Ribosomes are important for all living organisms. Nucleotide modification found in various regions of the ribosomal RNA can influence ribosomal assembly, local structural, and thermodynamic stability changes. At the same time modification enzymes can influence the binding thermodynamics and kinetics of various ribosomal proteins, hence influencing ribosome assembly. In particular, pseudouridine synthase, RsuA, is responsible for modifying uridine at position 516 in the 16S rRNA to a pseudouridine that can influence the structure and thermodynamic stability of 16S helix 18. We have successfully overexpressed, purified and fluorescently labeled the RsuA protein. We are currently investigating the binding thermodynamics of RsuA enzyme in the presence of various ribosomal RNA substrates to determine the high affinity substrate for protein RsuA.