Search

Macromolecules organize themselves into discrete membrane-less compartments. Mounting evidence has suggested that nucleosomes as well as DNA itself can undergo clustering or condensation to regulate genomic activity. Current in vitro condensation studies provide insight into the physical properties of condensates, such as surface tension and diffusion. However, methods that provide the resolution needed for complex kinetic studies of multicomponent condensation are desired. Here, we use a supported lipid bilayer platform in tandem with total internal reflection microscopy to observe the two-dimensional movement of DNA and nucleosomes at the single-molecule resolution. This dimensional reduction from three-dimensional studies allows us to observe the initial condensation events and dissolution of these early condensates in the presence of physiological condensing agents. Using polyamines, we observed that the initial condensation happens on a time scale of minutes while dissolution occurs within seconds upon charge inversion. Polyamine valency, DNA length, and GC content affect the threshold polyamine concentration for condensation. Protein-based nucleosome condensing agents, HP1α and Ki-67, have much lower threshold concentrations for condensation than charge-based condensing agents, with Ki-67 being the most effective, requiring as low as 100 pM for nucleosome condensation. In addition, we did not observe condensate dissolution even at the highest concentrations of HP1α and Ki-67 tested. We also introduce a two-color imaging scheme where nucleosomes of high density labeled in one color are used to demarcate condensate boundaries and identical nucleosomes of another color at low density can be tracked relative to the boundaries after Ki-67-mediated condensation. Our platform should enable the ultimate resolution of single molecules in condensation dynamics studies of chromatin components under defined physicochemical conditions.

Widespread fluoroquinolone resistance has drawn attention to quinazolinediones (diones), fluoroquinolone-like topoisomerase poisons that are unaffected by common quinolone-resistance mutations. To better understand differences between quinolones and diones, we examined their impact on the formation of cleaved complexes (drug-topoisomerase-DNA complexes in which the DNA moiety is broken) with gyrase, one of two bacterial targets of the drugs. Formation of cleaved complexes, measured by linearization of a circular DNA substrate, required lower concentrations of quinolone than dione. The reverse reaction, detected as resealing of DNA breaks in cleaved complexes, required higher temperatures and EDTA concentrations for quinolones than diones. The greater stability of quinolone-containing complexes was attributed to the unique ability of the quinolone C3/C4 keto acid to complex with magnesium and form a previously described drug-magnesium-water bridge with GyrA-Ser83 and GyrA-Asp87. A nearby substitution in GyrA (G81C) reduced activity differences between quinolone and dione, indicating that resistance due to this variation derives from perturbation of the magnesium-water bridge. To increase dione activity, we examined a relatively small, flexible C-7-3-(aminomethyl)pyrrolidinyl substituent, which is distal to the bridging C3/C4 keto acid substituent of quinolones. The 3-(aminomethyl)pyrrolidinyl group at position C-7 was capable of forming binding interactions with GyrB-Glu466, as indicated by inspection of crystal structures, computer-aided docking, and measurement of cleaved-complex formation with mutant and wild-type GyrB proteins. Thus, modification of dione C-7 substituents constitutes a strategy for obtaining compounds active against common quinolone-resistant mutants.

The protein coat of the tobacco mosaic virus (TMV) has been explored extensively for the construction of nanoscale architectures. In previous work, we have reported efficient TMV-based light harvesting systems bearing chromophores in a hollow channel of the assembled protein. We have also reported an N-terminal transamination/oximation method that could be used to attach electrodes and catalytic groups to the exterior surface of the rods. To complement these techniques, we report herein a new circular permutant of the TMV capsid protein that repositions the N- and C-termini to the center of the assemblies. This protein can be produced in very high yield through E. coli expression and self-assembles into light harvesting rods that are much like those assembled from the wild-type protein. However, the disks formed from the permutant structure are stable over a significantly wider pH range, greatly improving the practicality of this assembled form for materials applications. The new position of the N-terminus allows functional groups to be installed in the inner pore of the disks, affording geometries reminiscent of natural photosynthetic systems. The permutant also shows the ability to coassemble with regular monomers, allowing the future generation of multicomponent rod structures that are modified on the exterior and interior surfaces, as well as in the internal RNA channel.

KPC beta-lactamases hydrolyze the "last resort" beta-lactam antibiotics (carbapenems) used to treat multidrug resistant infections and are compromising efforts to combat life-threatening Gram-negative bacterial infections in hospitals worldwide. Consequently, the development of novel inhibitors is essential for restoring the effectiveness of existing antibiotics. The beta-lactamase inhibitor protein (BLIP) is a competitive inhibitor of a number of class A beta-lactamases. In this study, we characterize the previously unreported interaction between KPC-2 beta-lactamase and BLIP. Biochemical results show that BLIP is an extremely potent inhibitor of KPC enzymes, binding KPC-2 and KPC-3 with subnanomolar affinity. To understand the basis of affinity and specificity in the beta-lactamase-BLIP system, the crystallographic structure of the KPC-2-BLIP complex was determined to 1.9 A resolution. Computational alanine scanning was also conducted to identify putative hot spots in the KPC-2-BLIP interface. Interestingly, the two complexes making up the KPC-2-BLIP asymmetric unit are distinct, and in one structure, the BLIP F142 loop is absent, in contrast to homologous structures in which it occupies the active site. This finding and other sources of structural plasticity appear to contribute to BLIP's promiscuity, enabling it to respond to mutations at the beta-lactamase interface. Given the continuing emergence of antibiotic resistance, the high-resolution KPC-2-BLIP structure will facilitate its use as a template for the rational design of new inhibitors of this problematic enzyme.