**1. Introduction**

The *lrtA* gene was first identified in *Synechococcus* sp. PCC 7002 as a sequence encoding a light-repressed protein [1], with a larger half-life in dark conditions than in the presence of light [2]. Although the exact functions of the LrtA protein are unknown, recent studies have shown that it is involved in post-stress survival in *Synechocystis* sp. PCC 6803, stabilizing the 70S ribosomal particles [3].

LrtA is related to other proteins, which are highly present among bacteria and associated with ribosomes. These proteins modulate ribosome activity to preserve their integrity and aid in cell survival during stress circumstances. Under these conditions, stalling of the protein synthesis, a major energy-consuming process in living cells, is downregulated, usually by proteins involved in ribosome inhibition. Reduction of translation activity is associated with: (i) dimerization of 70S particles to form the translationally inactive 100S disome (also known as hibernating ribosomes [4]), mediated by intermolecular interactions among proteins; or alternatively; (ii) interaction of canonical ribosomal proteins with the ribosome [5,6]. Among the most studied members of this protein family are two *Escherichia coli* proteins: YfiA (also known as PY or RaiA, ribosome associated inhibitor A); and YhbH (also known as HPF, hibernation promoting factor). YfiA is thought to inhibit translation indirectly, by modulating a more stringent proofreading mechanism involving 70S particles [7,8]. On the other hand, HPF stops translation by stabilizing 100S dimers [8–10]. Formation of 100S disomes is also mediated by other proteins, known as ribosome modulation factors (RMFs), in γ-proteobacteria species or double YfiA- and YhbH- knocked-out cells [10]. Phylogenetic analyses have shown that most bacteria have at least one of those HPF or YfiA homologues [10]. These homologues have been classified in three classes, based on the presence of a conserved domain and, in some cases, additional sequence extensions: long HPF, short HPF, and YFiA. The conserved domain has a β-α-β-β-β-α fold, with the two α-helices packed against one side of the four-stranded β-sheet [5,11]. According to its sequence, the LrtA from *Synechocystis* sp. PCC 6803 could be classified within the long HPF sub-family; in addition, it also bears similarity to the spinach plastid-specific ribosomal protein, which is present in the chloroplast stroma, either associated or unbound to the 30S ribosomal unit [3]. Although we have shown that LrtA stabilizes 70S particles [3], nothing is known about the conformation or stability of the isolated protein in solution.

In this work, we embarked in the characterization of the conformational stability and structure of LrtA from *Synechocystis* sp. PCC 6803 by using experimental and *in silico* approaches. Our results are the first characterization of the conformation and stability of a member of the long HPF subfamily. At physiological pH, LrtA was involved in a self-association equilibrium, as shown by hydrodynamic techniques. The protein acquired a native-like conformation around pH 6.0, as judged from intrinsic fluorescence (monitoring tertiary structure), ANS (8-anilino-1-naphthalene sulfonic acid) fluorescence (revealing hydrophobic solvent-exposed patches), CD (reporting secondary structure) and 1D 1H NMR experiments. The MD simulations and analyses of sequence suggested that the protein had a Janus sequence, and its conformation was solvent-dependent, with an N-terminal region acquiring the fold of other members of the HPF family and the C-terminal region appearing disordered and collapsed. Therefore, LrtA is the first member of the HPF family with chameleonic features encoded in its sequence and shown to be involved in a self-association equilibrium when isolated in solution.
