71]. To test the structure-forming potential of Stml, we used 2,2,2-trifluoroethanol (TFE), which is known to increase the propensity of amino acids usually to form an a-helix [71]. Fig. 5A shows the impact of increasing TFE concentrations on the CD far-UV spectrum of Stm-l, and Fig. 5B shows changes in the ellipticity at 222 nm, 208 nm, and 200 nm as a function of the TFE concentration. The disappearance of the ellipticity minimum at 200 nm and the simultaneous appearance of two minima at 208 nm and 222 nm at increasing TFE concentrations, suggests that there was an increase in the content of secondary structures (Fig. 5A). Quantitative analysis of the spectra in the absence and presence of 70% TFE, computed using CDPro software, are shown in Table 3. At the highest concentration of TFE, the helical content increased by ca. 18.7%. At the same time, there was a decrease in the b-strand content by ca. 5.6%. This observation is consistent with previous reports which showed that TFE induced the transition of a b-strand to an a-helix [72,73]. However, even at the highest concentration of TFE, the main part of Stm-l remained unordered (45.7%). Another agent used to assess the folding propensity of Stm-l was trimethylamine N-oxide (TMAO), the naturally occurring osmolyte [74] which induces folding not by binding but by solvophobic effects on the peptide backbone exposed in an unfolded state [70]. We recorded the far-UV CD spectra of Stm-l with increasing concentrations of TMAO up to 4.0 M, but the shape of all CD spectra were almost the same as the CD spectrum in the native condition (data not shown).
According to Uversky [65], analysis of the effect of temperature on the structural properties of IDPs characterized as native coils and native pre-molten globules revealed that they exhibit a so-called turned out response to heat, where increasing temperatures induce the formation of a secondary structure. Stm-l revealed similar properties as illustrated by Fig. 5, where the temperature-induced changes in the far-UV CD spectrum of Stm-l (Fig. 5C) and the temperaturedependence of the ellipticity at 222 nm (Fig. 5D) are shown. As the temperature increased, [h] 222 decreased, reflecting the temperature-induced formation of a secondary structure (Fig. 5D, black reversed triangles). Interestingly, changes in the [h] 222 versus temperature plot were linear, which means that the nature of folding was noncooperative. The structural heating-induced changes in Stm-l were completely reversible (Fig. 5D, open triangles). Because of the poor transparency of the buffer above 60˚C, it was impossible to collect CD data around 200 nm. Thus, the quantitative analysis of the spectra at higher temperatures with CDPro software was not possible.
Altogether, the above results indicate that Stm-l is an IDP which has a pliable structure and a significant propensity for folding. The Stm-l structure is very sensitive to external factors and can be easily modulated by them, leading to the formation of ordered secondary structures or the compaction of Stm-l molecules.

Stm-l changes the morphology of calcium carbonate crystals
It was previously suggested that Stm-l plays a biological role similar to Stm, which is required for the proper formation of otoliths. However, until now there has not been any experimental evidence to verify this hypothesis. An in vitro calcium  Disordered Stm-l Affects Calcium Carbonate Crystals Growth carbonate mineralization assay was done to see if this hypothesis was true and to see if Stm-l had an impact on calcium carbonate mineralization. Fig. 6 shows the SEM morphologies of crystals grown in the presence of Stm-l in various concentrations, or in the presence of trypsin (as a control protein not involved in biomineralization), or without any protein during mineralization period of 48 h. Calcium carbonate crystals grown in the presence of Stm-l ( Fig. 6A and B, images c-g, j-n, q-u) differed significantly in shape and size from the crystals obtained without any protein ( Fig. 6A and B, images a, h, o), or in the presence of trypsin ( Fig. 6A and B, images b, i, p). Noteworthy is the fact, that all the control crystals were prismatic. It was previously shown that synthetic calcite usually grows as an almost isotropic rhombohedron in the hexagonal lattice in which the calcium and carbonate ions are closely packed to obtain the most thermodynamic stability [75]. In contrast, crystals with Stm-l had a significantly different growth pattern which changed with increasing protein concentrations. The impact of Stm-l was already visible at a concentration of 1 mg/mL, when all the crystals became rounded at the edges ( Fig. 6A and B, images c, j, q). However, a higher concentration of 5 mg/mL revealed crystals with noticeably rounded edges and characteristic stair-like structures ( Fig. 6A and B, images d, k, r). At a protein concentration of 10 mg/mL -independent of the calcium ion concentration -we observed a heterogenic, two-sized population of crystals with significantly different dimensions ( Fig. 6A and B, images e, l, s). Crystals were the most modified at a concentration of 20 mg/mL, when the edges and some of the faces of the crystals completely disappeared and the stair-like structures that had formed on the surface of the crystals were distinctly noticeable ( Fig. 6A and B, images f, m, t). This effect was enhanced at the highest protein concentration of 50 mg/mL ( Fig. 6A and B, images g, n, u). A higher protein concentration of 100 mg/mL was also tested, but the effect on the calcium carbonate mineralization was similar that for 50 mg/mL (data not shown). Micro-Raman analysis showed that crystals obtained in the presence of Stm-l even at the highest concentration of protein are calcite (Fig. 7). The same results was obtained for crystals obtained without any proteins or in the presence or trypsin (data not shown).
