The Role of Protein-Mineral Interactions for Protein Fragmentation.

The rate-limiting step in decomposition of soil organic matter (SOM) is depolymerization by extracellular enzymes. A major factor determining the activity of extracellular enzymes is their affinity to mineral surfaces. Our goal was (1) to establish the molecular range of modifications to protein integrity and protein structure as such protein becomes associated with mineral surfaces and (2) to determine what mechanisms of protein fragmentation and disintegration, such as oxidation and hydrolysis, occur depending on mineral surface type and on the conditions in the solution. We hypothesized that (1) the pedogenic oxides would generate more extensive alterations to the protein structure than phyllosilicates and (2) the mechanism of alteration by the pedogenic oxides would be oxidation of the polypeptide bonds. A well-characterized protein proxy (Gb1, IEP 4.0, 6.2 kDA) was adsorbed onto a variety of functionally different mineral surfaces (goethite, montmorillonite, kaolinite and birnesite) at pH 5 and pH 7. These systems were subsequently extracted using a recently developed protocol for metaproteomics analysis (alkaline SDS-TCA). We used two-dimensional Heteronuclear Single Quantum Coherence Nuclear Magnetic Resonance Spectroscopy (HSQC NMR) to observe structural modifications in the unadsorbed protein that was allowed to equilibrate during the adsorption process. Mass spectrometry was used in the unadsorbed and extracted proteins to observe any more pronounced fragmentation that could result in either hydrolytic or oxidative cleavage of Gb1. Preliminary results in the HSQC NMR spectra indicate that the pedogenic oxide Birnessite shows increasing structural modification of Gb1 with decreasing pH. No structural modifications from Gb1Ős native conformation were observed in kaolinite and goethite treatments. Our results suggest that not all mineral surfaces in soil act as sorption sites for extracellular enzymes. Decrease in extracellular enzyme activity may not be an indication of structural modification but of fragmentation of the protein itself.