It was revealed from the crystal structure analysis of S-ovalbumin (S-OVA) formed by alkaline treatment that Ser164, Ser236, and Ser320 take the D-amino acid residue configuration (Yamasaki et al., J Biol Chem 2003; 278:35524-35530). To address the implications of a D-configuration for these Ser residues in S-OVA formation, three mutant OVAs (S164A, S236A, and S320A) were generated to compare their thermostabilities before and after alkaline treatment. Following alkaline treatment, S236A showed a marked increase in melting temperature similar to the wild type (DeltaT(m), +9 degrees C) which corresponded to the formation of S-OVA, whereas the increment in T(m) for both S164A and S320A was only 4.5 degrees C. Furthermore, the T(m) value of the double mutant S164/320A remained unchanged after alkaline treatment, supporting the relevance of Ser164 and Ser320 for thermostabilization of OVA. As Arg142 was predicted to interact with D-Ser164 upon S-OVA formation, it was substituted to Ala to generate R142A. The resulting increment in T(m) of mutant R142A after alkaline treatment was 5.8 degrees C. The double mutant R142/S320A was therefore prepared to eliminate the participation of Ser320 in thermostabilization, and its T(m) value was compared before and after alkaline treatment. As expected, the increase in T(m) for the double mutant was only 1.2 degrees C. Taken together, the data suggest that D-configuration of Ser164 caused by alkaline treatment favors interaction with Arg142 through conformational changes of the side chain. These results strongly supported the participation of the configurational inversion of both Ser164 and Ser320 residues in the formation of S-OVA. Study holds ProTherm entries: 25617, 25618, 25619, 25620, 25621, 25622, 25623, 25624, 25625, 25626, 25627, 25628, 25629, 25630 Extra Details: ovalbumin; S-ovalbumin; D-amino acid configuration; alkaline treatment
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:55 p.m.
|Number of data points||26|
|Proteins||Ovalbumin ; Ovalbumin|
|Assays/Quantities/Protocols||Experimental Assay: Tm prot_conc:1.0 mg/mL, buffers:Glycine-NaOH: 0.1 M, details:Additives 0.02% sodium azide, pH:10.0 ; Experimental Assay: Tm buffers:Phosphate: 10 mM, details:Additives , prot_conc:0.25 mg/mL, pH:7.0 ; Derived Quantity: dTm prot_conc:1.0 mg/mL, buffers:Glycine-NaOH: 0.1 M, details:Additives 0.02% sodium azide, pH:10.0 ; Derived Quantity: dTm buffers:Phosphate: 10 mM, details:Additives , prot_conc:0.25 mg/mL, pH:7.0|
|Libraries||Mutations for sequence GSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSP|