Folding of bacterial luciferase involves a non-native heterodimeric intermediate in equilibrium with the native enzyme and the unfolded subunits.


Abstract

Bacterial luciferase is a heterodimeric enzyme that catalyzes the reaction of reduced FMN, O2 and an aliphatic aldehyde to yield the carboxylic acid and an excited flavin that emits blue-green light upon return to ground state. The two subunits of the luciferase from Vibrio harveyi, alpha and beta, have molecular weights of 40,108 and 36.349, respectively; the single active center resides primarily, if not exclusively, on the alpha subunit. We have found that bacterial luciferase can be unfolded in urea-containing 50 mM phosphate buffer, pH 7.0, and refolded by dilution of the urea with final luciferase concentrations of 5-25 micrograms/ml. We have analyzed the urea-induced equilibrium unfolding of bacterial luciferase by monitoring changes in both the far ultraviolet circular dichroism (predominantly secondary structure) and intrinsic fluorescence emission (predominantly tertiary structure) resulting from incubation in various concentrations of urea at 18 degrees C for 18-24 h. Both spectral methods indicated a biphasic unfolding transition; the first phase was protein concentration-independent, whereas the second phase was protein concentration-dependent. Equilibrium unfolding curves showed an increase in fluorescence up to 2 M urea followed by a decrease in intensity and red shift of the emission maximum. The ratio of the fluorescence emission in the presence of 2 M urea relative to that in the absence of urea was greater when fluorescence was excited at 295 nm than at 280 nm. The fluorescence increase in the 0-2 M urea range corresponded to the first phase of the biphasic unfolding process. The urea-induced loss of luciferase enzymatic activity appeared to correspond to the first transition observed with the spectroscopic methods, and likewise to be protein concentration-independent. These observations suggested a three-state unfolding mechanism in which the native heterodimeric enzyme rearranges to an inactive heterodimeric species that is well populated, followed by dissociation and unfolding of the alpha and beta subunits. The data were fit to a three-state mechanism using a nonlinear least squares method. At 18 degrees C in 50 mM phosphate, pH 7.0, the free energy change for the interconversion of the active heterodimer and the inactive heterodimeric species was estimated to be 4.52 +/- 0.30 kcal/mol; the free energy change for the interconversion of the inactive heterodimer and the individual subunits was 19.7 +/- 0.2 kcal/mol.(ABSTRACT TRUNCATED AT 400 WORDS) Study holds ProTherm entries: 5216, 5217 Extra Details: transition 1 biphasic unfolding transition; concentration-dependent;,enzymatic activity; three-state unfolding mechanism

Submission Details

ID: faWhLc7S4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:29 p.m.

Version: 1

Publication Details
Clark AC;Sinclair JF;Baldwin TO,J. Biol. Chem. (1993) Folding of bacterial luciferase involves a non-native heterodimeric intermediate in equilibrium with the native enzyme and the unfolded subunits. PMID:8496144
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1LUC 1996-12-07 1.5 BACTERIAL LUCIFERASE
1BSL 1997-11-12 1.95 STRUCTURE OF ALKANAL MONOOXYGENASE BETA CHAIN
3FGC 2009-05-26 2.3 Crystal Structure of the Bacterial Luciferase:Flavin Complex Reveals the Basis of Intersubunit Communication
1BRL 1996-04-01 2.4 THREE-DIMENSIONAL STRUCTURE OF BACTERIAL LUCIFERASE FROM VIBRIO HARVEYI AT 2.4 ANGSTROMS RESOLUTION
1XKJ 1997-07-07 2.5 BACTERIAL LUCIFERASE BETA2 HOMODIMER

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 B Alkanal monooxygenase alpha chain P07739 LUXB_VIBHA
100.0 A Alkanal monooxygenase alpha chain P07740 LUXA_VIBHA