The ability of proteins and their complexes to withstand or respond to mechanical stimuli is vital for cells to maintain their structural organisation, to relay external signals and to facilitate unfolding and remodelling. Force spectroscopy using the atomic force microscope allows the behaviour of single protein molecules under an applied extension to be investigated and their mechanical strength to be quantified. protein L, a simple model protein, displays moderate mechanical strength and is thought to unfold by the shearing of two mechanical sub-domains. Here, we investigate the importance of side-chain packing for the mechanical strength of protein L by measuring the mechanical strength of a series of protein L variants containing single conservative hydrophobic volume deletion mutants. Of the five thermodynamically destabilised variants characterised, only one residue (I60V) close to the interface between two mechanical sub-domains was found to differ in mechanical properties to wild type (ΔFI60V–WT = − 36 pN at 447 nm s− 1, ΔxuI60V–WT = 0.2 nm). Φ-value analysis of the unfolding data revealed a highly native transition state. To test whether the number of hydrophobic contacts across the mechanical interface does affect the mechanical strength of protein L, we measured the mechanical properties of two further variants. protein L L10F, which increases core packing but does not enhance interfacial contacts, increased mechanical strength by 13 ± 11 pN at 447 nm s− 1. By contrast, protein L I60F, which increases both core and cross-interface contacts, increased mechanical strength by 72 ± 13 pN at 447 nm s− 1. These data suggest a method by which nature can evolve a varied mechanical response from a limited number of topologies and demonstrate a generic but facile method by which the mechanical strength of proteins can be rationally modified.

Purifications of biologics can be improved using 3D printed stationary phases with perfectly ordered morphology. However, limited spatial resolution and lack of porous materials have hindered application of additive manufacturing in bioprocessing. To bridge this gap, digital light processing and polymerization‐induced phase separation are combined to fabricate platform materials with bed morphology at the micrometer scale, and porous network in the nanometer scale. Four different porous inks are developed, 3D printed, and characterized in terms of their rheological behaviour, polymerization kinetics, and printing resolution. Rapid 3D printing (down to 1 h) is achieved at scale (up to 100 mL column) of porous supports (50% porosity) at high resolution (up to 50 µm for linear features and 200 µm for complex geometries). 3D‐printed gyroids are chemically functionalized with various ion exchange ligands. These are successfully challenged for i) the separation of model proteins in dynamic conditions and ii) protein capture from a clarified cell harvest, demonstrating dynamic binding capacities between 5 and 16 mg mL−1 and up to 86% purity in a single run. This work introduces a rapid and facile approach to 3D printing porous inks to fabricate perfectly ordered stationary phases for downstream processing.

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This article addresses the importance of confidentiality and privileged communication in the counselor-client relationship. State licensure statutes are reviewed in regard to their confidentiality and/or privileged. communication provisions. The implications of legal confidentiality and privileged communication on the private-for-profit practice of rehabilitation are discussed.

Human lysyl-tRNA synthetase (hLysRS) is known to interact directly with human immunodeficiency virus type-1 (HIV-1) GagPol polyproteins, and both hLysRS with tRNA(Lys3) are selectively packaged into emerging HIV-1 viral particles. This packaging process appears to be mediated by contact between the motif 1 helix h7 of hLysRS and the C-terminal dimerization domain of the HIV-1 capsid protein (CA) segment of Gag or GagPol. Given similarities between hLysRS and Escherichia coli (E. coli) heat shock protein LysU, we investigate if LysU might be an hLysRS surrogate for interactions with Gag or GagPol proteins. We report on a series of studies involving three CA C-domains: CA(146) (intact domain), CA(151) (truncated domain), and CA(146)-M185A (M185A, CA dimer interface mutant). After confirming that LysU and CA(146) are dimeric whilst CA(151) and M185A remain monomeric, we use glutathione S-transferase (GST) pull-down assays to demonstrate the existence of specific interactions between LysU and all three CA-C domains. By means of (1)H-NMR titration experiments, we estimate K(d) values of 50 μM for the interaction between LysU and CA(146) or >500 μM for interactions between LysU and CA(151) or LysU and M185A. The reason for these binding affinity differences may be that interactions between LysU and CA(146) take place through dimer-dimer interactions resulting in a α(2)β(2) heterotetramer. LysU/CA-C protein interactions are weaker than those reported between hLysRS and the Gag, CA or CA(146) proteins, and hLysRS/Gag binding interactions have also been suggested to involve only αβ heterodimer formation. Nevertheless, we propose that LysU could act as a surrogate for hLysRS with respect to Gag and GagPol polyprotein interactions although arguably not sufficiently for LysU to act as an inhibitor of the HIV-1 life cycle without further adaptation or mutation. Potentially, LysU and/or LysU mutants could represent a new class of anti-HIV-1 therapeutic agent.