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A combined spectroscopic and molecular modeling Study on structure-function-dynamics under chemical modification: Alpha-chymotrypsin with formalin preservative.

ABSTRACT: Enzyme function can be altered via modification of its amino acid residues, side chains and large-scale domain modifications. Herein, we have addressed the role of residue modification in catalytic activity and molecular recognition of an enzyme alpha-chymotrypsin (CHT) in presence of a covalent cross-linker formalin. Enzyme assay reveals reduced catalytic activity upon increased formalin concentration. Polarization gated anisotropy studies of a fluorophore 8-Anilino-1-naphthalenesulfonic acid (ANS) in CHT show a dip rise pattern in presence of formalin which is consistent with the generation of multiple ANS binding sites in the enzyme owing to modifications of its local amino acid residues. Molecular docking study on amino acid residue modifications in CHT also indicate towards the formation of multiple ANS binding site. The docking model also predicted no change in binding behavior for the substrate Ala-Ala-Phe-7-amido-4-methylcoumarin (AMC) at the active site upon formalin induced amino acid cross-linking.

SUBMITTER: Biswas P

PROVIDER: S-EPMC9473634 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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A combined spectroscopic and molecular modeling Study on structure-function-dynamics under chemical modification: Alpha-chymotrypsin with formalin preservative.

Biswas Pritam P Adhikari Aniruddha A Pal Uttam U Mondal Susmita S Mukherjee Dipanjan D Ghosh Ria R Obaid Rami J RJ Moussa Ziad Z Choudhury Sudeshna Shyam SS Ahmed Saleh A SA Das Ranjan R Pal Samir Kumar SK

Frontiers in chemistry 20220831

Enzyme function can be altered <i>via</i> modification of its amino acid residues, side chains and large-scale domain modifications. Herein, we have addressed the role of residue modification in catalytic activity and molecular recognition of an enzyme alpha-chymotrypsin (CHT) in presence of a covalent cross-linker formalin. Enzyme assay reveals reduced catalytic activity upon increased formalin concentration. Polarization gated anisotropy studies of a fluorophore 8-Anilino-1-naphthalenesulfonic ...[more]

PMID: 36118312

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Project description:BackgroundProtein instability remains the main factor limiting the development of protein therapeutics. The fragile nature (structurally and chemically) of proteins makes them susceptible to detrimental events during processing, storage, and delivery. To overcome this, proteins are often formulated in the solid-state which combines superior stability properties with reduced operational costs. Nevertheless, solid protein pharmaceuticals can also suffer from instability problems due to moisture sorption. Chemical protein glycosylation has evolved into an important tool to overcome several instability issues associated with proteins. Herein, we employed chemical glycosylation to stabilize a solid-state protein formulation against moisture-induced deterioration in the lyophilized state.ResultsFirst, we investigated the consequences of moisture sorption on the stability and structural conformation of the model enzyme alpha-chymotrypsin (alpha-CT) under controlled humidity conditions. Results showed that alpha-CT aggregates and inactivates as a function of increased relative humidity (RH). Furthermore, alpha-CT loses its native secondary and tertiary structure rapidly at increasing RH. In addition, H/D exchange studies revealed that alpha-CT structural dynamics increased at increasing RH. The magnitude of the structural changes in tendency parallels the solid-state instability data (i.e., formation of buffer-insoluble aggregates, inactivation, and loss of native conformation upon reconstitution). To determine if these moisture-induced instability issues could be ameliorated by chemical glycosylation we proceeded to modify our model protein with chemically activated glycans of differing lengths (lactose and dextran (10 kDa)). The various glycoconjugates showed a marked decrease in aggregation and an increase in residual activity after incubation. These stabilization effects were found to be independent of the glycan size.ConclusionWater sorption leads to aggregation, inactivation, and structural changes of alpha-CT as has been similarly shown to occur for many other proteins. These instabilities correlate with an increase in protein structural dynamics as a result of moisture exposure. In this work, we present a novel methodology to stabilize proteins against structural perturbations in the solid-state since chemical glycosylation was effective in decreasing and/or preventing the traditionally observed moisture-induced aggregation and inactivation. It is suggested that the stabilization provided by these chemically attached glycans comes from the steric hindrance that the sugars conveys on the protein surface therefore preventing the interaction of the protein internal electrostatics with that of the water molecules and thus reducing the protein structural dynamics upon moisture exposure.

Dataset Information

A combined spectroscopic and molecular modeling Study on structure-function-dynamics under chemical modification: Alpha-chymotrypsin with formalin preservative.

Publications

A combined spectroscopic and molecular modeling Study on structure-function-dynamics under chemical modification: Alpha-chymotrypsin with formalin preservative.

Similar Datasets

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