Labeling of Specific Cysteines in Proteins Using Reversible Metal Protection  Michael C. Puljung, William N. Zagotta  Biophysical Journal  Volume 100, Issue 10, Pages 2513-2521 (May 2011) DOI: 10.1016/j.bpj.2011.03.063 Copyright © 2011 Biophysical Society Terms and Conditions

Figure 1 Design of the cysteine protection method. (A) Schematic diagram depicting the specific cysteine protection method, CyMPL. The Cd2+ binding affinity of a desired cysteine is increased by placing an additional metal-binding amino acid nearby. Cd2+ is used to protect the desired cysteine while background cysteines are reacted with a nonfluorescent modifying reagent. Upon removal of Cd2+, the specific cysteine is available to react with fluorophore. (B) Reaction scheme for cysteine reacting with bimane C3-maleimide and competitive binding by Cd2+. (C) Time course of fluorescence increase upon reaction of the Cys Only peptide with bimane C3-maleimide at different [CdCl2]. (D) Concentration-response curve showing the rate (normalized to 0 Cd2+) of the reaction of the Cys Only peptide with bimane C3-maleimide as a function of [CdCl2]total. Data were fit with a single-site binding curve (see Materials and Methods). (E) Box plot showing the apparent Kd and ΔG for Cd2+ binding to Cys Only peptide, GSH, and the cysteine-free C-terminal region of the HCN2 ion channel with a single cysteine inserted at position 563 (HCN2 S563C) or position 570 (HCN2 K570C). Biophysical Journal 2011 100, 2513-2521DOI: (10.1016/j.bpj.2011.03.063) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 2 Cd2+ affinity of helical peptides with introduced minimal metal-binding sites. (A) Cartoons depicting peptides with a single cysteine (Cys-only), a cys-his pair (Cys/His i+3), and a cys-cys pair (Cys/Cys i+3) spaced at residues i and i+3 as well as a helix from MBP E22C,K25C. (B) Time course of fluorescence increase upon reaction of Cys/His i+3 peptide with bimane C3-maleimide at different [CdCl2]. (C) Concentration-response curve showing the rate (normalized to 0 Cd2+) of the reaction of Cys Only peptide, Cys/His i+3, and Cys/Cys i+3 with bimane C3-maleimide as a function of [CdCl2]total. Data were fit as indicated in Materials and Methods. (D) Box plot showing the apparent Kd and ΔG for Cd2+ binding to peptides or proteins containing a single cysteine, cys-his binding sites, and cys-cys binding sites. Biophysical Journal 2011 100, 2513-2521DOI: (10.1016/j.bpj.2011.03.063) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 3 Cd2+ binding to a β-strand metal-binding site. (A) Cartoons depicting β-strand Cd2+-binding sites containing a single cysteine (HCN2cys-free S563C) and a cys-his pair (HCN2cys-free S563C,K565H). (B) Box plot showing the apparent Kd and ΔG for Cd2+ binding to Cys-only peptide, and the constructs depicted in A. Biophysical Journal 2011 100, 2513-2521DOI: (10.1016/j.bpj.2011.03.063) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 4 Application of protection of a cys-his motif to label individual components in a mixture of soluble proteins. (A) Cartoon showing HCN2 with a cys-his pair in the β roll (HCN2cys-free S563C,K565H; HCN-cys,his) and a fusion of MBP and HCN2 with a single cysteine (MBP-HCN2cys-free K570C; MBP-HCN-cys). (B) Schematic diagram representing an experiment in a mixture of proteins in solution in which MBP-HCN-cys is labeled with Flc while HCN-cys,his is protected from reaction with Flc by binding Cd2+. (C) SDS-PAGE showing Flc labeling of MBP-HCN-cys and HCN-cys,his at increasing [CdCl2]. A Coomassie stain of the same gel is shown below. (D) Schematic diagram representing an experiment in which HCN-cys,his is selectively labeled with Flc by first protecting it with Cd2+ while MBP-HCN-cys is blocked with NEM, and then removing Cd2+ and labeling the deprotected HCN-cys,his with Flc. (E) SDS-PAGE showing Flc labeling of MBP-HCN-cys and HCN-cys,his after reaction of the proteins with NEM in increasing [CdCl2]. The final lane shows labeling of both proteins in the absence of Cd2+ or NEM. A Coomassie stain of the same gel is shown below. Biophysical Journal 2011 100, 2513-2521DOI: (10.1016/j.bpj.2011.03.063) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 5 Application of the cysteine protection method to reduce the fluorescence background when labeling a protein in a cellular environment. (A) Cartoon depicting the fusion protein expressed in Xenopus laevis oocytes for extracellular labeling experiments. The protein is based on the single-pass membrane protein Syntaxin1A with GFP fused to the N-terminus (intracellular) and MBP K25C,K26C fused to the C-terminus (extracellular). (B) Confocal images of Xenopus laevis oocytes injected with mRNA encoding GFP-Syx-MBP or water (control). Oocytes were labeled with Alexa Fluor 546 C5 maleimide after incubation in buffer, buffer containing 100 μM NEM, or buffer containing 100 μM NEM + 1 mM CdCl2. Scale bar = 500 μm. (C) Plot showing the intensity of AlexaFluor 546 C5 maleimide labeling versus GFP fluorescence for oocytes injected with RNA encoding GFP-Syx-MBP with no block (cyan circles), after 100 μM NEM block (gray triangles), and after 100 μM NEM block in the presence of 100 μM (magenta squares) or 1 mM (green diamonds) CdCl2. (D) Box plot showing (from left to right) AlexaFluor 546 labeling intensity of water-injected oocytes with no block (cyan), after 100 μM NEM block (gray), and after 100 μM NEM block in the presence of 100 μM (magenta) or 1 mM (green) CdCl2. Biophysical Journal 2011 100, 2513-2521DOI: (10.1016/j.bpj.2011.03.063) Copyright © 2011 Biophysical Society Terms and Conditions