Wheat germ lipase is a cereal lipase which is a monomeric protein. In the present study we sought to structurally characterize this protein along with equilibrium unfolding in solution. Conformational changes occurring in the protein with varying pH, were monitored by circular dichroism (CD) spectroscopy, fluorescence emission spectroscopy, binding of hydrophobic dye, 1-anilino 8-naphthalenesulfonic acid (ANS) and dynamic light scattering (DLS). Our study showed that acid denaturation of lipase lead to characterization of multiple monomeric intermediates. Native protein at pH 7.0 showed far-UV spectrum indicating mixed structure with both alpha and beta-type of characteristics. Activity of lipase was found to fall on either sides of pH 7.0-8.0. Acid-unfolded state was characterized at pH 4.0 with residual secondary structure, disrupted tertiary spectrum and red-shifted fluorescence spectrum with decreased intensity. Further decrease in pH lead to formation of secondary structure and acid-induced molten globule state was found to be stabilized at pH 1.4, with exposed tryptophan residues and hydrophobic patches. Notably, interesting finding of this study was characterization of acid-induced state at pH 0.8 with higher secondary structure content than native lipase, regain in tertiary spectrum and induction of compact conformation. Although enzymatically inactive, acid-induced state at pH 0.8 was found to be structurally more stable than native lipase, as shown by chemical and thermal denaturation profiles. Study holds ProTherm entries: 25795, 25796, 25797, 25798, 25799, 25800 Extra Details: Acid-inducedstate; Enzyme activity; Molten globule pH-induced denaturation; Wheat germlipase
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:56 p.m.
|Number of data points||6|
|Assays/Quantities/Protocols||Experimental Assay: Tm buffers:KCl-HCl: 20 mM, pH:0.8 ; Experimental Assay: Tm buffers:Sodium phosphate: 20 mM, pH:7.0 ; Experimental Assay: Cm buffers:KCl-HCl: 20 mM, pH:0.8, prot_conc:0.1 mg/mL ; Experimental Assay: Cm prot_conc:0.2 mg/mL, buffers:KCl-HCl: 20 mM, pH:0.8 ; Experimental Assay: Cm pH:7.0, buffers:Sodium phosphate: 20 mM, prot_conc:0.1 mg/mL ; Experimental Assay: Cm prot_conc:0.2 mg/mL, buffers:Sodium phosphate: 20 mM, pH:7.0|
|Libraries||Mutations for sequence MRRFRLVGFLSSLVLAAGAALTGAATAQAAQPAAADGYVALGDSYSSGVGAGSYISSSGDCKRSTKAHPYLWAAAHSPSTFDFTACSGARTGDVLSGQLGPLSSGTGLVSISIGGNDAGFADTMTTCVLQSESSCLSRIATAEAYVDSTLPGKLDGVYSAISDKAPNAHVVVIGYPRFYKLGTTCIGLSETKRTAINKASDHLNTVLAQRAAAHGFTFGDVRTTFTGHELCSGSPWLHSVNWLNIGESYHPTAAGQSGGYLPVLNGAA|