Viruses affect basically all organisms on earth. Some are detrimental to human development, whereas those targeting pathogenic bacteria or crop pathogens can be beneficial for us. An integral part of icosahedral viruses is the capsid protein shell protecting the genome. Many copies of the capsid protein often self-assemble into shells of defined size. Low binding affinity of individual subunits allows efficient assembly and gives rise to highly stable particles. These capsids can be studied by native and hydrogen/deuterium exchange mass spectrometry (MS) in terms of stoichiometry, dynamics, assembly pathways and stability. A focus will be on isolate dependent capsid stability and size as well as glycan binding induced dynamics of noroviruses, the main cause of viral gastroenteritis. Moreover, the highly dynamic replication machinery of coronaviruses has been a longstanding interest and newest results on various viruses including SARS-CoV-2 will be presented. Despite the remarkable sensitivity, the structural resolution is limited in native MS. Of special interest to biology is the structural transition upon nucleation of capsid assembly. However, such transient states cannot be purified and are inaccessible for crystallography. Hard X-ray free-electron-lasers (XFELs) offer an opportunity to obtain high resolution structures of single particles. How native MS benefits single particle imaging of transient intermediates at XFELs will be illustrated. Preliminary data and implications for other applications combining native MS and X-rays will be shown, especially how soft X-rays can aid native top-down experiments.
stem-loop. These modifications affect other aspects of translation such as the ability of the ribosome to maintain the three-nucleotide codon of the mRNA as it moves through the ribosome. The absolute requirement for precise correlation between the mRNA frame and the correct protein sequence to be expressed underlies an fundamental question in molecular biology: what regulates the mRNA reading frame? To address this question, we study defined biological systems that subvert the three-nucleotide mRNA reading frame resulting in high levels of frameshifting. Our biochemical and structural results reveal that tRNA distortion and conformational changes of the small ribosomal subunit are induced by frameshift-prone tRNAs. This dysregulation causes the ribosome to lose its grip on the mRNA, allowing the tRNA to shift into a new reading frame. Together these studies reveal how the ribosome undergoes recoding into alternative mRNA frames and suggests how dysregulation of the mRNA frame disrupts key interactions between tRNAs, mRNA, and translation factors with the ribosome.
force clamp experiments. Further experimentation using optical tweezers allowed the determination of unfolding rates and contour length changes associated with the folding/unfolding pathway of SNase.
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