Role of His101 in the Protein Folding/Unfolding of a Goose-Type Lysozyme from Ostrich (Struthio camelus) Egg White.
To understand the role of His101 in protein structure stabilization of goose-type (G-type) lysozyme, we conducted thermal unfolding/refolding experiments using native G-type lysozyme from ostrich egg white (nOEL), the recombinant G-type lysozyme (rOEL), and the mutant lysozyme, in which His101 is mutated to alanine (H101A-OEL). Thermal stability on lytic activity and in-gel refolding experiments provided similar profiles for all three OELs. Circular dichroism (CD) spectroscopy was used to determine the secondary structure of three OELs as a function of temperature. Unfolding/refolding experiments (30-90 °C) monitored by CD spectroscopy revealed an unfolding transition at 65-67 °C and a complete refolding at almost the same temperature. Notably, a slightly lower thermal stability was observed for H101A-OEL, corresponding to the calculated difference in transition free energy of thermal unfolding (∆∆Gm) between rOEL and H101A-OEL of -0.63 kcal/mol. To assess the effects of H101A mutation on the electrostatic behavior, we examined the pH-activity profile of the three OELs. nOEL and rOEL exhibit bimodal relationship between pH and lytic activity showing optima at pH 3.0 and 7.0, while optima for H101A-OEL activity were pH 4.0 and 6.0. Electrostatic environment surrounding His101 was affected by the H101A mutation resulting in the slightly lower thermal stability.
Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand.|Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen, Thailand.
Chalermchai
C
Somboonpatarakun
Department of Advanced Bioscience, Kindai University, 3327-204 Nakamachi, Nara, 631-8505, Japan.
Tamo
T
Fukamizo
Department of Bioscience, School of Agriculture, Tokai University, Aso, Kumamoto, 869-1404, Japan.
Tomohiro
T
Araki
Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand. somkly@kku.ac.th.|Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen, Thailand. somkly@kku.ac.th.
Sompong
S
Klaynongsruang
D000818:Animals; D030161:Avian Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004529:Egg White / Q000737:chemistry*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D020310:Struthioniformes; D013816:Thermodynamics
D030161:Avian Proteins; D011994:Recombinant Proteins; D006639:Histidine; D009113:Muramidase
Goose-type lysozyme; Histidine residue; Protein folding/unfolding; Thermal unfolding; pH-activity relationship