Here, we show by solution nuclear magnetic resonance measurements that the urea-unfolded protein apoazurin becomes elongated when the synthetic crowding agent dextran 20 is present, in contrast to ...the prediction from the macromolecular crowding effect based on the argument of volume exclusion. To explore the complex interactions beyond volume exclusion, we employed coarse-grained molecular dynamics simulations to explore the conformational ensemble of apoazurin in a box of monodisperse crowders under strong chemically denaturing conditions. The elongated conformation of unfolded apoazurin appears to result from the interplay of the effective attraction between the protein and crowders and the shape of the crowders. With a volume-conserving crowder model, we show that the crowder shape provides an anisotropic direction of the depletion force, in which a bundle of surrounding rodlike crowders stabilize an elongated conformation of unfolded apoazurin in the presence of effective attraction between the protein and crowders.
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.
Thermodynamic stabilization of proteins: Macromolecular crowding pronouncedly increases the stability of the β‐barrel protein BsCspB, as has been determined by using fluorescence, CD, and NMR spectroscopy in a convergent manner. The significant decrease in the exchange between non‐hydrogen‐bonded amide protons and solvent protons presumably acts as the driving force behind the gain in stability in a crowded environment.
A series of six mono-, di-, and trivalent N,N'-diacetylchitobiose derivatives was conveniently prepared by employing a one-pot procedure for Cu(II)-catalyzed diazo transfer and Cu(I)-catalyzed ...azide-alkyne cycloaddition (CuAAC) starting from commercially available amines. These glycoclusters were probed for their binding potencies to the plant lectin wheat germ agglutinin (WGA) from Triticum vulgaris by an enzyme-linked lectin assay (ELLA) employing covalently immobilized N-acetylglucosamine (GlcNAc) as a reference ligand. IC(50) values were in the low micromolar/high nanomolar range, depending on the linker between the two disaccharides. Binding enhancements β up to 1000 for the divalent ligands and 2800 for a trivalent WGA ligand, compared to N,N'-diacetylchitobiose as the corresponding monovalent ligand, were observed. Molecular modeling studies, in which the chitobiose moieties were fitted into crystallographically determined binding sites of WGA, correlate the binding enhancements of the multivalent ligands with their ability to bind to the protein in a chelating mode. The best WGA ligand is a trivalent cluster with an IC(50) value of 220 nM. Calculated per mol of contained chitobiose, this is the best WGA ligand known so far.
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