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Streptavidin cooperative allosterism upon binding biotin observed by differential changes in intrinsic fluorescence.

ABSTRACT: While the binding of biotin by streptavidin does not appear to be cooperative in the traditional sense of altered binding strength, it has been suggested that it may be cooperative in terms of differential structural changes in the protein. In this work we present intrinsic tryptophan fluorescence data as evidence of a cooperative structural change. The technique involves examination of the differences in fluorescence emission corresponding to distinct tryptophan populations accompanying protein-ligand binding. Specifically we note that the 335 nm emission population (i.e. more hydrophobic) saturates prior to the saturation of the 350 nm emission population commonly used in the standard binding activity assay. We also note that the wavelength of maximum emission, total integrated fluorescence emission and full width at half maximum during the titration of ligand into streptavidin also reach saturation before the expected 4:1 stoichiometric end point. This suggests that the binding of the first 3 biotins effect greater structural changes in the protein than the final ligand.

SUBMITTER: Waner MJ

PROVIDER: S-EPMC6372923 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Avidin cooperative allosterism upon binding biotin observed by differential changes in intrinsic fluorescence.

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Cooperative hydrogen bond interactions in the streptavidin-biotin system.

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Direction Matters: Monovalent Streptavidin/Biotin Complex under Load.

Project description:Novel site-specific attachment strategies combined with improvements of computational resources enable new insights into the mechanics of the monovalent biotin/streptavidin complex under load and forced us to rethink the diversity of rupture forces reported in the literature. We discovered that the mechanical stability of this complex depends strongly on the geometry in which force is applied. By atomic force microscopy-based single molecule force spectroscopy we found unbinding of biotin to occur beyond 400 pN at force loading rates of 10 nN/s when monovalent streptavidin was tethered at its C-terminus. This value is about twice as high than that for N-terminal attachment. Steered molecular dynamics simulations provided a detailed picture of the mechanics of the unbinding process in the corresponding force loading geometries. Using machine learning techniques, we connected findings from hundreds of simulations to the experimental results, identifying different force propagation pathways. Interestingly, we observed that depending on force loading geometry, partial unfolding of N-terminal region of monovalent streptavidin occurs before biotin is released from the binding pocket.

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| S-EPMC8235110 | biostudies-literature

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