*3.6. Tb3+ Binding Fluorescence*

Binding of Tb3+ ions to hOtolC1q and its mutants was assessed using steady-state fluorescence. Terbium (III) chloride was dissolved in MilliQ water to a final concentration of approximately 0.5 M. Exact concentration of TbCl3 was determined by titration of diluted stock solution with EDTA in the presence of xylenol orange. Aliquots of diluted TbCl3 were added to 2 mL 3.7 μM protein solution in a 10 × 10 mm quartz SUPRASIL® cuvette (Hellma Analytics, Müllheim, Germany) and incubated for 15 min at room temperature. Subsequently, fluorescence emission at 520-580 nm was recorded using an excitation wavelength of 280 nm using a Fluorolog-SPEX fluorimeter (HORIBA Scientific, Jobin-Yvon, Kyoto, Japan) equipped with a Peltier heating accessory set at 20 ◦C. The bandwidth was set at 5 nm for both excitation and emission monochromators. A cut-off filter absorbing below 350 nm was installed in the emission path. Obtained fluorescence intensities were processed and fitted to a model based on work by Gonzalez et al. [62,63]. Data analysis was conducted as described [27].

#### *3.7. Circular Dichroism*

Circular dichroism of 0.2 mg/mL proteins in H10Na500G5 with 1 mM EDTA, 0.1 mM CaCl2, 1 mM CaCl2, 10 mM CaCl2, 100 mM CaCl2 or 7-fold excess of TbCl3 was measured in 1 mm quartz SUPRASIL® cuvettes (Hellma Analytics, Müllheim, Germany) using Jasco J-815 spectropolarimeter (Jasco, Easton, MD, USA) with a Peltier temperature control accessory set at 20 ◦C. The proteins were incubated with the additives at room temperature for at least 1 h before the measurements. The spectra were collected between 200 and 260 nm every 1 nm at scanning speed of 50 nm/min with five accumulations. Data, for which photomultiplier voltage was below 600 V, were analyzed. CD spectra of the proteins were corrected for buffer background signal and normalized for protein composition and concentration using an equation [64]:

$$\theta\_{mrw} = \frac{\theta \cdot MRW}{10 \cdot c \cdot l} \left[\frac{\text{deg} \cdot \text{cm}^2}{\text{dmol}}\right] \tag{3}$$

where *θmrw* is a mean residue ellipticity, *θ*—ellipticity [degrees], *MRW—*mean residual weight of a protein [g/mol], *c—*protein concentration [g/L] and *l—*optical pathlength of a cuvette [cm]. The secondary structure content was estimated using CDPro [65].

#### *3.8. Analytical Ultracentrifugtion*

Sedimentation velocity analytical ultracentrifugation (SV AUC) was conducted in a Beckman Coulter ProteomeLab XLI analytical ultracentrifuge (Beckman Coulter, Brea, CA, USA) with an An60Ti rotor and assembled cells with two-channel 12 mm charcoal filled Epon® centerpieces and quartz windows, or sapphire windows for samples containing DTT. The proteins were analyzed at concentrations of 0.1, 0.25 and 0.5 mg/mL in H10Na500G5 with 1 mM EDTA or 10 mM CaCl2. Additional measurements were made for 0.25 mg/mL protein with EDTA and CaCl2 supplemented with 1 mM DTT. Effect of Tb3+ was analyzed by centrifuging 0.25 mg/mL protein with 7-fold molar excess of TbCl3. Assembled cells with the samples were preincubated in the ultracentrifuge for 3 h at 20 ◦C and then centrifuged at 50,000 rpm (approximately 200,000× *g* at the bottom of the cell) overnight. The absorbance scans at 280 nm were collected continuously with 0.003 cm resolution. The scans were time-corrected [66] and analyzed in SEDFIT (version, 16.1c, October 2018, available at https://sedfitsedphat.nibib.nih.gov/, accessed 16 August 2021) using a continuous *c*(*s*) distribution model [67] with at least 20 points per 1 S. Partial specific volumes of the proteins, densities and dynamic viscosities of the solvents were calculated using SEDNTERP (version 3.0.3, 14 March 2021, available at http://www.jphilo.mailway.com/download.htm, accessed 16 August 2021). Maximum entropy regularization with *p* = 0.95 was used. Simplex and Marquardt–Levenberg algorithms were alternately used until the RMSD converged. Among the results of the calculations were sedimentation coefficients (*s*), sedimentation coefficients corrected for water at 20 ◦C (s20,w), weight-averaged sedimentation coefficients (*s*20,*w*), apparent molecular weights (*MW*app) and frictional ratios (*f*/*f* 0). *c*(*s*) distributions were visualized using GUSSI (version 1.4.2, 24 July 2018, available at https://www.utsouthwestern.edu/labs/mbr/software/, accessed 16 August 2021) [68] and Origin Pro 9.0 software.

