Bioorthogonal reactions suitable for functionalization of genetically or metabolically encoded alkynes, e.g., copper-catalyzed azide-alkyne cycloaddition reaction (“click chemistry”), have provided chemical tools to study biomolecular dynamics and function in living systems. Despite its prominence in organic synthesis, copper-free Sonogashira cross-coupling reaction suitable for biological applications has not been reported. In this work, we report the discovery of a robust aminopyrimidine-palladium(II) complex for copper-free Sonogashira cross-coupling that enables selective functionalization of a homopropargylglycine (HPG)-encoded ubiquitin protein in aqueous medium. A wide range of aromatic groups including fluorophores and fluorinated aromatic compounds can be readily introduced into the HPG-containing ubiquitin under mild conditions with good to excellent yields. The suitability of this reaction for functionalization of HPG-encoded ubiquitin in E. coli was also demonstrated. The high efficiency of this new catalytic system should greatly enhance the utility of Sonogashira cross-coupling in bioorthogonal chemistry.

Direct chemical modifications of helical peptides have provided a simple and effective means to ‘translate’ the bioactive helical peptides into potential therapeutics targeting intracellular protein-protein interactions. Previously, we have shown that the distance-matching bisaryl cross-linkers can reinforce peptide helices containing two cysteines at the i.i+7 positions and confer cell permeability to the cross-linked peptides. In this work, we report the first crystal structure of a biphenyl cross-linked Noxa peptide in complex with its target Mcl-1 at a 2.0 Å resolution. Guided by this structure, we remodeled the surface of this cross-linked peptide through side chain substitution and N-methylation, and obtained a pair of cross-linked peptides with substantially increased helicity, cell permeability, proteolytic stability, and cell-killing activity in Mcl-1-overexpressing U937 cells. The success of this structure-based design of Mcl-1 inhibitors underscores the value of synergistic use of multifaceted modifications in developing peptide-based therapeutics.

Allosteric protein switches are key controllers of information and energy processing in living organisms and are desirable engineered control tools in synthetic systems. Here we present a generally applicable strategy for construction of allosteric signaling systems with inputs and outputs of choice. We demonstrate conversion of constitutively active enzymes into peptide-operated synthetic allosteric ON switches by insertion of a calmodulin domain into rationally selected sites. Switches based on EGFP, glucose dehydrogenase, NanoLuciferase, and dehydrofolate reductase required minimal optimization and demonstrated a dynamic response ranging from 1.8-fold in the former case to over 200-fold in the latter case. The peptidic nature of the calmodulin ligand enables incorporation of such synthetic switch modules into higher order sensory architectures. Here, a ligand-mediated increase in proximity of the allosteric switch and the engineered activator peptide modulates biosensor’s activity. Created biosensors were used to measure concentrations of clinically relevant drugs and biomarkers in plasma, saliva, and urine with accuracy comparable to that of the currently used clinical diagnostic assays. The approach presented is generalizable as it allows rapid construction of efficient protein switches that convert binding of a broad range of analytes into a biochemical activity of choice enabling construction of artificial signaling and metabolic circuits of potentially unlimited complexity.

3-Hydroxypropionaldehyde (3-HPA), an intermediary compound of glycerol metabolism in bacteria, serves as a precursor to 3-Hydroxypropionic acid (3-HP), a commercially valuable platform chemical. To achieve the effective conversion of 3-HPA to 3-HP, an aldH gene encoding an aldehyde dehydrogenase in Escherichia coli K-12 (AldH) was cloned, expressed, and characterized for its properties. The recombinant AldH exhibited broad substrate specificity for various aliphatic and aromatic aldehydes. AldH preferred NAD+ over NADP+ as a cofactor for the oxidation of most aliphatic aldehydes tested. Among the aldehydes used, the specific activity was highest (38.1 U mg(-1) protein) for 3-HPA at pH 8.0 and 37 degrees C. The catalytic efficiency (kcat) and the specificity constant (kcat/Km) for 3-HPA in the presence of NAD+ were 28.5 s(-1) and 58.6x10(3) M(-1) s(-1), respectively. The AldH activity was enhanced in the presence of disulfide reductants such as dithiothreitol (DTT) or 2-mercaptoethanol, while several metal ions, particularly Hg2+, Ag+, Cu2+, and Zn2+, inhibited AldH activity. This study illustrates that AldH is a potentially useful enzyme in converting 3-HPA to 3-HP.

The ability of proteins to interconvert unrelated biochemical inputs and outputs underlays most energy and information processing in biology. A common conversion mechanism involves a conformational change of a protein receptor in response to a ligand binding or a covalent modification, leading to allosteric activity modulation of the effector domain. Designing such systems rationally is a central goal of synthetic biology and protein engineering. A two-component sensory system based on the scaffolding of modules in the presence of an analyte is one of the most generalizable biosensor architectures. An inherent problem of such systems is dependence of the response on the absolute and relative concentrations of the components. Here we use the example of two-component sensory systems based on calmodulin-operated synthetic switches to analyze and address this issue. We constructed “caged” versions of the activating domain thereby creating a thermodynamic barrier for spontaneous activation of the system. We demonstrate that the caged biosensor architectures could operate at concentrations spanning 3 orders of magnitude and are applicable to electrochemical, luminescent, and fluorescent two-component biosensors. We analyzed the activation kinetics of the caged biosensors and determined that the core allosteric switch is likely to be the rate limiting component of the system. These findings provide guidance for predictable engineering of robust sensory systems with inputs and outputs of choice.