Characterization of Stabilization Conditions for PahZ2KT

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Gonzalez, Marriah
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Middle Tennessee State University
Poly(aspartic acid) (PAA) is a biodegradable water-soluble synthetic polypeptide often used as a replacement for environmentally persistent polycarboxylates used in a wide-range of industrial and biomedical applications.(1,2,3) PAA was found to be degraded by two bacterial strains isolated from river water, Sphingomonas sp. KT-1 and Pedobacter sp. KP-2.(1,3) Two enzymes purified from Sphingomonas sp. KT-1 are responsible for the hydrolytic degradation of PAA into smaller aspartic acid units.4 Poly(aspartic acid) hydrolase-1 (PahZ1KT-1) is an endopeptidase that cleaves β-amide linkages, peptide bonds between β-amino acids, while Poly(aspartic acid) hydrolase-2 (PahZ2KT-1) is an exopeptidase responsible for both α-amide, peptide bond between α-amino acids, and β-amide cleavage.2 Understanding conditions that stabilize PahZ2KT-1 may allow for protein engineering efforts that can enhance catalytic efficiency of tPAA degradation. It is reported here that with the introduction of NaCl, PahZ2KT-1 was seen to sample a larger variety of conformational states through size-exclusion chromatography and dynamic light scattering (DLS) experiments. Limited proteolysis and protein unfolding experiments visualized by fluorescence spectroscopy demonstrated that protein stability was independent of NaCl concentration. However, reactions containing Zn(II) were found, through limited proteolysis and unfolding experiments, to display increased stability. DLS also demonstrated an increased PahZ2KT-1 particle diameter from 9 ± 1 nm without the addition of zinc and 157 ± 13 nm with the addition of zinc. Lastly, binding affinities were determined by varying concentrations of zinc and produced a Kd within the nanomolar range.
M28 Metalloprotease, PahZ2KT-1, Poly(aspartic acid) (PAA), Sphingomonas sp. KT-1, Biophysics