Anotop 10

The iSCAMS measurement was carried out using a Refeyn TwoMP mass photometer (Refeyn Ltd.). Cleaned coverslips were assembled into flow chambers. All the buffers used for analysis were filtered through a syringe filter with 0.45-μm pore size (Anotop 10, Whatman). For measurements, all samples were freshly diluted from stock solutions. Sample proteins were diluted to the final concentration of 20 nM in 20 mM HEPES (pH 7.5) buffer containing 200 mM or 400 mM NaCl. The protein solution was loaded into the sample well after finding the focus with the buffer. Obtained data were processed with the DiscoverMP program (Refeyn Ltd.)

The iSCAMS measurement was carried out using a Refeyn TwoMP mass photometer (Refeyn Ltd.). Cleaned coverslips were assembled into flow chambers. All the buffers used for analysis were filtered through a syringe filter with 0.45-μm pore size (Anotop 10, Whatman). For measurements, all samples were freshly diluted from stock solutions. Sample proteins (100 nM) were diluted to the final concentration of 20 nM in 20 mM HEPES (pH 7.5) buffer containing 200 mM or 400 mM NaCl. The protein solution was loaded into the sample well after finding the focus with the buffer. Obtained data were processed with the DiscoverMP program (Refeyn Ltd.)

The iSCAMS measurement was carried out using the Refeyn TwoMP mass photometer (Refeyn Ltd.). Cleaned coverslips were assembled into flow chambers. All the buffers were filtered through a syringe filter with 0.02 µm pore size (Anotop 10, Whatman). All samples were diluted from stock solutions, and measurements were taken either immediately after diluting the samples (i.e., before reaching an association-dissociation equilibrium) or after keeping the diluted sample at 4 °C for various lengths of time. To detect the hexadecameric Cep63 (424–541)•Cep152 (1205–1295) complex, the sample (110 µM) prepared in a buffer [20 mM sodium citrate, 150 mM NaCl (pH 5.5), and 1 mM TCEP] was diluted to 200 nM and immediately analyzed. To examine the reversible nature of the hexadecameric complex, the sample was diluted to 100 nM, equilibrated for various lengths of time, and analyzed.

DLS measurements were performed at 25°C using a DynaPro MS-X instrument (Wyatt Technology Corporation, Santa Barbara, CA, USA) in a thermostated 30 μL quartz cuvette. The protein solutions and the buffer were filtered through 0.02 μm Anotop 10 filters (Whatman plc, Brentford, Middlesex, UK) before the measurements. Sets of DLS data were acquired every 5 s until at least 20 sets of data were obtained. The measurements were performed in triplicate. Dynamics V6 software (Wyatt Technology Corporation, Santa Barbara, CA, USA) was used in data collection and processing. The experimental autocorrelation curves were analyzed to obtain the particle size distributions using the implemented regularization fit.

DLS measurements were obtained on a Zetasizer Nano ZS instrument (Malvern), using disposable ZEN0112 polystyrene cuvettes. Settings used for particle size measurements were the following: solvent refractive index 1.330, viscosity 0.8872 cP, protein refractive index 1.450, protein absorption 0.001, temperature 25 °C, equilibration 2 min, measurement angle 173 Å backscatter, analysis model multiple narrow modes. Samples were prepared at 3–4 μM protein concentration in 100 mM potassium phosphate, pH 7.4. Native holo forms were preincubated for 30 min with additional 100 μM PLP and 2 mM cDopa-bound species for 30 min before analysis. All samples were filtered using a 0.02 μm Anotop 10 filter (Whatman). Data of particle size are reported as mean ± SEM of three independent replicates. Each single value derives from at least 40 measurements, each one consisting of 12–18 repetitions.

DLS measurements were carried out for rWT/C18S Ocm at 20.0 °C using a Zetasizer Nano ZS system (Malvern Instruments Ltd.). The backscattered light from a 4 mW He-Ne laser (632.8 nm) was collected at an angle of 173°. Protein concentration was 1.3–1.7 mg/mL. The buffer conditions: 20 mM H₃BO₃-KOH, pH 8.4, 1.5 mM EDTA or 1 mM CaCl₂/MgCl₂. The protein samples were preliminarily passed through 0.02 µM Whatman Anotop 10 syringe filters. The acquisition time for a single autocorrelation function was 100 s. The resulting autocorrelation function was an average of ten measurements. The intensity-weighted size distributions were calculated using preset parameters for distilled water at 20 °C (refractive index of 1.33 and a viscosity value of 1.0031 cP). The estimates of mean hydrodynamic radii (R_h) of Ocm were derived from these distributions.