Cy3b maleimide

Detailed information about the primary antibodies against CEP164, FBF1, SCLT1, CEP89, CEP83, Cby1, CP110, CEP97, CEP162, CEP290, C2CD3, IFT88, TTBK2, ODF2, EHD1, ARL13B, and Centrin is provided in Supplementary Table 3. Antibodies conjugated to Alexa Fluor 488 (mouse A21202 and rabbit A21206; Thermo Fisher Scientific, Waltham, MA, USA) and Alexa Fluor 647 (anti-mouse A21236, anti-rabbit A21245, anti-rat A21247, and anti-goat A21447; Thermo Fisher Scientific) were used as secondary antibodies. Cy3B-conjugated secondary antibody was custom-made by conjugating Cy3B maleimide (PA63131, GE, Pittsburgh, PA, USA) to IgG antibodies (rabbit 711-005-152 and rat 712-005-153; Jackson ImmunoResearch, West Grove, PA, USA).

To randomly incorporate both donor and acceptor fluorophores into the channel, substoichiometric labeling with a mixture corresponding to protein/donor/acceptor ratios of 10⁴:8:4 was performed. A reaction mixture of 200 μL KirBac1.1, 1 μL Cy3B maleimide (GE Healthcare), and 0.5 μL Alexa Fluor 647 maleimide (Invitrogen, Carlsbad, CA) was made and incubated in the dark at room temperature for 1 h. It was then added to Amintra CoHIS resin (equilibrated with 20 volumes of 20 mM HEPES pH 7.5, 150 mM KCl) and incubated at 4°C for 1 h. Affinity purification was performed by washing with 20 volumes of wash buffer (20 mM HEPES pH 7.5, 150 mM KCl, 5 mM DM, 10 mM imidazole) followed by elution with 10 volumes of elution buffer (20 mM HEPES pH 7.5, 150 mM KCl, 5 mM DM, 500 mM imidazole) after a 15 min incubation. The eluate was run through an equilibrated NAP-5 column (GE Healthcare) and six 0.5 mL fractions were collected. Wild-type KirBac1.1 contains no endogenous cysteines, and consistent with previous reports (26 (link)), no background labeling was observed. The labeling efficiency of reporter cysteine mutants was estimated by fluorescent visualization of proteins on an SDS-PAGE gel (Nu-PAGE 4-12% Bis-Tris; Novex, Waltham, MA) and visualized using a Pharos FXTM molecular imager (BioRad, Hercules, CA) with Discovery Series Quantity One v4.6.9 software to determine labeled fractions.

For double-labeling H1, both dyes (dissolved in dimethylsulfoxide) were added to the protein in a 1:1:1 molar ratio. Reactions were incubated at room temperature for 2 h, and stopped by adding 20 mM dithiothreitol. Products were purified by RP-HPLC^{12 (link)}. Lyophilized Avi-tagged ProTα E56C/D110C was dissolved under nitrogen atmosphere at a concentration of 200 μM in 100 mM potassium phosphate buffer, pH 7.0. The protein was then labeled for 3 h at room temperature with a 0.7:1 molar ratio of Cy3B maleimide (GE Healthcare) to protein. Labeled protein was separated from unlabeled protein by RP-HPLC on a Reprosil Gold C18 column (Dr. Maisch). The second labeling reaction was carried out for 3 h at room temperature with a 2:1 molar ratio of LD650 maleimide⁵³ (Lumidyne) to protein. For preparing doubly labeled variants of ProTα, the protein in 100 mM potassium phosphate buffer, pH 7.0, was incubated with Alexa Fluor 488 maleimide (Invitrogen) at a dye-to-protein ratio of 0.8:1 for 1 h at room temperature and then with Alexa Fluor 594 maleimide (Invitrogen) at 1:1 molar ratio overnight at 4 °C. The labeled protein was separated and purified by RP-HPLC on a Reprosil Gold C18 column. The correct mass of the labeled protein was confirmed by electrospray ionization mass spectrometry.

pVIc (GVQSLKRRRCF), streptavidin Alexa Fluor 546 conjugate, 5′-fluorescein-labelled 12-mer ssDNA (5′-GACGACTAGGAT-3′), 5′-fluorescein-labelled 18-mer ssDNA (5′-CAGGAAACAGCTATGACC-3′), and 5′-fluorescein-labelled-36-mer ssDNA (5′-GATTGCATGATTAGAGTGTGCTGGATGTGATAGTGA-3′) were purchased from Invitrogen (Carlsbad, CA). Labelled ssDNAs were annealed to their complimentary strands according to standard protocols. 8-Actin-C (SIVHRKCF) and 11-Actin-C (SGPSIVHRKCF) were purchased from Research Genetics (Huntsville, AL, USA). Cy3B-maleimide was purchased from GE Healthcare (Piscataway, NJ). Biotin PEG-3K maleimide was purchased from Nektar. Octylglucoside was purchased from Fisher Scientific (Faden, NJ). n-dodecyl-β-D-maltopyranoside (DDM) was purchased from Anatrace (Maumee, OH). The concentrations of pVIc, 8-Actin-C and 11-Actin-C were determined by titration of the cysteine residue with Ellman's reagent61 (link)62 (link) using an extinction coefficient of 14,150 M⁻¹ cm⁻¹ at 412 nm for released thionitrobenzoate. The N-terminal TMR-labelled human p53 CTD segment (AA 376–388) was purchased from the Biopolymer & Proteomics Laboratory at MIT (>85% purity).

The primary antibodies used in this study are listed in Supplementary file 1 Table 2. Secondary antibodies used in this work were Alexa Fluor 488 (mouse A21202 and rabbit A21206; Thermo Fisher Scientific, Waltham, MA, USA), Alexa Fluor 647 (anti-mouse A21236, anti-rabbit A21245, anti-rat A21247; Thermo Fisher Scientific) and Cy3B-conjugated antibody, which was custom-made as described previously (Yang et al., 2018 (link)). Briefly, 10 mg/ml Cy3B maleimide (PA63131, GE Healthcare, Pittsburgh, PA, USA) dissolved in DMOS/DMF (1:1) was mixed with IgG antibodies (rabbit 711-005-152, rat 712-005-153; Jackson ImmunoResearch, West Grove, PA, USA) at a 1:1 ratio by volume. 0.67 M borate buffer (1859833, Thermo Fisher Scientific) was then added to the mixture, achieving a final concentration of 4%. The reaction mixture was protected from light and incubated at room temperature for 1 hr. The mixture was cleaned up using purification resin (1860513, Thermo Fisher Scientific) to remove excess dye and stored at 4°C until later use.