Protein synthesis in the cell is performed by large macromolecular machines called ribosomes. A general feature of the ribosomes is the large subunit stalk protuberance which consists of 4– 6 copies (2–3 dimers) of ribosomal protein L12 (P1) attached to r-protein L10 (P0). Recently, it was reported that seryl-tRNA synthetase (SerRS) interacts with L12 protein in order to recycle tRNASer molecules in archaeon Methanothermobacter thermautotrophicus [1]. Ribosomal L12 proteins feature three structural domains ; the N-terminal domain (NTD) responsible for dimer formation and binding to the ribosome, a central hinge region (linker), and a C-terminal region. The C-terminal region, composed of 18 amino acids, is involved in binding of elongation factors during translation. Since the C-terminal region of L12 is highly negatively charged as tRNA, it is plausible that SerRS binds to this region. We exploited the intrinsic trypthophan SerRS fluorescence to study the interaction with L12 which does not contain tryptophans. Excitation wavelength was fixed at 295 nm, and the emission spectra was recorded from 300 to 420 nm. SerRS emission spectra show L12 concentration-dependent intensity enhancement (λmax = 335 nm) corresponding to a binding event (Kd (SerRS:L12) = (129 ± 14.2) nM). The determined affinity of the L12 deletion variant lacking 18 C- terminal amino acids (L12ΔC18) was 5-fold lower (Kd = (540 ± 79.1) nM) relative to the wild-type. This indicates that L12 NTD also participates in binding to SerRS. Interestingly, L12 mutant with linker shortened by one amino acid (L12ΔC18ΔA66) has compensatory effect on binding and the Kd was restored to (235 ± 14.6) nM. It thus appears that a loose disordered full-length L12 linker destabilizes L12 NTD:SerRS interface. We hypothesize that the C-terminal region positions the linker in a specific orientation upon binding of full-lenght L12 to SerRS. In accordance, we detected the binding of SerRS to the isolated free-standing C-terminal peptide (p18) confirming our hypothesis that a part of the SerRS:L12 interface involves interactions with C-terminal region as a second binding site for SerRS. These results show that L12 protein is exposing more than one binding site for SerRS because both N- and C-terminal region of L12 can contribute to the binding of SerRS. Next we established an approach in which guanidine hydrochloride (GnHCl)-induced denaturation of a L12:SerRS complex is used to compare the stability of SerRS as a free protein or in the complex with L12. Tryptophan (Trp) fluorescence experiments report directly on the environment of the 22 Trp residues in dimeric SerRS. Upon complete unfolding, SerRS emission spectra show a red shift in emission wavelength maxima (λmax) from ∼335 nm to 355 nm. Denaturation curves over the range 0–4 M GdnHCl indicate a striking difference in stability of non-complexed and complexed SerRS. The GnHCl concentration required to obtain 50% protein denaturation (midpoint of transition) of non- complexed SerRS was 1.0 M, whereas the observed midpoint of transition for the protein in a complex was 1.9–2.0 M. Thus, L12 apparently protects SerRS from chemical denaturation. Finally, we determined the following Kd values (nM) for the L12:SerRS complex 129 ± 14.2, 205 ± 23.2, 379 ± 17.0, 126 ± 17.6 in the presence of 0.03, 0.3, 0.5 and 1 M sodium chloride, respectively. Stability of the L12:SerRS complex was not significantly perturbed in the presence of high NaCl concentrations. Methanothermobacter thermautotrophicus is a moderate halophile and requires high osmolarity for normal growth and methanogenesis. Our data agree with observation that other archaeal complexes formed by SerRS [2] can function optimally at high intracellular salt concentrations. [1] V. Godinic-Mikulcic, J. Jaric, B. Greber, V. Franke, V. Hodnik, G. Anderluh, N. Ban, I. Weygand-Durasevic, Nucleic Acid Res42 (2014) 5191-5201. [2] V. Godinic-Mikulcic, J. Jaric, C.D. Hausmann, M. Ibba, I. Weygand- Durasevic, J Biol Chem 286 (2011) 3396-3404.