Evidence for an initiation site for hen lysozyme folding from the reduced form using its dissected peptide fragments

We prepared two dissected fragments of hen lysozyme and examinedwhether or not these two fragments associated to form a native-likestructure. One (Fragment I) is the peptide fragment Asn59–homoserine-105containing Cys64–Cys80 and Cys76–Cys94. The other(Fragment II) is the peptide fragment Lys1–homoserine-58connected by two disulfide bridges, Cys6–Cys127 and Cys30–Cys115,to the peptide fragment Asn106–Leu129. It was found thatthe Fragment I immobilized in the cuvette formed an equimolarcomplex with Fragment II (K_d = 3.3x10^–4 M at pH 8 and25°C) by means of surface plasmon resonance. Moreover, fromanalyses by circular dichroism spectroscopy and ion-exchangechromatography of the mixture of Fragments I and II at pH 8under non-reducing conditions, it was suggested that these fragmentsassociated to give the native-like structure. However, the mutantFragment I in which Cys64–Cys80 and Cys76–Cys94are lacking owing to the mutation of Cys to Ala, or the mutantfragment in which Trp62 is mutated to Gly, did not form thenative-like species with Fragment II, because the mutant FragmentI derived from mutant lysozymes had no local conformation dueto mutations. Considering our previous results where the preferentialoxidation of two inside disulfide bonds, Cys64–Cys80 andCys76–Cys94, occurred in the refolding of the fully reducedFragment I, we suggest that the peptide region correspondingto Fragment I is an initiation site for hen lysozyme folding.     

Increased thermal stability against irreversible inactivation of 3-isopropylmalate dehydrogenase induced by decreased van der Waals volume at the subunit interface

We have investigated factors affecting stability at the subunit–subunitinterface of the dimeric enzyme 3-isopropylmalate dehydrogenase(IPMDH) from Bacillus subtilis. Site-directed mutagenesis wasused to replace methionine 256, a key residue in the subunitinteraction, with other amino acids. Thermal stability againstirreversible inactivation of the mutated enzymes was examinedby analyzing the residual activity after heat treatment. Themutations M256V and M256A increased thermostability by 2.0 and6.0°C, respectively, whereas the mutations M256L and M256Ihad no effect. Thermostability of the M256F mutated enzyme was4.0°C lower than that of the wild-type enzyme. To our surprise,increasing the hydrophobicity of residue 256 within the hydrophobiccore of the enzyme resulted in a lower thermal stability. Themutated enzymes showed an inverse correlation between thermostabilityand the volume of the side chain at position 256. Based on theX-ray crystallographic structure of Escherichia coli IPMDH,the environment around M256 in the B.subtilis homolog is predictedto be sterically crowded. These results suggest that Met256prevents favorable packing. Introduction of a smaller aminoacid at position 256 improves the packing and stabilizes thedimeric structure of IPMDH. The van der Waals volume of theamino acid residue at the hydrophobic subunit interface is animportant factor for maintaining the stability of the subunit–subunitinterface and is not always optimized in the mesophilic IPMDHenzyme. Received September 3, 2002; revised June 13, 2003; accepted June 20, 2003.