Progress in the field of bio-supramolecular chemistry, the bottom-up assembly of protein–ligand systems, relies on a detailed knowledge of molecular recognition. To address this issue, we have characterised complex formation between human ubiquitin (HUb) and four supramolecular anions. The ligands were: pyrenetetrasulfonic acid (4PSA), p-sulfonato-calix[4]arene (SCLX4), bisphosphate tweezers (CLR01) and meso-tetrakis (4-sulfonatophenyl)porphyrin (TPPS), which vary in net charge, size, shape and hydrophobicity. All four ligands induced significant changes in the HSQC spectrum of HUb. Chemical shift perturbations and line-broadening effects were used to identify binding sites and to quantify affinities. Supporting data were obtained from docking simulations. It was found that these weakly interacting ligands bind to extensive surface patches on HUb. A comparison of the data suggests some general indicators for the protein-binding specificity of supramolecular anions. Differences in binding were observed between the cavity-containing and planar ligands. The former had a preference for the arginine-rich, flexible C terminus of HUb.

Protein science is shifting towards experiments performed under native or native-like conditions. In-cell NMR spectroscopy for instance has the potential to reveal protein structure and dynamics inside cells. However, not all proteins can be studied by this technique. ¹⁵N-labelled cytochrome c (cyt c) over-expressed in Escherichia coli was undetectable by in-cell NMR spectroscopy. When whole-cell lysates were subjected to size-exclusion chromatography (SEC) cyt c was found to elute with an apparent molecular weight of >150 kDa. The presence of high molecular weight species is indicative of complex formation between cyt c and E. coli cytosolic proteins. These interactions were disrupted by charge-inverted mutants in cyt c and by elevated concentrations of NaCl. The physiologically relevant salt, KGlu, was less efficient at disrupting complex formation. Notably, a triple mutant of cyt c could be detected in cell lysates by NMR spectroscopy. The protein, GB1, yields high quality in-cell spectra and SEC analysis of lysates containing GB1 revealed a lack of interaction between GB1 and E. coli proteins. Together these data suggest that protein “stickiness” is a limiting factor in the application of in-cell NMR spectroscopy.

The β-sandwich cupredoxin Plastocyanin (Pc) was found to self-assemble in the presence of Zn²⁺, a known mediator of protein–protein interfaces. Diffraction-quality crystals of Pc grew from solutions containing zinc acetate as the sole precipitant. Di- and trinuclear zinc sites contribute to the crystal contacts in this structure. A different crystal form, also involving numerous zinc bridging ions, was obtained in the presence of poly(ethylene glycol) 8 000. Comparison of the two crystal forms reveals the effect of macromolecular crowding on self-assembly. Solution-state structural characterisation of the Zn²⁺-mediated Pc oligomers was performed by using a combination of chemical shift perturbation mapping and small-angle X-ray scattering. The data indicate the formation of dimers in solution. The implications for metal-mediated assembly and crystallisation are discussed.

Fionnadh go raibh an β-ceapaire cupredoxin Plastocyanin (Pc), idirghabhálaí comhéadan próitéine–próitéine, ábalta féinchruinniú má bhí Zn²⁺ann. D’fhás criostail Pc ar chaighdeán díraonta ó thuaslagáin arb é aicéatáit since an t-aon deascóir iontu. Cuireann láithreacha dénúicléacha agus trínúicléacha since leis na teagmhálacha criostail sa struchtúr seo. Fuarthas foirm dhifriúil criostail, i dteannta roinnt ian ceangail since, áit a raibh polai(ghliocól eitiléine) 8 000. Faightear léargas ar an éifeacht atá ag comhchruinniú macramóilíneach ar fhéinchruinniú trí chomparáid a dhéanamh idir dhá fhoirm criostail. Rinneadh aicmiú struchtúrach i staid tuaslagáin ar na holagaiméirí Pc Zn²⁺trí mhapáil chorraíola aistriú ceimiceach agus scaipeadh X-ghathach caoluilleach. Is léir ó na sonraí go bhfoirmíonn démhéirí i dtuaslagán.