Triple point mutation Asp10 -> His, Asn101 -> Asp, Argl48 -> Ser in T4 phage lysozyme leads to the molten globule

The triple amino acid replacement (Asp10 His, Asn101 Asp,Arg148 Ser) in T4 phage lysozyme was carried out by site-directedmutagenesis. At acid pH (2.7) the mutant is in a confonnationalstate with the properties of the molten globule: (i) the mutantprotein molecule is essentially compact; (ii) its CD spectrumin the near UV region is drastically reduced in intensity ascompared with the wild type protein spectrum; (iii) the CD spectrumin the far UV region indicates the presence of pronounced secondarystructure in the mutant; (iv) unlike the wild type protein themutant protein can bind the hydrophobic fluorescent probe, ANS.