Dataset DOI: 10.5061/dryad.f1vhhmh8p

Description of the data and file structure

Microscale thermophoresis

Microscale thermophoresis (MST) was performed in the presence of the fluorescently labeled FrhA_Split-PBD using unlabeled ligands such as AGYTD. FrhA_Split-PBD was labeled using the primary amine-based labeling kit of Red-NHS 2nd Generation (NanoTemper Technologies, San Francisco, CA, USA). The labeling reaction was performed in MST buffer (50 mM HEPES pH 8, 150 mM NaCl, 5 mM CaCl₂) with a protein concentration of 20 µM (molar ratio dye:protein ≈ 3:1). The reaction mixture was incubated at room temperature for 1 h in a dark environment for protection from photobleaching. Subsequently, the unreacted dye was removed from the labeled protein using the supplied desalting column, which was equilibrated with MST buffer. The degree of labeling (DOL) was determined using UV/Vis spectrophotometry at 650 and 280 nm—a DOL of 0.5 was typically achieved.

After dialysis of the labeled protein against a buffer containing 50 mM Tris-HCl pH 9, 150 mM NaCl, 5 mM CaCl₂, and 0.05% v/v Tween-20, the post-dialysis buffer was filtered and used to solubilize the ligands. A series of sixteen 1:1 dilutions of the ligands was prepared, resulting in ligand concentrations ranging from 440 μM to 13.4 nM. After 1 h incubation, the samples were loaded into standard monolith NT.115 capillaries from NanoTemper Technologies. The MST measurements were conducted using a Monolith NT.115 instrument at an ambient temperature of ~ 22 °C, with instrument parameters set to 20% excitation power and 40% MST power. Three independently prepared replicates were performed for each ligand. The NanoTemper MO.Affinity Analysis software (version 2.3, NanoTemper Technologies) was used to analyze the fluorescence data, fit sigmoidal binding models, and provide the dissociation constants. In the software, the K_d values were determined by plotting the change in normalized fluorescence [ΔF_norm(‰) = F₁/F₀] against the logarithm of the concentrations of peptide ligands, where F₁ and F₀ respectively correspond to the heated and baseline regions of the thermophoresis traces [43]. The time between F₁ and F₀ was set to 20 seconds for analysis.

Multiple Sequence Alignment

Protein sequence alignment of the PBD and split domains of FrhA with their homologs in MpIBP. The sequences forming the domains were determined by X-ray crystal structures (FrhA_Split_PBD PDB ID: 9YBQ; MpIBP _Split_PBD PDB ID: 6X5W). An asterisk (*) indicates positions which have a fully conserved residue. A colon (:) indicates conservation between groups of strongly similar properties, and a period (.) indicates conservation between groups of weakly similar properties. A blank space indicates no similarity at the position. Residues lining the ligand-binding pocket are indicated by red boxes. The alignment was generated by Clustal Omega on the EMBL-EBI Job Dispatcher. The sequences share 60.1% identity, and the residues lining the ligand-binding pocket are conserved.

Files and variables

File: Data_FEBSL-25-0854.zip

Description: 

This ZIP file contains two directories.

The MST directory contains the following files:

• FrhA_PBD 20s_CompareResultsRawData.xlsx
• Export-Table 1.csv
• Fit-Values-Table 1.csv

The XLSX file contains two sheets. CSV files are respectively exported from sheet 1 and sheet 2 from the ELSX file.  The CSV files contain no additional information, but the XLSX contains graphs of plotted data. 

The XLSX file contains data of MST experiments of FrhA_Split-PBD with all ligands shown in the paper. Both analysis results and raw measurements are included.

The following variables are relevant for data re-processing:

• Peptide Names (AGYTD, GYTD, YTD ...): The ligands tested and described in the main publication.
• Target (FrhA 38 kDa): The macromolecule/protein construct described in the main publication.
• TargetConcentration: The concentration of FrhA 38 kDa in M.
• Bound: The fluorescence level in the Kd model is considered as the ligand fully bound in arbitrary units.
• Unbound: The fluorescence level in the Kd model is considered as the ligand fully unbound in arbitrary units.
• Kd: The dissociation constant determined by the Kd model in M.
• TargetConc: Same as TargetConcentration. The value for all runs should be 0.00000008 M.
• Standard Deviation: The standard deviation of Kd.
• Dose: Concentration of the ligand/peptide in M.
• [Peptide name]: The raw fluorescence level in arbitrary units.
• DeltaFNorm: normalized change in fluorescence in arbitrary units.

Note that Dose, [peptide name], and DeltaFNorm in Export-Table 1.csv show the fitted data, while these variables show the raw data in Fit-Values-Table 1.csv.

The Multiple Sequence Alignment directory contains the following:
Clustal Omega _ Job Dispatcher _ EMBL-EBI.html 

The HTML file can be opened by most browsers and requires the files in the directory to display the multiple sequence alignment results. The HTML file, when opened, displays the full sequence of FrhA_Split_PBD and MpIBP_Split_PBD.

This serves as a permanent record of the multiple sequence alignment. The main data are the protein sequence and their alignment. Under the amino acid sequences, a row of symbols shows the amino acid matching. The symbol "*" indicates an exact amino acid match. The symbol " ": indicates strong amino acid similarity. The symbol ". indicates weak amino acid similarity. The space between them shows no amino acid similarity.