Fluorometric Measurement of Calmodulin-Dependent Peptide–Protein Interactions Using Dansylated Calmodulin

Cite as: Nuñez, E. et al. (2024). Fluorometric Measurement of Calmodulin-Dependent Peptide–Protein Interactions Using Dansylated Calmodulin. Bio-protocol 14(7): e4963. DOI: 10.21769/BioProtoc.4963. Copyright: © 2024 The Authors; exclusive licensee Bio-protocol LLC. This is an open access article under the CC BY 4.0 license (https://creativecommons.org/licenses/by/4.0/). 1 Published: Apr 05, 2024 Open Access Fluorometric Measurement of Calmodulin-Dependent Peptide–Protein Interactions Using Dansylated Calmodulin Eider Nuñez*, Arantza Muguruza-Montero, Sara M. Alicante, and Alvaro Villarroel Instituto Biofisika, CSIC-UPV/EHU, Leioa, Spain *For correspondence: eider[email protected] Abstract Key features • Dynamic fluorescence and real-time monitoring: dansyl-modified CaM enables sensitive, real-time fluorescence, providing valuable insights into the dynamics of molecular interactions and ligand binding. • Selective interaction and stable fluorescent adducts: DNSC selectively interacts with primary amino groups, ensuring specific detection and forming stable fluorescent sulfonamide adducts. • Versatility in research and ease of identification: D-CaM is a versatile tool in biological research, facilitating identification, precise quantification, and drug assessment for therapeutic development. • Sensitivity to surrounding alterations: D-CaM exhibits sensitivity to its surroundings, particularly ligand-induced changes, offering subtle insights into molecular interactions and environmental influences. Keywords: Dansyl-Calmodulin, CaM-target, Steady-state fluorescence spectroscopy, Calcium dependency, Dansylchloride, Peptide, Protein This protocol is used in: eLife (2023), DOI: 10.7554/eLife.81961 The assessment of peptide–protein interactions is a pivotal aspect of studying the functionality and mechanisms of various bioactive peptides. In this context, it is essential to employ methods that meet specific criteria, including sensitivity, biocompatibility, versatility, simplicity, and the ability to offer real-time monitoring. In cellular contexts, only a few proteins naturally possess inherent fluorescence, specifically those containing aromatic amino acids, particularly tryptophan. Nonetheless, by covalently attaching fluorescent markers, almost all proteins can be modified for monitoring purposes. Among the early extrinsic fluorescent probes designed for this task, dansyl chloride (DNSC) is a notable option due to its versatile nature and reliable performance. DNSC has been the primary choice as a fluorogenic derivatizing reagent for analyzing amino acids in proteins and peptides for an extended period of time. In our work, we have effectively utilized the distinctive properties of dansylated-calmodulin (D-CaM) for monitoring the interaction dynamics between proteins and peptides, particularly in the context of their association with calmodulin (CaM), a calcium-dependent regulatory protein. This technique not only enables us to scrutinize the affinity of diverse ligands but also sheds light on the intricate role played by calcium in these interactions. Cite as: Nuñez, E. et al. (2024). Fluorometric Measurement of Calmodulin-Dependent Peptide–Protein Interactions Using Dansylated Calmodulin. Bio-protocol 14(7): e4963. DOI: 10.21769/BioProtoc.4963. 