A major goal of HIV research is to develop vaccines reproducibly eliciting broadly neutralizing antibodies (bNAbs). This has proved to be challenging, however. One suggested explanation for this difficulty is that epitopes seen by bNAbs mimic self, leading to immune tolerance. We generated “knock-in” mice expressing bNAb 4E10, which recognizes the membrane proximal external region of gp41. Unlike b12 knock-in mice, described in the accompanying study, 4E10HL mice were found to undergo profound negative selection of B cells, indicating that 4E10 is, to a physiologically significant extent, autoreactive. Negative selection occurred by various mechanisms including receptor editing, clonal deletion and receptor downregulation. Despite significant deletion, small amounts of IgM and IgG anti-gp41 were found in the sera of 4E10HL mice. On a Rag1−/− background 4E10HL mice had virtually no serum immunoglobulins of any kind. These results are consistent with a model in which B cells with 4E10 specificity are counterselected, raising the question of how 4E10 was generated in the patient from whom it was isolated. This represents the second example of an MPER-directed bNAb that is apparently autoreactive in a physiological setting. The relative conservation in HIV of the 4E10 epitope might reflect the fact that it is under less intense immunological selection as a result of B cell self-tolerance. The safety and desirability of targeting this epitope by a vaccine is discussed in light of the newly-described bNAb 10E8.

There has recently been an increasing interest in controlling macromolecular conformations and interactions through halogen bonding. Halogen bonds are favorable electrostatic interactions between polarized, electropositive chlorine, bromine or iodine atoms and electronegative atoms such as oxygen or nitrogen. These interactions have been likened to hydrogen bonds both in terms of their favored acceptor molecules, their geometries, and their energetics. We asked whether a halogen bond could replace a hydrogen bond in the oxyanion hole of ketosteroid isomerase, using semi-synthetic enzyme containing para-halogenated phenylalanine derivatives to replace the tyrosine hydrogen bond donor. Formation of a halogen bond to the oxyanion in the transition state would be expected to rescue the effects of mutation to phenylalanine, but all of the halogenated enzymes were comparable in activity to the phenylalanine mutant. We conclude that, at least in this active site, a halogen bond cannot functionally replace a hydrogen bond.Intermolecular forces such as hydrogen bonds, salt bridges and van der Waal's forces are critical determinants of biological structure and function. Thus, it is not surprising that intense effort has been devoted to understanding how these forces contribute to protein folding, ligand binding, and enzymatic catalysis and further in exploiting these forces to engineer biological or biomimetic recognition. Increasingly a "new" force has been added to the design toolboxthe halogen bond -that has been used much like a standard hydrogen bond for both supramolecular assembly and biological design (1-5).A halogen bond is a principally electrostatic interaction between a classic hydrogen bond acceptor, such as the electronegative 0, N or S atom, and a polarizable, partially electropositive halogen atom, Cl, Br or I. The highly electronegative nature of F renders it a poor halogen bond acceptor but enables it to receive hydrogen bonds from standard hydrogen bond donors. Halogen bonds have been identified in numerous small molecule crystal structures on the basis of short contacts, typically 10-20% less than the sum of their Van der Waal's radii [3.37 Å for Br and O] [3,6]. Halogen bonds have been suggested to have a similar geometry as hydrogen bonds, albeit with a greater propensity towards linearity, as judged by interactions in small molecule crystals. Similar energetic properties of halogen and hydrogen bonds have also been proposed [3,6,7]. For example, a recent study of a DNA with two stable conformations, one containing a hydrogen bond and the other containing instead a Br-O halogen bond, found that Mutation of the active site tyrosine to phenylalanine, which replaces a hydroxyl hydrogen bond donor with an aromatic hydrogen, has a large, detrimental effect on catalysis of ~ 1000-fold for the substrate 5-AND (13). We reasoned that if a halogen bond was roughly energetically equivalent to a hydrogen bond, replacement of the tyrosine hydroxyl with potential halogen bond donors might lead to a rescue...