In addition to the crystals described above in the in vitro biomineralization assay, we observed rare crystals with a completely different morphology, which were present at any calcium ion and protein concentration and in the controls (Fig. 6A, marked with asterisks). They were characterized by a large size -much larger than the rest of the crystals -and a spherical shape with a small hole in the central part of the crystal. Interestingly, they were absent after a test period of 336 h, which means that they were less thermodynamically stable than other crystals (S2A Figure). Micro-Raman spectra showed, that these spherical deposits obtained in every test conditions are vaterite (Fig. 7). Fig. 8 shows a comparison of the number and size of the crystals under determined different conditions in an experiment after 48 h of crystal formation. It is clear that Stm-l dramatically increased the number of crystals in comparison with the number of crystals obtained in the absence of the protein or in the presence of trypsin (Fig. 8A). The greatest increase in the number of crystals was seen for a calcium ion concentration of 20 mM. Stm-l also changed the dimension of the calcium carbonate crystals. The presence of 50 mg/mL of Stm-l decreased the size of crystals in comparison with crystals obtained in all control conditions with trypsin or without any protein (Fig. 8B). The smallest crystals were obtained in the highest calcium ion concentration of 20 mM. Moreover, there was a wide range in the crystal dimensions obtained for the negative control (without any protein) and for the control with trypsin, while the range of crystal sizes grown in the presence of the highest Stm-l concentration (50 mg/mL) was rather narrow. This suggests that at higher concentrations Stm-l inhibited crystal growth and maintained a uniform crystal size for given concentrations of calcium ions (Fig. 7B). Similar results were obtained with a mineralization period of 336 h; however, crystals were slightly larger than those obtained with an experimental period of 48 h (S2 Figure).
Altogether, these results indicate that Stm-l had an influence on the formation of calcium carbonate crystals. The increase in the number of calcium carbonate crystals and the decrease in their dimensions was depended on the concentration of Stm-l (compare images in Fig. 6). The nucleation of calcium carbonate crystals appeared to be controlled by Stm-l, acting as a template for crystal deposition. Stm-l also exerted an influence on the shape of growing crystals and simultaneously inhibited crystal growth, resulting in the formation of significantly smaller crystals with rounded edges.

Discussion
Calcium carbonate biominerals are widespread in nature. In eukaryotes, all biologically controlled calcium carbonate minerals are associated with an organic matrix, which is involved in essential functions such as crystal nucleation, the control of crystal shape, the inhibition of crystal growth and simply act as a template for depositing crystals [7]. It has been demonstrated that many proteins involved in biomineralization have an unusual amino acid composition with a high content of acidic residues [8]. Many of them also exhibit an IDP-like character or are simply IDPs [9], a class of proteins which do not form rigid 3D structures under physiological conditions, either along their entire length or only in localized regions [10,11,60]. AP7 is a mollusk shell IDP. The N-terminus of AP7 is a calcite binging, random-coil region, which affects calcium carbonate crystal