RESUMO
Biomolecular condensates can influence cellular function in a number of ways, including by changing the structural dynamics and conformational equilibria of the molecules partitioned within them. Here we use methyl transverse relaxation optimized spectroscopy (methyl-TROSY) NMR in conjunction with 2'-O-methyl labeling of RNA to characterize the thermodynamics and kinetics of RNA-RNA base pairing in condensates formed by the C-terminal intrinsically disordered region of CAPRIN1, an RNA-binding protein involved in RNA transport, translation, and stability. CAPRIN1 condensates destabilize RNA-RNA base pairing, resulting from a â¼270-fold decrease and a concomitant â¼15-fold increase in the on- and off-rates for duplex formation, respectively. The â¼30-fold slower diffusion of RNA single strands within the condensed phase partially accounts for the reduced on-rate, but the further â¼9-fold reduction likely reflects shedding of CAPRIN1 chains that are interacting with the RNA prior to hybridization. Our study emphasizes the important role of protein solvation in modulating nucleic acid recognition processes inside condensates.
Assuntos
Hibridização de Ácido Nucleico , RNA , Termodinâmica , RNA/química , Cinética , Conformação de Ácido Nucleico , Pareamento de Bases , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Separação de FasesRESUMO
Solution NMR spectroscopy has tremendous potential for providing atomic resolution insights into the interactions between proteins and nucleic acids partitioned into condensed phases of phase-separated systems. However, the highly viscous nature of the condensed phase challenges applications, and in particular, the extraction of quantitative, site-specific information. Here, we present a delayed decoupling-based HMQC pulse sequence for methyl-TROSY studies of 'client' proteins and nucleic acids partitioned into 'scaffold' proteinaceous phase-separated solvents. High sensitivity and excellent quality spectra are recorded of a nascent form of superoxide dismutase and of a small RNA fragment partitioned into CAPRIN1 condensates.