2 Published: Apr 05, 2024 Graphical overview Fluorescence emission profiles of dansylated-calmodulin (D-CaM) in different states. Fluorescence emission spectra of D-CaM upon excitation at 320 nm are depicted. Conditions include apo-D-CaM (gray), holo-D-CaM (red), apo-D-CaM bound to peptide (blue), and holo-D-CaM bound to peptide (purple). Corresponding structural representations of D-CaM next to each condition are superimposed on the respective spectra along with the hydrophobicity of the dansyl environment, which increases upon binding of peptide or Ca2+ to D-CaM. Upon peptide binding to D-CaM, there is an enhancement in the fluorescent intensity of the spectra; upon Ca2+ binding, there is an enhancement of the intensity and a leftward shift of the spectra. Background Calmodulin (CaM), a pivotal Ca2+-binding protein, intricately regulates essential biological functions by binding to the cation, thereby exerting meticulous control over an array of effector proteins. This interaction induces conformational changes in CaM, critically influencing cellular processes such as muscle contraction and neurotransmitter release, as exhaustively elucidated by Chin and Means [1] and Rhoads and Friedberg [2]. The trajectory of unraveling CaM's multifaceted role spans decades, commencing in the 1970s with the identification of cyclic nucleotide phosphodiesterase as one of the initial proteins binding to CaM, as underscored by Rasmussen et al. [3]. Subsequently, Klee and Vanaman's seminal work in 1982 laid the foundational understanding of CaM's centrality in cellular signal transduction. The ongoing delineation of over 300 target peptides for CaM, meticulously documented by Klee and Vanaman [4], accentuates its indispensability in diverse cellular processes. Technological advancements, such as dansylation [5] and fluorogenesis [6], have significantly contributed to the precise identification and characterization of CaM targets, thereby enhancing our understanding of fundamental cellular processes. Among these tools, dansyl-CaM (D-CaM), a derivative of CaM conjugated with dansyl chloride (DNSC) [5-(dimethylamino)naphthalene-1-sulfonyl chloride], emerges as a distinctive and powerful instrument for analyzing interactions with peptides and proteins. DNSC specifically interacts with primary amino groups, forming stable blue or blue-green fluorescent sulfonamide adducts with aliphatic and aromatic amines (Tyr, Phe, Trp, etc.). The incorporation of dansyl into CaM enables the sensitive detection and thorough examination of this modified protein using fluorescence-based techniques. High-resolution structural scrutiny of apo-CaM and holo-CaM has unveiled intricate Ca2+-induced structural changes, laying bare hydrophobic interfaces, aligning with the observations of Chin and Means [1] and Rhoads and Cite as: Nuñez, E. et al. (2024). Fluorometric Measurement of Calmodulin-Dependent Peptide–Protein Interactions Using Dansylated Calmodulin. Bio-protocol 14(7): e4963. DOI: 10.21769/BioProtoc.4963. 