to be the association of two folding units. To probe the role of diffusion in this rate-limiting step, the urea-induced unfolding and refolding of the protein was examined in the presence of a number of viscosity-enhancing agents. The analysis was simplified by studying the effect of these agents on folding unit dissociation, the rate-limiting unfolding reaction, and the reverse of the rate-limiting step in refolding. In the presence of ethylene glycol, the relaxation times for unfolding to the same final conditions increased with increasing concentration of the cosolvent. When the effects of the cosolvent on protein stability were taken into account, the rates were found to show a unitary linear dependence on the viscosity of the solution. Similar results were obtained with glycerol and low concentrations of glucose, demonstrating that the effect is general and not specific to any viscogenic agent. These results clearly demonstrate that the rate-limiting folding unit association/dissociation reaction in the alpha subunit of tryptophan synthase involves a diffusional process.     

Structural requirements for the stabilization of metarhodopsin II by the C terminus of the alpha subunit of transducin

The retinal receptor rhodopsin undergoes a conformational change upon light excitation to form metarhodopsin II (Meta II), which allows interaction and activation of its cognate G protein, transducin (G(t)). A C-terminal 11-amino acid peptide from transducin, G(talpha)-(340-350), has been shown to both bind and stabilize the Meta II conformation, mimicking heterotrimeric G(t). Using a combinatorial library we identified analogs of G(talpha)-(340-350) that bound light-activated rhodopsin with high affinity (Martin, E. L., Rens-Domiano, S., Schatz, P. J., and Hamm, H. E. (1996) J. Biol. Chem. 271, 361-366). We have made peptides with key substitutions either on the background of the native G(talpha)-(340-350) sequence or on the high affinity sequences and used the stabilization of Meta II as a tool to determine which amino acids are critical in G protein-rhodopsin interaction. Removal of the positive charge at the N termini by acylation or delocalization of the charge by K to R substitution enhances the affinity of the G(talpha)-(340-350) peptides for Meta II, whereas a decrease was observed following C-terminal amidation. Cys-347, a residue conserved in pertussis toxin-sensitive G proteins, was shown to interact with a hydrophobic site in Meta II. These studies provide further insight into the mechanism of interaction between the G(talpha) C terminus and light-activated rhodopsin.     

Evidence for the generation of myristylated FMN by bacterial luciferase

The genes responsible for the light production in bioluminescent bacteria are present as an operon, luxCDABEG. Many strains of Photobacteria carry an additional gene, termed luxF. X‐ray crystallographic analysis of LuxF revealed the presence of four molecules of a flavin derivative, i.e. 6‐(3′‐(R)‐myristyl) flavin adenine mononucleotide (myrFMN) non‐covalently bound to the homodimer. In the present study, we exploited the binding of myrFMN to recombinant apo‐LuxF to explore the occurrence of myrFMN in various bioluminescent bacteria. MyrFMN was detected in all bacterial strains tested including Vibrio and Aliivibrio indicating that it is more widely occurring in bioluminescent bacteria than previously assumed. We also show that apo‐LuxF captures myrFMN and thereby relieves the inhibitory effect on luciferase activity. Thus our results provide support for the hypothesis that LuxF acts as a scavenger of myrFMN in bioluminescent bacteria. However, the source of myrFMN remained obscure. To address this issue, we established a cofactor regeneration enzyme‐catalyzed cascade reaction that supports luciferase activity in vitro for up to 3 days. This approach enabled us to unambiguously demonstrate that myrFMN is generated in the bacterial bioluminescent reaction. Based on this finding we postulate a reaction mechanism for myrFMN generation that is based on the luciferase reaction.     

细菌荧光素酶体外发光体系的建立及应用于NADH定量检测

摘要：【目的】本研究旨在建立鳆发光杆菌（Photobacterium leiognathi）YL 荧光素酶：FMN-NADH氧化还原酶体外发光双酶体系,并对荧光素酶：FMN-NADH氧化还原酶体外发光双酶体系应用于NADH的定量检测进行初步探索。【方法】利用从鳆发光杆菌提取并经部分纯化的荧光素酶和FMN-NADH氧化还原酶,通过优化体系中各底物的添加量,实现荧光素酶的体外发光。【结果】荧光素酶：FMN-NADH氧化还原酶体外发光双酶体系为：1 mL酶液中添加100 μL十二烷醛（27 mmol/L）、0.5 μL FMN-Na（10 mmol/L）、300 μL NADH（0.14 mmol/L）。NADH与荧光素酶：FMN-NADH氧化还原酶体系的发光强度呈良好的线性关系,其线性范围为1.0×10-10 ～1.0×10-8 mol/L。【结论】荧光素酶：FMN-NADH氧化还原酶体外发光双酶体系可以简便、灵敏、快速的定量检测NADH,为其进一步应用于环境检测、食品卫生与安全等领域活细菌数量的检测奠定了基础。