Structural analysis of phospholipase A(2) from functional perspective. 2. Characterization of a molten globule-like state induced by site-specific mutagenesis

Previous NMR studies have shown that many phospholipase A(2) (PLA2, from bo vine pancreas, overexpressed in Escherichia coli) mutants display some prop erties reminiscent of a molten globule state. Further NMR analyses for some of the mutants indicated that formation of the "molten globule-like state" is a pH-dependent phenomenon. The mutants I9Y and I9F showed perturbed NMR properties throughout the pH range studied, while the mutants H48A and C44 A/C105A displayed native-like spectra at neutral pH but molten globule-like ones under acidic conditions, with a "transition pH" around 4. On the othe r hand, wild-type PLA2 exhibits exceptional pH stability and turns into a s imilar molten globule-like state only under highly acidic conditions such a s 1 M HCl. The H48A mutant was used to rigorously establish the property of the molten globule-like state of PLA2 mutants. The results of far-UV CD, n ear-UV CD, and ANS-binding fluorescence suggest that H48A retains native-li ke secondary structures but loses tertiary structure during the conformatio nal transition. However, the tertiary structure is not completely lost, as evidenced by the retention of some long-range NOEs in two-dimensional NOESY spectra. The conclusion was further substantiated by three-dimensional NOE SY-HSQC experiments on a N-15-labeled H48A sample. It was revealed that the molten globule-like state at mildly acidic pH retained some rigid tertiary structure, which consisted of partial alpha-helix II (Y52-L58), alpha-heli x III (D59-V63), beta-wing (S74-S85) and partial alpha-helix TV (A90-N97). These residual tertiary structures grouped in half of the protein could be attributed to stabilization by some of the disulfide bonds. The extreme sen sitivity of the PLA2 structure to site-directed mutagenesis is unprecedente d. It is interesting to note that most of the functional residues (the acti ve site, the hydrophobic channel, the interfacial binding site, and the cal cium-binding loop) are located in the remainder of the protein, which is we ll disrupted in tertiary interactions.