3 Published: Apr 05, 2024 Friedberg [2]. Structural analyses of CaM–peptide complexes reveal a commonality in interaction patterns, particularly in the hydrophobic domains of the CaM protein, resembling interactions observed with classical proteins and inhibitors [2]. Significantly, these peptides manifest a positively charged amphiphilic alpha-helical structure, irrespective of their amino acid sequences, as expounded upon by O’Neil and DeGrado [7]. The observed high affinity (Kd) of these peptides, falling in the range of 10-9–10-12 M, positions them as putative bioactive entities, exceeding the affinity of traditional organic CaM inhibitors (Kd ~10-3 M), as demonstrated by Chen et al. [8] and Peersen et al. [9]. Nevertheless, the conventional understanding of CaM function encounters challenges with the discovery of proteins preferentially binding to apo-CaM, influencing their Ca2+ affinity, as exemplified by Smith et al. [10]. Additionally, CaM-binding partners exhibit minimal or no sequence similarity, posing a conundrum for attempts at structural categorization. In navigating this intricate terrain, the elucidated method emerges as a potent tool, facilitating expeditious, costeffective, and reliable identification of CaM targets even in instances of deviation from canonical binding patterns. This approach not only sheds light on diverse interactions involving D-CaM but also imparts invaluable insights into the nuanced relationships between proteins within the dynamic realm of CaM. Materials and reagents Biological materials 1. Calmodulin in pET14b plasmid or other bacterial protein expression vector 2. Peptides from Proteogenix (https://www.proteogenix.science/) or another source Peptides exhibiting sequences, structures, or functions favorable for CaM binding are recommended, with an advised minimum length of 5 and a maximum of 50 amino acids to avoid potential challenges in purity and yield. See Note 1. 3. E. coli BL21 DE3 (Sigma-Aldrich, catalog number: 69450-M) (other strains may work as well) Reagents 1. Dansyl chloride (DNSC) (Merck, Supelco, catalog number: 03641) 2. Sephadex G-25 (Merck, Sigma-Aldrich, catalog number: S5772) 3. Sepharose CL-4B (Merck, Sigma-Aldrich, catalog number: 4B200) 4. Base Trizma (Tris) (Merck, Sigma-Aldrich, catalog number: T1503) 5. HEPES (Thermo Fisher, Thermo Scientific Chemicals, catalog number: J16926.A1) 6. Potassium chloride (KCl) (Merck, Sigma-Aldrich, catalog number: P9541) 7. Ethyleneglycol-bis(β-aminoethyl)-N,N,Nʹ,Nʹ-tetraacetic acid (EGTA) (Merck, Millipore, catalog number: 324626) 8. Calcium chloride dihydrate (CaCl2) (Merck, Millipore, catalog number: 208291) 9. Sodium chloride (NaCl) (Merck, Sigma-Aldrich, catalog number: S3014) 10. 30% Acrylamide/Bis solution, 37.5:1 (Bio-Rad, catalog number: 1610158) 11. Sodium dodecyl sulfate (SDS) (Merck, Sigma-Aldrich, catalog number: 436143) 12. Ammonium persulfate (APS) (Merck, Sigma-Aldrich, catalog number: A3678) 13. Tetrametiletilendiamina (TEMED) (Thermo Fisher, catalog number: 17919) 14. Isopropanol (Merck, Sigma-Aldrich, catalog number: I9516) 15. Coomassie Brilliant Blue R-250 powder (CBB R-250) (Bio-Rad, catalog number: 1610400) 16. Methanol (Merck, Sigma-Aldrich, catalog number: 1060351000) 17. Acetic acid (glacial) 100% (Merck, Supelco, catalog number: 100066) 18. Phenyl-Sepharose CL-4B (Merck, Cytiva, catalog number: GE17-0150-01) 19. Dimethyl Sulfoxide (DMSO) (Merck, Sigma-Aldrich, catalog number: D2650) 20. N,N-Dimethylformamide (DMF) (Merck, Supelco, catalog number: DX1730) Cite as: Nuñez, E. et al. (2024). Fluorometric Measurement of Calmodulin-Dependent Peptide–Protein Interactions Using Dansylated Calmodulin. Bio-protocol 14(7): e4963. DOI: 10.21769/BioProtoc.4963. 