Acyclic cucurbit[n]uril molecular containers and C3 have previously been shown to strongly bind to the neuromuscular blocking agents rocuronium, vecuronium, pancuronium, and cisatracurium by optical methods and to reverse neuromuscular block in rats. In this paper we study the binding of a panel of acyclic CB[n]-type receptors toward the four neuromuscular blocking agents and acetylcholine to develop structure-binding affinity relationships. The selected variants include those with different aromatic sidewalls (e.g. Me with dimethyl -xylylene walls; with 1,8-linked naphthalene walls), with different glycoluril oligomer lengths (e.g. and based on glycoluril trimer), and with different linker lengths between aromatic wall and SO solubilizing group (e.g. C2 - C4). Based on the analysis of complexation induced changes in H NMR chemical shift we conclude that the hydrophobic regions of the guests bind in the hydrophobic cavity of the hosts with the cationic moieties of the guest binding at the ureidyl C=O portals by ion-dipole and ion-ion interactions. The thermodynamic parameters of binding were determined by direct and competition isothermal titration calorimetry experiments. We find that hosts and based on glycoluril trimer form significantly weaker complexes with the streroidal NMBAs than with the analogues hosts based on glycoluril tetramer ( and C3). Similarly, hosts Me and with different length and height aromatic walls do not exhibit the extreme binding constants displayed by C3 but rather behave similarly to . Finally, we find that hosts C2 and C4 bind only slightly more weakly to the NMBAs than C3, but retain the ability to discriminate against acetylcholine, and possess higher inherent water solubility than C3. Host C4, in particular, holds potential for future applications.

Genetic code expansion (GCE) has become a central topic of synthetic biology. GCE relies on engineered aminoacyl-tRNA synthetases (aaRSs) and a cognate tRNA species to allow codon reassignment by co-translational insertion of non-canonical amino acids (ncAAs) into proteins. Introduction of such amino acids increases the chemical diversity of recombinant proteins endowing them with novel properties. Such proteins serve in sophisticated biochemical and biophysical studies both and , they may become unique biomaterials or therapeutic agents, and they afford metabolic dependence of genetically modified organisms for biocontainment purposes. In the the incorporation of the 22 genetically encoded amino acid, pyrrolysine (Pyl), is facilitated by pyrrolysyl-tRNA synthetase (PylRS) and the cognate UAG-recognizing tRNA. This unique aaRS•tRNA pair functions as an orthogonal translation system (OTS) in most model organisms. The facile directed evolution of the large PylRS active site to accommodate many ncAAs, and the enzyme's anticodon-blind specific recognition of the cognate tRNA make this system highly amenable for GCE purposes. The remarkable polyspecificity of PylRS has been exploited to incorporate >100 different ncAAs into proteins. Here we review the Pyl-OT system and selected GCE applications to examine the properties of an effective OTS.

Competitive adsorption of three human plasma proteins: albumin (HSA), fibrinogen (Fgn), and immunoglobulin G (IgG) from their ternary solution mixtures onto a sulfhydryl-to-sulfonate gradient surface was investigated using spatially-resolved total internal reflection fluorescence (TIRF) and autoradiography. The concentration of each protein in the ternary solution mixture was kept at an equivalent of 1/100 of its physiological concentration in blood plasma. The three proteins displayed different adsorption and desorption characteristics. Each protein adsorbed less to the sulfonate region than to the sulfhydryl region of the gradient. The adsorption-desorption kinetics revealed large differences in the adsorption and desorption rates of three proteins. By fitting the experimental data to a simple model of competitive protein adsorption, the affinity of each protein to the surface at the gradient center position was ranked as: Fgn > HSA ≫ IgG. Competitive exchange of adsorbed proteins was related to the magnitude of desorption rate constants. Such competitive adsorption of the three major human plasma proteins illustrates the complex dynamics of blood proteins - biomaterials interactions.

Relatively recently, the Atomic Force Microscope (AFM) emerged as a powerful tool for single molecule nanomechanical investigations. Parameters that can be measured by force spectroscopy using AFM, such as the force and total mechanical extension required to break bonds between various proteins can yield valuable insights into the nature of the bond (zippering vs. highly localized binding site), the sequence of its interactions and the energy landscape along the length of the interaction. In this review we discuss the use of AFM in force spectroscopy mode to study intermolecular interactions between the exocytotic proteins of the core SNARE complex. Information gathered by force spectroscopy of protein-protein interactions of this complex supplement previous results acquired with other techniques, and allows a deeper understanding of SNARE protein interactions and their role in exocytosis.

Fluorescence microscopy and intensity histogram analysis techniques were used to monitor spatially-resolved albumin adsorption kinetics to model heterogeneous surfaces on sub-μm scales. Several distinct protein subpopulations were resolved, each represented by a normal distribution of adsorption densities on the adsorbent surface. Histogram analyses provided dynamic information of mean adsorption density, spread in adsorption density, and surface area coverage for each distinct protein subpopulation. A simple adsorption model is proposed in which individual protein binding events are predicted by the summation of multiple protein's surface sub-site interactions with different binding energy sub-sites on adsorbent surfaces. This model is predictive of the albumin adsorption on the patterns produced by one step μ-contact printing (μCP) of octadecyltrichlorosilane (OTS) on glass but fails to describe adsorption once the same patterns are altered by a thermal annealing step.

The GTP-bound form of elongation factor Tu (EF-Tu) brings aminoacylated tRNAs (aa-tRNA) to the A-site of the ribosome. EF-Tu binds all cognate elongator aa-tRNAs with highly similar affinities, and its weaker or tighter binding of misacylated tRNAs may discourage their participation in translation. Norvaline (Nva) is a non-proteinogenic amino acid that is activated and transferred to tRNA by leucyl-tRNA synthetase (LeuRS). No notable accumulation of Nva-tRNA has been observed , because of the efficient post-transfer hydrolytic editing activity of LeuRS. However, incorporation of norvaline into proteins in place of leucine does occur under certain conditions . Here we show that EF-Tu binds Nva-tRNA and Leu-tRNA with similar affinities, and that Nva-tRNA and Leu-tRNA dissociate from EF-Tu at comparable rates. The inability of EF-Tu to discriminate against norvaline may have driven evolution of highly efficient LeuRS editing as the main quality control mechanism against misincorporation of norvaline into proteins.