Dissecting the role of substrate folding in enzymatic digestion.

IF 1.9 4区 医学 Q4 BIOPHYSICS
Biointerphases Pub Date : 2025-09-01 DOI:10.1116/6.0004803
Nilimesh Das, Tanmoy Khan, Soumya Chaudhury, Bhaswati Sengupta, Pratik Sen
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引用次数: 0

Abstract

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.

剖析底物折叠在酶消化中的作用。
酶解蛋白的效率通常归因于酶本身的性质,而底物通常被视为被动参与者。在这项研究中,我们证明了底物的构象状态严重影响蛋白水解效率。以人血清白蛋白(HSA)为模型底物,木瓜蛋白酶为酶,尿素为受控变性剂,我们系统地研究了底物构象如何影响蛋白水解。虽然木瓜蛋白酶在不同的尿素浓度下保持其结构和功能的完整性,但HSA通过明确的构象状态(天然,紧凑的中间和未折叠)转变,使我们有机会分离底物结构的影响。利用位点特异性荧光标记和单分子荧光相关光谱,我们监测蛋白质水解的进展。我们的研究结果表明,与天然HSA相比,在3M尿素时消化速度减慢,其中HSA采用致密形式,而在6M时消化速度加快,其中HSA呈现未折叠状态。这些结果表明,底物折叠可能通过控制蛋白酶的可及性来影响消化动力学,并强调其在机械酶学和蛋白质组学工作流程中的重要性。
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来源期刊
Biointerphases
Biointerphases 生物-材料科学:生物材料
自引率
0.00%
发文量
35
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.
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