4 Published: Apr 05, 2024 21. Acetone (Merck, Sigma-Aldrich, catalog number: 179124) 22. Ethanol (Merck, Sigma-Aldrich, catalog number: 34852-M) 23. Glycerol (Merck, Sigma-Aldrich, catalog number: G5516) 24. Nitrocellulose membrane (Merck, Cytiva, catalog number: GE10600001) 25. Bradford (Merck, Supelco, catalog number: B6916) 26. Ponceau Red (Ponceau 4R) (Merck, Supelco, catalog number: 18137) 27. Trifluoroacetic acid (TFA) (Merck, Sigma-Aldrich, catalog number: 302031) 28. Ammonium hydroxide solution (NH4OH) (Merck, Sigma-Aldrich, catalog number: 221228) 29. Acetonitrile (Merck, Sigma-Aldrich, catalog number: 34851) 30. LB broth (Lennox) (Merck, Sigma-Aldrich, catalog number: L3022) 31. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Merck, Sigma-Aldrich, catalog number: I5502) 32. Phenylmethanesulfonyl fluoride (PMSF) (Merck, Roche, catalog number: 10837091001) Solutions 1. Dansylation buffer (D buffer) (see Recipes) 2. Lysis buffer (L buffer) (see Recipes) 3. Equilibration buffer (CQ buffer) (see Recipes) 4. Wash buffer (CW buffer) (see Recipes) 5. High salt wash buffer (CHSW buffer) (see Recipes) 6. Elution buffer (CE buffer) (see Recipes) 7. Fluorescence buffer (F buffer) (see Recipes) 8. Calcium buffer (Ca buffer) (see Recipes) 9. APS 10% (see Recipes) 10. SDS 10% (see Recipes) 11. Tris-HCl 1.5 M, pH 8.8 (see Recipes) 12. Tris-HCl 1 M, pH 6.8 (see Recipes) 13. 15% Acrylamide electrophoresis gel RESOLVING (see Recipes) 14. Stacking acrylamide gel (see Recipes) 15. Running buffer 10× (see Recipes) 16. Loading buffer 5× (see Recipes) 17. Coomassie Blue (see Recipes) 18. Fast De-staining solution (see Recipes) 19. De-staining solution (see Recipes) 20. Ampicillin 100 μg/mL (see Recipes) 21. Ponceau Red (see Recipes) Recipes 1. Dansylation buffer (D buffer) Reagent Final concentration Quantity CaCl2 (1 M) 20 mM 2 mL Tris-HCl (1 M, pH 8.5) 100 mM 10 mL H2O n/a 88 mL Total n/a 100 mL 2. Lysis buffer (L buffer) Reagent Final concentration Quantity EDTA (1 M) 2 mM 0.2 mL Tris-HCl (1 M, pH 7.5) 50 mM 5 mL PMSF (0.1 M) 2 mM 2 mL Cite as: Nuñez, E. et al. (2024). Fluorometric Measurement of Calmodulin-Dependent Peptide–Protein Interactions Using Dansylated Calmodulin. Bio-protocol 14(7): e4963. DOI: 10.21769/BioProtoc.4963. 5 Published: Apr 05, 2024 H2O n/a 83.8 mL Total n/a 100 mL 3. Equilibration buffer (Q buffer) Reagent Final concentration Quantity CaCl2 (1 M) 5 mM 0.5 mL Tris-HCl (1 M, pH 7.5) 50 mM 5 mL NaCl (1 M) 100 mM 10 mL H2O n/a 84.5 mL Total n/a 100 mL 4. Wash buffer (W buffer) Reagent Final concentration Quantity CaCl2 (1 M) 0.1 mM 10 µL Tris-HCl (1 M, pH 7.5) 50 mM 5 mL NaCl (1 M) 100 mM 10 mL H2O n/a 84.90 mL Total n/a 100 mL 5. High salt wash buffer (CHSW buffer) Reagent Final concentration Quantity CaCl2 (1 M) 0.1 mM 10 µL Tris-HCl (1 M, pH 7.5) 50 mM 5 mL NaCl (1 M) 500 mM 50 mL H2O n/a 44.90 mL Total n/a 100 mL 6. Elution buffer (E buffer) Reagent Final concentration Quantity EGTA (1 M) 1 mM 100 µL Tris-HCl (1 M, pH 7.5) 50 mM 5 mL Total n/a 94.9 mL 7. Fluorescence buffer (F buffer) Reagent Final concentration Quantity KCl (1 M) 120 mM 12 mL HEPES (1 M, pH 7.4) 50 mM 5 mL NaCl (1 M) 5 mM 0.5 mL EGTA (1 M) 5 mM 0.5 mL H2O n/a 82 mL Total n/a 100 mL See Note 2. 8. Calcium buffer (Ca buffer) Reagent Final concentration Quantity KCl (1 M) 120 mM 12 mL HEPES (1 M, pH 7.4) 50 mM 5 mL NaCl (1 M) 5 mM 0.5 mL EGTA (1 M) 5 mM 0.5 mL CaCl2 (1 M) 20 mM 2 mL H2O n/a 80 mL Cite as: Nuñez, E. et al. (2024). Fluorometric Measurement of Calmodulin-Dependent Peptide–Protein Interactions Using Dansylated Calmodulin. Bio-protocol 14(7): e4963. DOI: 10.21769/BioProtoc.4963. 6 Published: Apr 05, 2024 Total n/a 100 mL See Note 3. 