Anion exchanger 1 (AE1, Band 3) is the predominant membrane protein of erythrocytes. Its 52 kDa C-terminal domain functions as a chloride-bicarbonate exchanger, while its 43 kDa N-terminal cytosolic domain (cdb3) anchors the cytoskeleton to the membrane. Several proteins bind to cdb3, including protein 4.2, a cytoskeletal protein. Three mutations in cdb3 are associated with hereditary spherocytosis (HS) and decreased levels of protein 4.2 in erythrocytes. In this study, these cdb3 mutants (E40K, G130R, and P327R) were expressed in and purified from Escherichia coli. Sedimentation experiments showed that the wild-type and mutant proteins are dimers. No difference in secondary structure between mutant and wild-type proteins was detected using circular dichroism (CD) analysis. The wild-type and mutant proteins underwent similar pH-dependent conformational changes when monitored by intrinsic tryptophan fluorescence. Urea denaturation of proteins monitored by intrinsic fluorescence showed no significant differences in the sensitivity of the proteins to this chemical denaturant. Thermal denaturation monitored by CD and by calorimetry revealed that only the P327R mutant had a significantly lower midpoint of transition (approximately 5 degrees C) than the wild-type protein, suggesting a modest decrease in stability. The results show that the HS mutant cdb3 proteins do not differ to any great extent in structure from the wild-type protein, suggesting that the HS mutations may directly affect protein 4.2 binding. Study holds ProTherm entries: 19788, 19789, 19790, 19791, 19792, 19793, 19794, 19795, 19796, 19797, 19798, 19799, 19800, 19801, 19802 Extra Details: 1 mM DTT was added in the experiment membrane protein; secondary structure; conformational changes; binding
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:52 p.m.
|Number of data points||23|
|Proteins||Band 3 anion transport protein ; Band 3 anion transport protein|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: Tm prot_conc:1.3 mg/mL, pH:7.5, ionic:sodium chloride: 50 mM, details:Additives ; Experimental Assay: Tm ionic:sodium fluoride: 50 mM, prot_conc:0.3 mg/mL, details:Additives DTT (1 mM),, pH:7.0 ; Derived Quantity: dTm prot_conc:1.3 mg/mL, pH:7.5, ionic:sodium chloride: 50 mM, details:Additives ; Derived Quantity: dTm ionic:sodium fluoride: 50 mM, prot_conc:0.3 mg/mL, details:Additives DTT (1 mM),, pH:7.0|
|Libraries||Mutations for sequence MEELQDDYEDMMEENLEQEEYEDPDIPESQMEEPAAHDTEATATDYHTTSHPGTHKVYVELQELVMDEKNQELRWMEAARWVQLEENLGENGAWGRPHLSHLTFWSLLELRRVFTKGTVLLDLQETSLAGVANQLLDRFIFEDQIRPQDREELLRALLLKHSHAGELEALGGVKPAVLTRSGDPSQPLLPQHSSLETQLFCEQGDGGTEGHSPSGILEKIPPDSEATLVLVGRADFLEQPVLGFVRLQEAAELEAVELPVPIRFLFVLLGPEAPHIDYTQLGRAAATLMSERVFRIDAYMAQSRGELLHSLEGFLDCSLVLPPTDAPSEQALLSLVPVQRELLRRRYQSSPAKPDSSFYKGLDLNGGPDDPLQQTGQLF|