Substrate accessibility of bovine milk proteins to a Pseudomonas metalloprotease reveals why caseins are more readily hydrolysed than whey proteins
- 1University College Cork
Abstract
Structure-aware proteolysis prediction often ranks candidate cleavage sites using solvent accessibility from static protein structures, yet this assumption is rarely tested against native-state cleavage data. We examined this problem using AprX, a serralysin-family protease that readily hydrolyses caseins but shows lower activity toward native whey proteins. Across six bovine milk proteins, sequence compatibility and local scissile-bond accessibility did not separate caseins from whey proteins. In contrast, accessibility fell progressively in whey proteins as the required contiguous substrate window increased, reaching 0% across the working P4-P4' footprint, while all evaluable casein windows remained accessible in isolated-chain ensembles. Local remodelling showed that whey segments could still adopt AprX-compatible backbone geometry. An external thermolysin benchmark rejected a rigid whole-protein clash metric, but did not alter the contiguous-accessibility result. These findings identify footprint-scale accessibility as a distinct structural variable and show why single-residue solvent exposure can overestimate protease accessibility in folded proteins.
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BibTeX
@misc{sinha2026substrate,
title = {{Substrate accessibility of bovine milk proteins to a Pseudomonas metalloprotease reveals why caseins are more readily hydrolysed than whey proteins}},
author = {A. Sinha and T. Racca},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.02.756230},
url = {https://www.biorxiv.org/content/10.64898/2026.10.02.756230v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Substrate accessibility of bovine milk proteins to a Pseudomonas metalloprotease reveals why caseins are more readily hydrolysed than whey proteins
AU - A. Sinha
AU - T. Racca
PY - 2026
DA - 2026/10/02
PB - bioRxiv
DO - 10.64898/2026.10.02.756230
UR - https://www.biorxiv.org/content/10.64898/2026.10.02.756230v1
AB - Structure-aware proteolysis prediction often ranks candidate cleavage sites using solvent accessibility from static protein structures, yet this assumption is rarely tested against native-state cleavage data. We examined this problem using AprX, a serralysin-family protease that readily hydrolyses caseins but shows lower activity toward native whey proteins. Across six bovine milk proteins, sequence compatibility and local scissile-bond accessibility did not separate caseins from whey proteins. In contrast, accessibility fell progressively in whey proteins as the required contiguous substrate window increased, reaching 0% across the working P4-P4' footprint, while all evaluable casein windows remained accessible in isolated-chain ensembles. Local remodelling showed that whey segments could still adopt AprX-compatible backbone geometry. An external thermolysin benchmark rejected a rigid whole-protein clash metric, but did not alter the contiguous-accessibility result. These findings identify footprint-scale accessibility as a distinct structural variable and show why single-residue solvent exposure can overestimate protease accessibility in folded proteins.
ER -