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Structural analysis of phospholipase A2 from functional perspective. 2. Characterization of a molten globule-like
1Department of Chemistry, Campus Chemical Instrument Center, The Ohio State University, Columbus 43210, USA.
Abstract:
Previous NMR studies have shown that many phospholipase A2 (PLA2, from bovine pancreas, overexpressed in Escherichia coli) mutants display some properties 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 C44A/C105A displayed native-like spectra at neutral pH but molten globule-like ones under acidic conditions, with a "transition pH" around 4. On the other hand, wild-type PLA2 exhibits exceptional pH stability and turns into a similar molten globule-like state only under highly acidic conditions such as 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, near-UV CD, and ANS-binding fluorescence suggest that H48A retains native-like secondary structures but loses tertiary structure during the conformational 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 NOESY-HSQC experiments on a 15N-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-helix III (D59-V63), beta-wing (S74-S85) and partial alpha-helix IV (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 sensitivity of the PLA2 structure to site-directed mutagenesis is unprecedented. It is interesting to note that most of the functional residues (the active site, the hydrophobic channel, the interfacial binding site, and the calcium-binding loop) are located in the remainder of the protein, which is well disrupted in tertiary interactions.
Insights
Site-directed mutagenesis of phospholipase A2 (PLA2) reveals pH-dependent molten globule states in mutants. Specific mutations like H48A show altered pH stability, retaining some tertiary structure under acidic conditions.
Area of Science:
- Biochemistry
- Structural Biology
- Protein Folding
Background:
- Previous Nuclear Magnetic Resonance (NMR) studies indicated that several phospholipase A2 (PLA2) mutants exhibit molten globule-like properties.
- The formation of this molten globule-like state was observed to be pH-dependent in some PLA2 mutants.
Purpose of the Study:
- To investigate the pH-dependent conformational transitions of PLA2 mutants.
- To characterize the structural properties of the molten globule-like state in PLA2 mutants, particularly the H48A mutant.
Main Methods:
- Nuclear Magnetic Resonance (NMR) spectroscopy (including 2D NOESY and 3D NOESY-HSQC) was employed to study PLA2 mutants.
- Circular Dichroism (CD) spectroscopy (far-UV and near-UV) and ANS-binding fluorescence were used to assess secondary and tertiary structure.
- Site-directed mutagenesis was used to generate specific PLA2 variants (e.g., I9Y, I9F, H48A, C44A/C105A).
Main Results:
- Mutants H48A and C44A/C105A showed native-like spectra at neutral pH but transitioned to molten globule-like spectra under acidic conditions (pH ~4).
- The H48A mutant retained native-like secondary structures but lost significant tertiary structure at acidic pH, confirmed by CD and fluorescence.
- NMR data revealed that the molten globule-like state of H48A retained some rigid tertiary structure, including specific alpha-helices and a beta-wing, stabilized by disulfide bonds.
Conclusions:
- The molten globule-like state in PLA2 mutants is a pH-dependent phenomenon, with some mutants exhibiting transitions at mildly acidic pH.
- The H48A mutant's molten globule state retains partial, stabilized tertiary structures, indicating incomplete unfolding.
- Site-directed mutagenesis profoundly impacts PLA2 structure, affecting stability and potentially functional sites located in less disrupted regions.