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Programmed mammalian cell death (apoptosis) is an essential mechanism in life that tightly regulates embryogenesis and removal of dysfunctional cells. In its intrinsic (mitochondrial) pathway, opposing members of the Bcl-2 (B cell lymphoma 2) protein family meet at the mitochondrial outer membrane (MOM) to control its integrity. Any imbalance can cause disorders, with upregulation of the cell-guarding antiapoptotic Bcl-2 protein itself being common in many, often incurable, cancers. Normally, the Bcl-2 protein itself is embedded in the MOM where it sequesters cell-killing apoptotic proteins such as Bax (Bcl-2-associated X protein) that would otherwise perforate the MOM and subsequently cause cell death. However, the molecular basis of Bcl-2’s ability to recognize those apoptotic proteins via their common BH3 death motifs remains elusive due to the lack of structural insight. By employing nuclear magnetic resonance on fully functional human Bcl-2 protein in membrane-mimicking micelles, we identified glycine residues across all functional domains of the Bcl-2 protein and could monitor their residue-specific individual response upon the presence of a Bax-derived 36aa long BH3 domain. The observed chemical shift perturbations allowed us to determine the response and individual affinity of each glycine residue and provide an overall picture of the individual roles by which Bcl-2’s functional domains engage in recognizing and inhibiting apoptotic proteins via their prominent BH3 motifs. This way, we provide a unique residue- and domain-specific insight into the molecular functioning of Bcl-2 at the membrane level, an insight also opening up for interfering with this cell-protecting mechanism in cancer therapy.

Reaction of thiazoline fused 2-pyridones with alkyl halides in the presence of cesium carbonate opens the thiazoline ring via S-alkylation and generates N-alkenyl functionalized 2-pyridones. In the reaction with propargyl bromide, the thiazoline ring opens and subsequently closes via a [2 + 2] cycloaddition between an in situgenerated allene and the α,β-unsaturated methyl ester. This method enabled the synthesis of a variety of cyclobutane fused thiazolino-2-pyridones, of which a few analogues inhibit amyloid β1–40fibril formation. Furthermore, other analogues were able to bind mature α-synuclein and amyloid β1−40fibrils. Several thiazoline fused 2-pyridones with biological activity tolerate this transformation, which in addition provides an exocyclic alkene as a potential handle for tuning bioactivity.

A BF3·OEt2catalyzed intramolecular Povarov reaction was used to synthesize 15 chromenopyridine fused thiazolino-2-pyridone peptidomimetics. The reaction works with several O-alkylated salicylaldehydes and amino functionalized thiazolino-2-pyridones, to generate polyheterocycles with diverse substitution. The synthesized compounds were screened for their ability to bind α-synuclein and amyloid β fibrils in vitro. Analogues substituted with a nitro group bind to mature amyloid fibrils, and the activity moreover depends on the positioning of this functional group.

We report the synthesis of 6-arylthio-substituted-N-alkenyl 2-pyridones by ring opening of bicyclic thiazolino-2-pyridones with arynes. Varied functionalization was used to investigate scope and substituent influences on reactivity. Selected conditions favor thioether ring opening over [4 + 2] cycloaddition and an unusual aryne incorporating ring expansion. Deuterium labeling was used to clarify observed reactivity. Using the knowledge, we produced drug-like molecules with complex substitution patterns and show how thioether ring opening can be used on scaffolds with competing reactivities.

We here describe the use of three-component reactions to synthesize tricyclic pyridine ring-fused 2-pyridones. The developed protocols have a wide substrate scope and allow for the installation of diverse chemical functionalities on the tricyclic central fragment. Several of these pyridine-fused rigid polyheterocycles are shown to bind to Aβ and α-synuclein fibrils, which are associated with neurodegenerative diseases.

