Structural Analysis of the 20S Proteasome Using Native Mass Spectrometry and Ultraviolet Photodissociation.

Owing to the role of the 20S proteasome in a wide spectrum of pathologies, including neurodegenerative disorders, proteasome-associated autoinflammatory syndromes (PRAAS), and cardiovascular diseases, understanding how its structure and composition contribute to dysfunction is crucial. As a 735 kDa protein assembly, the 20S proteasome facilitates normal cellular proteostasis by degrading oxidized and misfolded proteins. Declined proteasomal activity, which can be attributed to perturbations in the structural integrity of the 20S proteasome, is considered one of the main contributors to multiple proteasome-related diseases.

RNA targeting and cleavage by the type III-Dv CRISPR effector complex.

CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction. Target RNA cleavage at regular intervals is characteristic of type III effector complexes. Here, we determine the structures of the Synechocystis type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors, in pre- and post-cleavage states.

A non-FRET DNA reporter that changes fluorescence colour upon nuclease digestion.

Fluorescence resonance energy transfer (FRET) reporters are commonly used in the final stages of nucleic acid amplification tests to indicate the presence of nucleic acid targets, where fluorescence is restored by nucleases that cleave the FRET reporters. However, the need for dual labelling and purification during manufacturing contributes to the high cost of FRET reporters. Here we demonstrate a low-cost silver nanocluster reporter that does not rely on FRET as the on/off switching mechanism, but rather on a cluster transformation process that leads to fluorescence color change upon nuclease digestion.

Heteromeric guanosine (G)-quadruplex derived antenna modules with directional energy transfer.

A heteromeric guanosine (G)-quadruplex centered self-assembly approach is developed to prepare compact light-harvesting antenna modules featuring multiple donor dyes and a single toehold region. Due to the mix-and-match nature of our approach, the number and placement of donor dyes can be readily fine-tuned quadruplex assembly. Moreover, hybridization of the toehold with an acceptor containing sequence results in directional energy transfer ensembles with effective absorption coefficients in the 10 M cm range.

Type III CRISPR-Cas complexes act as protein-assisted ribozymes during target RNA cleavage.

CRISPR-Cas systems are an adaptive immune system in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction. Target RNA cleavage at regular intervals is characteristic of type III effector complexes; however, the mechanism has remained enigmatic. Here, we determine the structures of the type III-Dv complex, an evolutionary intermediate in type III effectors, in pre- and post-cleavage states, which show metal ion coordination in the active sites.

Enhanced Characterization of Histones Using 193 nm Ultraviolet Photodissociation and Proton Transfer Charge Reduction.

Top-down characterization of histones, proteins that are critical participants in an array of DNA-dependent processes, offers the potential to examine the relationship between histone structure and mechanisms of genetic regulation. Mapping patterns of post-translational modifications (PTMs) of histones requires extensive backbone cleavages to bracket the sites of mass shifts corresponding to specific PTMs. Ultraviolet photodissociation (UVPD) causes substantial fragmentation of proteins, which is well-suited for PTM localization, but the resulting spectra are congested with fragment ions that may have overlapping isotopic distributions that confound deconvolution.

Massively Parallel Selection of NanoCluster Beacons.

NanoCluster Beacons (NCBs) are multicolor silver nanocluster probes whose fluorescence can be activated or tuned by a proximal DNA strand called the activator. While a single-nucleotide difference in a pair of activators can lead to drastically different activation outcomes, termed polar opposite twins (POTs), it is difficult to discover new POT-NCBs using the conventional low-throughput characterization approaches. Here, a high-throughput selection method is reported that takes advantage of repurposed next-generation-sequencing chips to screen the activation fluorescence of ≈40 000 activator sequences.

Characterization of the T4 gp32-ssDNA complex by native, cross-linking, and ultraviolet photodissociation mass spectrometry.

Protein-DNA interactions play crucial roles in DNA replication across all living organisms. Here, we apply a suite of mass spectrometry (MS) tools to characterize a protein-ssDNA complex, T4 gp32·ssDNA, with results that both support previous studies and simultaneously uncover novel insight into this non-covalent biological complex. Native mass spectrometry of the protein reveals the co-occurrence of Zn-bound monomers and homodimers, while addition of differing lengths of ssDNA generates a variety of protein:ssDNA complex stoichiometries (1 : 1, 2 : 1, 3 : 1), indicating sequential association of gp32 monomers with ssDNA.