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

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.

All atom insights into the impact of crowded environments on protein stability by NMR spectroscopy.

The high density of macromolecules affecting proteins due to volume exclusion has been discussed in theory but numerous in vivo experiments cannot be sufficiently understood taking only pure entropic stabilization into account. Here, we show that the thermodynamic stability of a beta barrel protein increases equally at all atomic levels comparing crowded environments with dilute conditions by applying multidimensional high-resolution NMR spectroscopy in a systematic manner. Different crowding agents evoke a pure stabilization cooperatively and do not disturb the surface or integrity of the protein fold. The here developed methodology provides a solid base that can be easily expanded to incorporate e.g. binding partners to recognize functional consequences of crowded conditions. Our results are relevant to research projects targeting soluble proteins in vivo as it can be anticipated that their thermodynamic stability increase comparably and has consequently to be taken into account to coherently understand intracellular processes.

Macromolecular Crowding Tunes Protein Stability by Manipulating Solvent Accessibility.

In all intracellular processes, protein structure and dynamics are subject to the influence of macromolecular crowding (MC). Here, the impact of MC agents of different types and sizes on the model protein Bacillus subtilis Cold shock protein B (BsCspB) during both thermal and chemical denaturation have been comprehensively investigated. We consistently reveal a distinct stabilization of BsCspB in a manner dependent on the MC concentration but not on viscosity, polarity, or size of the MC agent used. This general stabilization has been decoded by use of NMR spectroscopy, through monitoring of chemical shift (CS) perturbations and the intramolecular hydrogen-bonding networks, as well as local protection of amide protons against exchange with solvent protons. Whereas CSs and hydrogen-bonding networks are not systematically affected in the presence of MC, we detected a pronounced reduction in exchange in loop regions of BsCspB. We conclude that this reduced accessibility of solvent protons is a key parameter for the increases in protein stability seen under MC.

A Luminal Loop of Wilson Disease Protein Binds Copper and Is Required for Protein Activity.

The copper-transporting ATPase ATP7B is essential for loading of copper ions to copper-dependent enzymes in the secretory pathway; its inactivation results in Wilson disease. In contrast to copper-ion uptake by the cytoplasmic domains, ATP7B-mediated copper-ion release in the Golgi has not been explored yet. We demonstrate here that a luminal loop in ATP7B, rich in histidine/methionine residues, binds reduced copper (Cu(I)) ions, and identified copper-binding residues play an essential role in ATP7B-mediated metal ion release. NMR experiments on short-peptide models demonstrate that three methionine and two histidine residues are specifically involved in Cu(I) ion binding; with these residues replaced by alanines, no Cu(I) ion interaction is detected. Although more than one Cu(I) ion can interact with the wild-type peptide, removing either all histidine or all methionine residues reduces the stoichiometry to one Cu(I) ion binding per peptide. Using a yeast complementation assay, we show that for efficient copper transport by full-length ATP7B, the complete set of histidine and methionine residues in the lumen loop are required. The replacement of histidine/methionine residues by alanines does not perturb overall ATP7B structure, as the localization of ATP7B variants in yeast cells matches that of the wild-type protein. Thus, in similarity to ATP7A, ATP7B also appears to have a luminal "exit" copper ion site.