BMB reports 102 BMB reports http://bmbreports.org *Corresponding author. Tel: +989123840600; Fax: +982188007598; E-mail: ranjbarb@modares.ac.ir, saman_h@modares.ac.ir DOI 10.5483/BMBRep.2011.44.2.102 Received 4 October 2010, Accepted 14 December 2010 Keywords: Aggregation, Kinetics, Luciferase, Refolding, Thermo- dynamic stability The effect of surface charge balance on thermodynamic stability and kinetics of refolding of firefly luciferase Khosrow Khalifeh, Bijan Ranjbar * , Bagher Said Alipour & Saman Hosseinkhani * Department of Biophysics & Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, P.O. Box, 14115-175 Tehran, Iran Thermodynamic stability and refolding kinetics of firefly luci- ferase and three representative mutants with depletion of neg- ative charge on a flexible loop via substitution of Glu by Arg (ER mutant) or Lys (EK mutant) as well as insertion of another Arg in ER mutants (ERR mutant) was investigated. According to thermodynamic studies, structural stability of ERR and ER mu- tants are enhanced compared to WT protein, whereas, these mutants become prone to aggregation at higher temperatures. Accordingly, it was concluded that enhanced structural stabil- ity of mutants depends on more compactness of folded state, whereas aggregation at higher temperatures in mutants is due to weakening of intermolecular repulsive electrostatic inter- actions and increase of intermolecular hydrophobic interac- tions. Kinetic results indicate that early events of protein fold- ing are accelerated in mutants. [BMB reports 2011; 44(2): 102-106] INTRODUCTION It has been suggested that electrostatic interactions and surface charges play critical role in the folding kinetics and equili- brium stability of proteins (1-3). Garcia-Mira et al. (4) had per- formed comparative folding experiments with protein en- gineering strategies and found that long-range columbic inter- actions are important for organizing and stabilizing native-like structure in the early stage of protein folding. Moreover, it was shown that surface charges have important effects on the sta- bility of a variant of protein G B1 domain and such effects are not eliminated by salt screening (5). However, there are suggestions on destabilizing effects of charged and polar groups (6, 7); whereas some results indicate that surface charge-charge interactions are not essential for protein folding and stability (8). Luciferases are enzymes in bioluminescence organisms that produce light from conversion of chemical energy into an ex- cited electronic state which results in emission of photon in the visible region. The bioluminescence color of fireflies varies from green to red. It has been shown that only the railroad worm luciferase Phrixothrix hirtus can naturally emit red light. In order to elucidate the mechanisms of the bioluminescence color change, extensive studies were carried out and several mechanisms have been proposed (9, 10). Multiple sequence alignment of primary structure of Phrixothrix hirtus showed a missed Arg 353 in a flexible loop in green emitter firefly lu- ciferases. Accordingly, site directed mutagenesis technique were used for insertion of Arg in the corresponding position in the loop of firefly luciferase Lampyris turkestanicus (11) and it was shown that Arg 353 plays a critical role in the biolu- minescence color (12). A similar study on the North American firefly luciferase Photinus pyralis indicated that the presence of positive charges on the same loop has significant effects on the emitted light shift from green to red (13). Here, we investigated the refolding kinetics and thermody- namic stability of native and mutants of firefly luciferase (Lampyris turkestanicus, Accession No. AAU85360); where the surface charges of strands connecting loop were modified using site directed mutagenesis. It was shown that positive charge saturation on this flexible loop results in the change of color emission in firefly luciferase (14). RESULTS AND DISCUSSION In order to elucidate the relationships between structural prop- erties and color emission of bioluminescence, thermodynamic stability and kinetics of refolding of firefly luciferase and its mutants were investigated. The structures of WT and mutant luciferases are modeled with Swiss Model Program (15-17), which shows high struc- tural homology with Photinus pyralis firefly luciferase (18). Supplementary data 1 shows a part of protein containing β-strands connecting loop as well as a brief description of its sequence and mutations. It can be seen that a residue with negatively-R group (Glu) has been replaced by positively-R group (Arg and Lys) near two successive Asp to give ER, ERR and EK mutants. This loop is located between the 15 th and 16 th β-strands at N-terminal domain. Fig. 1 shows the normalized urea denaturation curves for