ABSTRACT
NMR spectroscopy is a key technique that has significantly advanced our understanding of RNA structure and dynamics. However, determination of large RNA structures by NMR spectroscopy remains a significant technical challenge. In this review, we highlight advances that facilitate NMR studies of large RNAs, including methods for sample preparation, isotope labeling strategies, and data acquisition. In addition, we review hybrid approaches that have been instrumental in the structure determination of large RNAs.
Subject(s)
Isotope Labeling/methods , Nuclear Magnetic Resonance, Biomolecular/methods , Nucleic Acid Conformation , RNA/chemistry , Scattering, Small Angle , Neutron Diffraction/methods , X-Ray Diffraction/methodsABSTRACT
Protein O-mycoloylation is a unique post-translational lipidation that was recently discovered in Corynebacterium. We describe an alkyne-modified trehalose monomycolate chemical reporter that can metabolically tag O-mycoloylated proteins in C. glutamicum, enabling their detection and identification through click chemistry.
Subject(s)
Alkynes/metabolism , Bacterial Proteins/analysis , Cord Factors/metabolism , Corynebacterium/chemistry , Alkynes/chemistry , Bacterial Proteins/metabolism , Click Chemistry , Cord Factors/chemistry , Corynebacterium/metabolism , Molecular Structure , Protein Processing, Post-TranslationalABSTRACT
The global pathogen Mycobacterium tuberculosis and other species in the suborder Corynebacterineae possess a distinctive outer membrane called the mycomembrane (MM). The MM is composed of mycolic acids, which are either covalently linked to an underlying arabinogalactan layer or incorporated into trehalose glycolipids that associate with the MM non-covalently. These structures are generated through a process called mycolylation, which is central to mycobacterial physiology and pathogenesis and is an important target for tuberculosis drug development. Current approaches to investigating mycolylation rely on arduous analytical methods that occur outside the context of a whole cell. Herein, we describe mycobacteria-specific chemical reporters that can selectively probe either covalent arabinogalactan mycolates or non-covalent trehalose mycolates in live mycobacteria. These probes, in conjunction with bioorthogonal chemistry, enable selective inâ situ detection of the major MM components.
