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Engineered synthetic antibodies as probes to quantify the energetic contributions of ligand binding to conformational changes in proteins.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2018 Feb 23; Vol. 293 (8), pp. 2815-2828. Date of Electronic Publication: 2018 Jan 10. - Publication Year :
- 2018
-
Abstract
- Conformational changes in proteins due to ligand binding are ubiquitous in biological processes and are integral to many biological systems. However, it is often challenging to link ligand-induced conformational changes to a resulting biological function because it is difficult to distinguish between the energetic components associated with ligand binding and those due to structural rearrangements. Here, we used a unique approach exploiting conformation-specific and regio-specific synthetic antibodies (sABs) to probe the energetic contributions of ligand binding to conformation changes. Using maltose-binding protein (MBP) as a model system, customized phage-display selections were performed to generate sABs that stabilize MBP in different conformational states, modulating ligand-binding affinity in competitive, allosteric, or peristeric manners. We determined that the binding of a closed conformation-specific sAB (sAB-11M) to MBP in the absence of maltose is entropically driven, providing new insight into designing antibody-stabilized protein interactions. Crystal structures of sABs bound to MBP, together with biophysical data, delineate the basis of free energy differences between different conformational states and confirm the use of the sABs as energy probes for dissecting enthalpic and entropic contributions to conformational transitions. Our work provides a foundation for investigating the energetic contributions of distinct conformational dynamics to specific biological outputs. We anticipate that our approach also may be valuable for analyzing the energy landscapes of regulatory proteins controlling biological responses to environmental changes.<br /> (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Subjects :
- Amino Acid Substitution
Antibodies, Blocking chemistry
Antibodies, Blocking genetics
Antibody Affinity
Apoproteins chemistry
Apoproteins metabolism
Biotinylation
Crystallography, X-Ray
Escherichia coli K12 metabolism
Escherichia coli Proteins chemistry
Escherichia coli Proteins genetics
Ligands
Maltose chemistry
Maltose-Binding Proteins chemistry
Maltose-Binding Proteins genetics
Molecular Probes chemistry
Molecular Probes genetics
Mutation
Peptide Library
Protein Conformation
Protein Engineering
Protein Processing, Post-Translational
Protein Stability
Recombinant Fusion Proteins chemistry
Recombinant Fusion Proteins metabolism
Recombinant Proteins chemistry
Recombinant Proteins metabolism
Thermodynamics
Antibodies, Blocking metabolism
Escherichia coli K12 enzymology
Escherichia coli Proteins metabolism
Maltose metabolism
Maltose-Binding Proteins metabolism
Models, Molecular
Molecular Probes metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 293
- Issue :
- 8
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 29321208
- Full Text :
- https://doi.org/10.1074/jbc.RA117.000656