9. APS 10% Reagent Final concentration Quantity APS 10% 1 g H2O n/a 10 mL 10. SDS 10% Reagent Final concentration Quantity SDS 10% 1 g H2O n/a 10 mL 11. Tris-HCl pH 8.8 1.5 M Reagent Final concentration Quantity Tris 1.5 M 36.3 g H2O n/a 200 mL *Note 4 12. Tris-HCl 1 M pH 6.8 Reagent Final concentration Quantity Tris 1 M 12.1 g H2O n/a 100 mL See Note 5. 13. 15% Acrylamide electrophoresis gel RESOLVING (for one gel) Reagent Final concentration Quantity Acrylamide 15% 2.5 mL Tris 1.5 M, pH 8.8 390 mM 1.3 mL 10% SDS 0.1% 50 µL 10% APS 0.1% 50 µL TEMED 0.004% 2 µL H2O n/a 1.2 mL Total n/a 5 mL 14. Electrophoresis gel STACKING (for one gel) Reagent Final concentration Quantity Acrylamide 4.95% 0.33 mL Tris 1 M, pH 6.8 125 mM 0.250 mL 10% SDS 0.1% 20 µL 10% APS 0.1% 20 µL TEMED 0.01% 2 µL H2O n/a 1.2 mL Total n/a 2 mL 15. Running buffer 10× Reagent Final concentration Quantity Tris 25 mM 33 g Glycine 1.92 M 144 g Cite as: Nuñez, E. et al. (2024). Fluorometric Measurement of Calmodulin-Dependent Peptide–Protein Interactions Using Dansylated Calmodulin. Bio-protocol 14(7): e4963. DOI: 10.21769/BioProtoc.4963. 7 Published: Apr 05, 2024 SDS 1% 10 g H2O n/a 1,000 mL Total n/a 1,000 mL 16. Loading buffer 5× Reagent Final concentration Quantity Tris HCl 1 M pH 6.8 250 mM 5 mL Glycerol 50% 10 mL SDS 10% 2 g Bromophenol blue 1% 0.125% 250 µL H2O n/a 4.75 mL Total n/a 10 mL 17. Coomassie Blue Reagent Final concentration Quantity Ethanol 50% 125 mL Acetic acid 10% 25 mL CBB R-250 0.25% 625 g H2O n/a 100 mL Total n/a 200 mL *Note 6 18. Fast Coomassie de-staining Reagent Final concentration Quantity Ethanol 10% 10 mL Acetic acid 20% 20 mL H2O n/a 70 mL Total n/a 100 mL 19. Coomassie de-staining Reagent Final concentration Quantity Acetic acid 10% 10 mL H2O n/a 90 mL Total n/a 100 mL 20. Ampicillin 100 µg/mL Reagent Final concentration Quantity Ampicillin 0.1 g/mL 1 g H2O n/a 10 mL Laboratory supplies 1. Standard dialysis tubing with 2000 MW cut off (e.g., Membra-Cel, Viskase, catalog number: 300911011) 2. Filters, 0.2 µm diameter (SARSTEDT, catalog number: 83.1826.001) 3. Protein concentrator (Amicon R-Ultra, 15 mL, 3 KDa) (Millipore, Merck, catalog number: UFC9003) 4. Magnetic stirrer (Fisherbrand, Fisher Scientific, catalog number: 11808892) 5. Small columns (1–5 mL bed volume) (empty PD-10 column for gravity flow purification) (Cytiva, catalog number: 17043501) 6. Electrophoresis chamber (Mini-PROTEAN Tetra Vertical Electrophoresis Cell) (Bio-Rad, catalog number: 1658004) Cite as: Nuñez, E. et al. (2024). Fluorometric Measurement of Calmodulin-Dependent Peptide–Protein Interactions Using Dansylated Calmodulin. Bio-protocol 14(7): e4963. DOI: 10.21769/BioProtoc.4963. 8 Published: Apr 05, 2024 7. Handcast electrophoresis gel accessories (for 1.00 mm thickness gels) (Bio-Rad, catalog number: 1658001FC) 8. Microtubes, 1.5 mL (Eppendorf, catalog number: 0030120086) 9. Centrifuge tube, 50 mL (Avantor, VWR, catalog number: 525-0634) Equipment 1. Orbital incubator (Sartorius, model: Certomat BS-1, catalog number: 20444445202) 2. Spectrophotometer (VWR, model: V-1200, catalog number: 634-60009 3. Balance (Fisher Scientific, model: FPOS622, catalog number: 8344272595) 4. Hot plate stirrer (Fisher Scientific, model: AREX, catalog number: 15369664) 5. pH meter (pH and ORP table-top bench meter laboratory Tester Hanna) (Servovendi, catalog number: 1965) 6. Centrifuge with fixed-angle rotor (Beckman, model: Avanti J-20 XP, catalog number: 