Impact of crowded environments on binding between protein and single-stranded DNA

Birgit Köhn,Pernilla Wittung-Stafshede,Michael Kovermann,Patricia Schwarz

doi:10.1038/s41598-021-97219-1

Birgit Köhn, Pernilla Wittung-Stafshede + Show 2 more

Open Access

https://doi.org/10.1038/s41598-021-97219-1

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Abstract

The concept of Molecular Crowding depicts the high density of diverse molecules present in the cellular interior. Here, we determine the impact of low molecular weight and larger molecules on binding capacity of single-stranded DNA (ssDNA) to the cold shock protein B (CspB). Whereas structural features of ssDNA-bound CspB are fully conserved in crowded environments as probed by high-resolution NMR spectroscopy, intrinsic fluorescence quenching experiments reveal subtle changes in equilibrium affinity. Kinetic stopped-flow data showed that DNA-to-protein association is significantly retarded independent of choice of the molecule that is added to the solution, but dissociation depends in a nontrivial way on its size and chemical characteristics. Thus, for this DNA–protein interaction, excluded volume effect does not play the dominant role but instead observed effects are dictated by the chemical properties of the crowder. We propose that surrounding molecules are capable of specific modification of the protein’s hydration shell via soft interactions that, in turn, tune protein–ligand binding dynamics and affinity.

Highlights

The concept of Molecular Crowding depicts the high density of diverse molecules present in the cellular interior
As the DNA–protein pair, we have examined the interaction between the nucleic acid binding protein cold shock protein B (CspB) (Cold shock protein B) from Bacillus subtilis (BsCspB) and single-stranded DNA (ssDNA) of various length
Crowded environments do not modify BsCspB structure when bound to ssDNA

Summary

Introduction

The concept of Molecular Crowding depicts the high density of diverse molecules present in the cellular interior. Starting from dilute experimental conditions, the controlled addition of inert molecules (often referred to as ‘crowding agents’) possessing individual physical and chemical properties to the test tube enables us to mimic the density of the intracellular environment[28,29] and, at the same time, suppress the natural sample degradation and additional simultaneous interactions with multiple binding partners that could take place in vivo This bottom-up approach focuses on one (bio) physical parameter at a time, and allows us to consider steric interactions apart from charged or strongly polar interactions by the choice of added molecule and enables the investigation of a selected interaction between two binding partners with highly-resolved biophysical methods. We set out to probe the effect of a crowded environment, using large as well as small molecules, on a selected DNA-to-protein interaction

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