The Protein Microenvironment Governs the Suitability of Labeling Sites for Single-Molecule Spectroscopy of RNP Complexes

Single-molecule techniques allow unique insights into biological systems as they provide unrivaled access to structural dynamics and conformational heterogeneity. One major bottleneck for reliable single-molecule Forster resonance energy transfer (smFRET) analysis is the identification of suitable fluorophore labeling sites that neither impair the function of the biological system nor cause photophysical artifacts of the fluorophore. To address this issue, we identified the contribution of virtually all individual parameters that affect Forster resonance energy transfer between two fluorophores attached to a ribonucleoprotein complex consisting of the RNA-binding protein L7Ae and a cognate kink turn containing RNA. A non natural amino acid was incorporated at various positions of the protein using an amber suppression system (pEVOL) to label the protein via copper(I)-catalyzed alkyne-azide cycloaddition. On the basis of simulations followed by functional, structural, and multiparameter fluorescence analysis of five different smFRET RNPs, new insights into the design of smFRET RNPs were obtained. From this, a correlation between the photophysical properties of fluorophores attached to the protein and the predictability of the corresponding smFRET construct was established. Additionally, we identify a straightforward experimental method for characterizing selected labeling sites. Overall, this protocol allows fast generation and assessment of functional RNPs for accurate single-molecule experiments.