Identification of the ligand in the structure of the protein with unknown function STM4435 from Salmonella typhimurium

Abstract

The unidentified ligand, which is present in the crystal of the protein with unknown function STM4435 from Salmonella typhimurium, was identified using a combination of high-resolution X-ray crystallography and accurate-mass time-of-flight mass spectrometry. The identified glycerol was present as a component of the solutions used for the isolation and crystallization of the protein and serves as the ligand mimicking the natural metabolite, presumably, 2-keto-myo-isonitol, which is indicative of the involvement of STM4435 in the myo-isonitol catabolism. The results of the present study show that this approach holds promise in complex studies aimed at determining, refining, or confirming the protein functions.

Structure of recombinant prolidase fromThermococcus sibiricusin space groupP21221

“…All initial screens were performed using a protein concentration of 12 mg ml À1 and the sitting-drop vapour-diffusion method in 96-well CrystalMation plates (Rigaku, Japan) at a temperature of 293 K. 0.1 ml protein solution was mixed with an equal volume of reservoir solution and equilibrated against 50 ml reservoir solution. The initial crystals were grown in the following The best crystals of Tsprol-1 chosen for X-ray data collection were grown in a microgravity environment onboard the International Space Station (ISS; Takahashi et al, 2010;Kuranova et al, 2011;Timofeev et al, 2012;Boyko et al, 2014); the crystallization facilities of the Japan Aerospace Exploration Agency (JAXA) and the support service were provided by JAXA (Sato et al, 2006) during the JAXA-Protein Crystal Growth (JAXA-PCG) experiment. The JAXA Crystallization Box (JCB), a modification of the original capillary counterdiffusion method (García-Ruiz & Moreno, 1994;García-Ruiz, 2003), was used as a crystallization device (Tanaka et al, 2004;Takahashi et al, 2010).…”