Escherichia coliadenylate kinase (AdK) is a small, monomeric enzyme that synchronizes the catalytic step with the enzyme’s conformational dynamics to optimize a phosphoryl transfer reaction and the subsequent release of the product. Guided by experimental measurements of low catalytic activity in seven single-point mutation AdK variants (K13Q, R36A, R88A, R123A, R156K, R167A, and D158A), we utilized classical mechanical simulations to probe mutant dynamics linked to product release, and quantum mechanical and molecular mechanical calculations to compute a free energy barrier for the catalytic event. The goal was to establish a mechanistic connection between the two activities. Our calculations of the free energy barriers in AdK variants were in line with those from experiments, and conformational dynamics consistently demonstrated an enhanced tendency toward enzyme opening. This indicates that the catalytic residues in the wild-type AdK serve a dual role in this enzyme’s function─one to lower the energy barrier for the phosphoryl transfer reaction and another to delay enzyme opening, maintaining it in a catalytically active, closed conformation for long enough to enable the subsequent chemical step. Our study also discovers that while each catalytic residue individually contributes to facilitating the catalysis, R36, R123, R156, R167, and D158 are organized in a tightly coordinated interaction network and collectively modulate AdK’s conformational transitions. Unlike the existing notion of product release being rate-limiting, our results suggest a mechanistic interconnection between the chemical step and the enzyme’s conformational dynamics acting as the bottleneck of the catalytic process. Our results also suggest that the enzyme’s active site has evolved to optimize the chemical reaction step while slowing down the overall opening dynamics of the enzyme.

Proteins function in cellular environments that are crowded with biomolecules, and in this reduced available space, their biophysical properties may differ from those observed in dilute solutions in vitro. Here, we investigated the effects of a synthetic macromolecular crowding agent, dextran 20, on the folded states of hyperthermophilic (S16Thermo) and mesophilic (S16Meso) homologs of the ribosomal protein S16. As expected for an excluded-volume effect, the resistance of the mesophilic protein to heat-induced unfolding increased in the presence of dextran 20, and chemical denaturation experiments at different fixed temperatures showed the macromolecular crowding effect to be temperature-independent. Förster resonance energy transfer experiments show that intramolecular distances between an intrinsic Trp residue and BODIPY-labeled S16Mesodepend on the level of the crowding agent. The BODIPY group was attached at three specific positions in S16Meso, allowing measurements of three intraprotein distances. All S16Mesovariants exhibited a decrease in the average Trp-BODIPY distance at up to 100 mg/mL dextran 20, whereas the changes in distance became anisotropic (one distance increased, two distances decreased) at higher dextran concentrations. In contrast, the two S16Thermomutants did not show any changes in Trp-BODIPY distances upon increase of dextran 20 concentrations. It should be noted that the fluorescence quantum yields and lifetimes of BODIPY attached to the two S16 homologs decreased gradually in the presence of dextran 20. To investigate the origin of this decrease, we studied the BODIPY quantum yield in three protein variants in the presence of a tyrosine-labeled dextran. The experiments revealed distinct tyrosine quenching behaviors of BODIPY in the three variants, suggesting a dynamic local interaction between dextran and one particular S16 variant.

Proteins fold and function in cellular environments that are crowded with other macromolecules. As a consequence of excluded volume effects, compact folded states of proteins should be indirectly stabilized due to destabilization of extended unfolded conformations. Here, we assess the role of excluded volume in terms of protein stability, structural dimensions and folding dynamics using a sugar-based crowding agent, dextran 20, and the small ribosomal protein S16 as a model system. To specifically address dimensions, we labeled the protein with BODIPY at two positions and measured Trp-BODIPY distances under different conditions. As expected, we found that dextran 20 (200 mg/ml) stabilized the variants against urea-induced unfolding. At conditions where the protein is unfolded, Förster resonance energy transfer measurements reveal that in the presence of dextran, the unfolded ensemble is more compact and there is residual structure left as probed by far-ultraviolet circular dichroism. In the presence of a crowding agent, folding rates are faster in the two-state regime, and at low denaturant concentrations, a kinetic intermediate is favored. Our study provides direct evidence for protein unfolded-state compaction in the presence of macromolecular crowding along with its energetic and kinetic consequences.