Nilimesh Das, Tanmoy Khan, Soumya Chaudhury, Bhaswati Sengupta, Pratik Sen
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Dissecting the role of substrate folding in enzymatic digestion.
The efficiency of enzymatic proteolysis is often attributed to the properties of the enzyme itself, with the substrate typically viewed as a passive participant. In this study, we demonstrate that the conformational state of the substrate critically influences proteolytic efficiency. Using human serum albumin (HSA) as a model substrate, papain as the enzyme, and urea as a controlled denaturing agent, we systematically investigated how substrate conformation might affect proteolysis. While papain maintains its structural and functional integrity across varying urea concentrations, HSA transitions through well-defined conformational states (native, compact intermediate, and unfolded), allowing us an opportunity to isolate the effects of the substrate structure. Utilizing site-specific fluorescent labeling and single-molecule fluorescence correlation spectroscopy, we monitor the progression of proteolysis. Our results show that digestion slows at 3M urea, where HSA adopts a compact form, and accelerates at 6M, where HSA takes on an unfolded state, compared to native HSA. These results reveal that substrate folding critically influences the digestion kinetics, probably by controlling protease accessibility and underscoring its importance in mechanistic enzymology and proteomics workflows.
期刊介绍:
Biointerphases emphasizes quantitative characterization of biomaterials and biological interfaces. As an interdisciplinary journal, a strong foundation of chemistry, physics, biology, engineering, theory, and/or modelling is incorporated into originated articles, reviews, and opinionated essays. In addition to regular submissions, the journal regularly features In Focus sections, targeted on specific topics and edited by experts in the field. Biointerphases is an international journal with excellence in scientific peer-review. Biointerphases is indexed in PubMed and the Science Citation Index (Clarivate Analytics). Accepted papers appear online immediately after proof processing and are uploaded to key citation sources daily. The journal is based on a mixed subscription and open-access model: Typically, authors can publish without any page charges but if the authors wish to publish open access, they can do so for a modest fee.
Topics include:
bio-surface modification
nano-bio interface
protein-surface interactions
cell-surface interactions
in vivo and in vitro systems
biofilms / biofouling
biosensors / biodiagnostics
bio on a chip
coatings
interface spectroscopy
biotribology / biorheology
molecular recognition
ambient diagnostic methods
interface modelling
adhesion phenomena.