8043-30-1171) 7. Rotor JA14 (Beckman, catalog number: 339247) 8. Microcentrifuge (Eppendorf, model: 5430R, catalog number: 5428000210) 9. Eppendorf® rotor F-35-6-30 (Eppendorf, Merck, catalog number: EP5427716009) 10. Eppendorf® rotor F-45-48-11 (Eppendorf, Merck, catalog number: EP5427755004) 11. Spectro fluorimeter (we used an SLM-Aminco 8100 Series 2, not commercially available) 12. Quartz cuvette with two transparent faces (we used 3 mm light path, 100 μL volume) (Hellma, catalog number: 105-251-15-40) Software and datasets 1. Sigmaplot 11.0 (Systat Software, Inc; 2008) (any other scientific data analysis and graphing software can be used) 2. Maxchelator (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/CaEGTA-TS.htm) Procedure A. CaM expression and purification The human CaM gene, inserted into the pET-14b expression vector, is introduced into BL21-DE3 E. coli (other bacteria strain to express non-toxic heterologous genes can also be used). The purification procedure for CaM has been adapted from existing literature [11] and results in substantial yields of soluble protein, as outlined below. 1. Protein expression a. Cultivate BL21-DE3 cells from glycerol stock in 1 L of LB medium at 37 °C, supplemented with 100 μg/mL ampicillin, until the optical density (A600) reaches 0.8–1. b. Induce protein expression by adding 0.4 mM IPTG and continue cultivation for 4–6 h at 37 °C or overnight at 20 °C. 2. Cell harvesting and resuspension a. Centrifuge the cells to collect them (9,000× g for 9 min at 4 °C) and wash the cell pellet twice with 50 mL of fresh lysis buffer. b. Resuspend the cell pellet in 30 mL of lysis buffer and store the sample in 10 mL aliquots at -20 °C. 3. Sample preparation a. Thaw an aliquot on ice and perform sonication (three cycles of 10 s at 50 kHz; keep the sample on ice). Cite as: Nuñez, E. et al. (2024). Fluorometric Measurement of Calmodulin-Dependent Peptide–Protein Interactions Using Dansylated Calmodulin. Bio-protocol 14(7): e4963. DOI: 10.21769/BioProtoc.4963. 9 Published: Apr 05, 2024 b. Subject the sample to three freeze–thaw cycles by alternating between a dry ice ethanol bath and a 37 °C water bath. c. Centrifuge the sample in a microcentrifuge at 14,000× g for 15 min. d. Heat the supernatant to 95 °C for 5 min, followed by centrifugation as previously described. This step leverages CaM's enhanced thermal stability. 4. Chromatography a. Introduce CaCl2 to the supernatant (final concentration: 5 mM). Load the sample at room temperature onto a 5 mL Phenyl–Sepharose column pre-equilibrated with CQ buffer. The chromatography can be achieved through gravity flow or by utilizing a peristaltic pump. Wash the column with 20 column volumes of CW buffer followed by 10 column volumes of CHSW buffer. b. Elute CaM with 20 column volumes of CE buffer, taking fractions of 500 µL. 5. Analysis and storage a. Mix 20 µL of CaM with 5 µL of 5× loading buffer. Load the mixture onto a 15% acrylamide gel and perform electrophoresis using a 1× dilution of the running buffer. Run the gel at a voltage of approximately 120–150 V for 90 min. Stain the gel with Coomassie Blue for 10 min and destain first using fast destaining for 15 min followed by regular destaining for 30 min. Concentrate the sample to have a final concentration of at least 1 mg/mL using an Amicon centrifugal filter of 3 kDa. Dialyze the fractions against MilliQ water, b. Store the purified CaM at -20 °C at 1 mg/mL in fractions of 1 mL or