Succinylation-induced conformational destabilization of lysozyme as studied by guanidine hydrochloride denaturation.
Using 100-fold molar excess of succinic anhydride, about 99% of lysine residues of hen egg white lysozyme (HEWL) were modified. Succinylated (S(99)) HEWL showed both charge and size homogeneity as judged by PAGE and gel filtration, respectively. Hydrodynamic parameters such as Stokes radius and frictional ratio (f/f(o)) showed more expanded conformation of S(99) HEWL compared to native HEWL as evident from the increase in Stokes radius (from 1.36 to 1.86 nm) and f/f(o) (from 0.86 to 1.15) values. Guanidine hydrochloride (GdnHCl) denaturation studies using fluorescence spectroscopy connoted a marked decrease in conformational stability of HEWL upon succinylation. Complete denaturation of S(99) HEWL was achieved at lower GdnHCl concentration ( approximately 3.8 M) compared to native HEWL ( approximately 5 M). Furthermore, free energy of stabilization (DeltaG(D)(H(2)O)) value also showed a notable decrease from 8,559 and 7,956 cal/mol (for native HEWL) to 4,404 and 4,669 cal/mol (for succinylated HEWL) using excitation at 280 and 295 nm, respectively. Both expanded conformation and decreased DeltaG(D)(H(2)O) can be attributed to the increase in the net negative charge on the protein upon succinylation. All these results manifested the importance of positively charged lysine residues in maintaining the conformational stability of HEWL through electrostatic interactions.
Ong
Hui Nee
HN
Biomolecular Research Group, Biochemistry Programme, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Arumugam
Bavani
B
Tayyab
Saad
S
D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D004795:Enzyme Stability / Q000187:drug effects*; D019791:Guanidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D019802:Succinic Acid / Q000494:pharmacology*; D013816:Thermodynamics
D019802:Succinic Acid; C421864:hen egg lysozyme; D009113:Muramidase; D019791:Guanidine