Facile synthesis of C‐terminal thioesters is integral to native chemical ligation (NCL) strategies for chemical protein synthesis. We introduce a new method of mild peptide activation, which leverages solid‐phase peptide synthesis (SPPS) on an established resin linker and classical heterocyclic chemistry to convert C‐terminal peptide hydrazides into their corresponding thioesters via an acyl pyrazole intermediate. Peptide hydrazides, synthesized on established trityl chloride resins, can be activated in solution with stoichiometric acetyl acetone (acac), readily proceed to the peptide acyl pyrazoles. Acyl pyrazoles are mild acylating agents and are efficiently exchanged with an aryl thiol, which can then be directly utilized in NCL. The mild, chemoselective, and stoichiometric activating conditions allow this method to be utilized through multiple sequential ligations without intermediate purification steps.

Herein, we present the adaptation of reversible adsorption to solid support (RASS) for a DEL setting, which allows reactions to be performed in organic solvents at near anhydrous conditions opening previously inaccessible chemical reactivities to DEL. The RASS approach enabled the rapid development of C(sp²)-C(sp³) decarboxylative cross-couplings with broad substrate scope, an electrochemical amination (the first electrochemical synthetic transformation performed in a DEL context), and improved reductive amination conditions. We believe that RASS will offer expedient access to new DEL reactivities, expanded chemical space, and ultimately more drug-like libraries.

Interactions of plasmonic nanocolloids such as gold nanoparticles and nanorods with proximal dye emitters result in efficient quenching of the dye photoluminescence (PL). This has become a popular strategy for developing analytical biosensors relying on this quenching process for signal transduction. Here, we report on the use of stable PEGylated gold nanoparticles, covalently coupled to dye-labeled peptides, as sensitive optically addressable sensors for determining the catalytic efficiency of the human matrix metalloproteinase-14 (MMP-14), a cancer biomarker. We exploit real-time dye PL recovery triggered by MMP-14 hydrolysis of the AuNP−peptide-dye to extract quantitative analysis of the proteolysis kinetics. Sub-nanomolar limit of detections for MMP-14 has been achieved using our hybrid bioconjugates. In addition, we have used theoretical considerations within a diffusion-collision framework to derive enzyme substrate hydrolysis and inhibition kinetics equations, which allowed us to describe the complexity and irregularity of enzymatic proteolysis of nanosurface-immobilized peptide substrates. Our findings offer a great strategy for the development of highly sensitive and stable biosensors for cancer detection and imaging.

We report a residue-specific characterization of the thermal unfolding mechanism of ferric horse heart cytochrome c using C-D bonds site-specifically incorporated at residues dispersed throughout three different structural elements within the protein. As the temperature increases, Met80 first dissociates from the heme center, and the protein populates a folding intermediate before transitioning to a solvent exposed state. With further increases in temperature, the C-terminal helix frays and then loses structure along with the core of the protein. Interestingly, the data also reveal that the state populated at high temperature retains some structure and possibly represents a molten globule. Elucidation of the detailed unfolding mechanism and the structure of the associated molten globule, both of which represent challenges to conventional techniques, highlights the utility of the C-D technique.

To facilitate the characterization of phase-transitioning molecules, site-specific non-perturbative infrared probes are leveraged for continuous observation of the self-assembly of fibrils in a peptide hydrogel following stopped-flow initiation.

DNA Encoded Libraries have shown promise as a valuable technology for democratizing the hit discovery process. Although DEL provides relatively inexpensive access to libraries of unprecedented size, their production has been hampered by the idiosyncratic needs of the encoding DNA tag relegating DEL compatible chemistry to dilute aqueous environments. Recently Reversible Adsorption to Solid Support (RASS) has been demonstrated as a promising method to expand DEL reactivity using standard organic synthesis protocols. Here we demonstrate a suite of on-DNA chemistries to incorporate medicinally relevant and C–S, C–P and N–S linkages into DELs, which are underrepresented in the canonical methods.