#### *3.9. Thermal Shift Assay*

Thermal shift assay (TSA) was conducted as described [27]. Five μM solutions of the proteins in H10Na500G5 were supplemented with SYPRO Orange at concentration of 5× (hOtolC1q, R339S, R402P, Q426R) or 10× (R342W). The measurements were done in the presence of 1 mM EDTA, 0.1 mM CaCl2, 1 mM CaCl2, 10 mM CaCl2, 100 mM CaCl2, and 7-fold molar excess of TbCl3. Final sample volume was 20 μL. The samples and the non-protein controls (Figure S4) were aliquoted into a 96-well plate in triplicate, covered with optically clear foil and incubated at room temperature for at least 1 h before the measurements. Fluorescence of SYPRO Orange was measured using Applied Biosystems ImageQuant5 qPCR thermal cycler (Thermo Fisher Scientific) with optical filters set as x1-m3 (excitation at 470 ± 15 nm, emission at 587 ± 10 nm) between 20 and 99 ◦C during heating at 0.033 ◦C/s. The data were analyzed using Protein Thermal Shift software (Thermo Fisher Scientific). Transition temperatures (*T*m) were determined from the derivative of fluorescence with increasing temperature (d*F*/d*T*).

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/ijms22169085/s1, Supplementary File S1.pdf—multiple sequence alignment of mammalian sequences of gC1q domain of otolin-1 found during NCBI BLAST search. Supplementary File S2.pdf contains Figures S1–S4, Tables S1 and S2. Figure S1. Dithiothreitol (DTT) does not affect the oligomerization of hOtolC1q R342W and R402P. Figure S2. Estimation of the secondary structure content of hOtolC1q and its mutants. Figure S3. Purification of hOtolC1q and its mutants. Figure S4. Background fluorescence in the thermal shift assay. Table S1. Parameters derived from the sedimentation velocity analytical ultracentrifugation. Table S2. Transition temperature (*T*m) values (in ◦C) determined using the thermal shift assay.

**Author Contributions:** Conceptualization, R.H., A.O. and P.D.; methodology, R.H., A.O. and P.D.; validation—R.H., A.O.; investigation—R.H., resources—R.H., A.O. and P.D., writing—original draft, R.H.; writing—review and editing—A.O. and P.D., visualization—R.H., supervision—A.O. and P.D., project administration—A.O., funding acquisition—R.H., A.O. and P.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Science Centre, Poland (grant number 2017/27/ N/NZ1/01319) and by a statutory activity subsidy from the Polish Ministry of Science and High Education for the Faculty of Chemistry of Wrocław University of Science and Technology.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data and materials underlying this article will be shared on request to one of the corresponding authors.

**Acknowledgments:** Sylwia Groborz and Martyna U´sciła are kindly acknowledged for preparing mutated constructs of hOtolC1q.

**Conflicts of Interest:** The authors declare no competing interest.

#### **Abbreviations**