lyophilize it in fractions of 1 mL. c. CaM concentration estimation can be done via absorbance at 276 nm, with ε276 = 3,030 M-1·cm-1. Alternatively, employ the Bradford method for quantification. B. Dansylation of calmodulin 1. CaM preparation Begin by diluting CaM in D buffer to achieve a final concentration of 1 mg/mL. 2. Dansyl chloride preparation a. Dissolve dansyl chloride in acetone at a concentration of 2.17 mg/mL. b. Store this dansyl chloride solution at either 4 °C or -20 °C in a dark environment. It remains stable for an extended period, often several months. 3. Dansylation process a. Add 12.5 μL of the prepared dansyl chloride solution to 1 mL of the CaM solution at 1 mg/mL. This results in a final concentration of dansyl chloride of approximately 100 μM. b. Incubate the mixture at room temperature, in darkness, for 2 h. During this time, gently vortex the mixture every 20 min. 4. Separation of dansylated CaM (D-CaM) a. To separate D-CaM from any unreacted dansyl chloride, you will need a disposable column packed with approximately 1 mL of Sephadex G-25. b. Equilibrate approximately 250 mg of dry resin with distilled water. c. Load the D-CaM mixture onto the column and collect fractions of 50–100 μL each. d. The initial fractions eluted, known as the "excluded fraction," contain the D-CaM conjugate. See Note 7. To determine the specific dansylated residues in CaM, tryptic digestion coupled with mass spectroscopy or gas-phase protein sequencers have been employed, reporting dansylation at either Lys75 or Lys 115 [12,13]. The mass spectrometry analysis revealed the binding of up to four dansyl molecules per CaM. Furthermore, tandem mass spectrometry of tryptic peptides strongly indicates dansylation at Ala1 and Lys148 [14]. The identification of the remaining two dansylated residues is pending further investigation; however, the data are consistent with the possibility of them being Lys75 and Lys115 [14]. 5. Fraction analysis Swiftly verify the presence of the protein in the collected fractions by performing dot blotting on nitrocellulose and staining it with Ponceau Red. Additionally, analyze the fractions using 15% SDS-PAGE gels. For a more detailed examination, record the emission spectra of each sample (as further explained Cite as: Nuñez, E. et al. (2024). Fluorometric Measurement of Calmodulin-Dependent Peptide–Protein Interactions Using Dansylated Calmodulin. Bio-protocol 14(7): e4963. DOI: 10.21769/BioProtoc.4963. 16 Published: Apr 05, 2024 Acknowledgments This article has been funded through the following research projects: the Ministry of Science and Innovation under the project PID2021-128286NB-100 funded by MCIN/AEI/10.13039/501100011033/FEDER, UE; support from the Basque Government under the project IT1707-22. S.M-A and E.N. received support from predoctoral (PRE_2021_1_0101) and postdoctoral (POS_2021_1_0017) contracts, respectively, provided by the Basque Government. This article has been previously described and validated by Alaimo et al. [14] and Nuñez et al. [23]. Subsequent verification has been conducted in various studies, as documented in the validation of the protocol section. Competing interests The authors declare that they have no competing interests. References 1. Chin, D. and Means, A. R. (2000). Calmodulin: a prototypical calcium sensor. Trends Cell Biol. 10(8): 322– 328. 2. Rhoads, A. R. and Friedberg, F. (1997). 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