Chemical methods: Commercially available compounds were used without further purification. All fluorogenic substrates for the labeling of SNAP-tag fusion proteins were prepared by reacting the building block CBG-NH2 (New England Biolabs) with commercially available N-hydroxysuccinimide esters of the corresponding fluorophores and amines of the corresponding quenchers. ATTO-488 NHS was purchased from ATTO-TEC GmbH (Siegen, Germany). Tide Fluor 3 (TF3) NHS, Tide Fluor 5 (TF5) NHS, Tide Quencher 2 (TQ2) acid, Tide Quencher 3 (TQ3) acid were purchased from AAT Bioquest, Inc. (Sunnyvale, CA). Dabcyl C2 amine and QXL670 C2 amine were purchased from AnaSpec, Inc. (Fremont, CA). DY-549 NHS and DY-647 NHS were purchased from Dyomics GmbH (Jena, Germany). Alexa Fluor 647 NHS, QSY-7 amine, and QSY-21 NHS were purchased from Life Technologies Co. (Carlsbad, CA). IRDye QC-1 NHS was provided by LI-COR Biosciences (Lincoln, NE). QSY-21 amine, TQ2 amine, TQ3 amine, and IRDye QC-1 amine were synthesized by reacting N-Fmoc-1,2-diaminoethane hydrobromide (Sigma–Aldrich) with commercially available QSY-21 NHS, TQ2 acid, TQ3 acid, and IRDye QC-1 NHS, respectively. Due to the confidential or proprietary nature of the majority of fluorophores and quenchers used in this study, very limited information about chemical structures is available from dye manufacturers.
Purification and analysis of substrates: Reversed-phase high-performance liquid chromatography (RP-HPLC) was performed on an Agilent LCMS Single Quad System 1200 Series (analytical) and Agilent 1100 Preparative-scale Purification System (semi-preparative). Analytical HPLC was performed on a Waters Atlantis T3 C18 column (2.1×150 mm, 5 μm particle size) at a flow rate of 0.5 mL min−1 with a binary gradient from solvent A (0.1 % aq. formic acid) to solvent B (acetonitrile with 0.1 % formic acid) and monitored by UV–visible absorbance at 280 nm and at the absorption maximum of each fluorophore. Semi-preparative HPLC was performed on a VYDAC 218TP series C18 polymeric reversed-phase column (22×250 mm, 10 μm particle size) at a flow rate of 20 mL min−1 using a water/acetonitrile gradient with trifluoroacetic acid (0.1 %) or 1 m triethyl ammonium bicarbonate buffer (0.1 %). Mass spectra were recorded by electrospray ionization (ESI) on Agilent 6210 Time-of-Flight (TOF) LC/MS System. UV spectra were recorded on a Beckman DU 640B Spectrophotometer.
Synthesis of fluorogenic substrates:
Reactions (1–2 μmol scale) were performed at room temperature in N,N
-dimethylformamide in the presence of CBG-NH2
(1.0 equiv), triethylamine (2.0 equiv), and the fluorophore N-hydroxysuccinimidyl ester (1.0 equiv). The mixture was stirred for 12 h. Then the corresponding quencher amine (1.1 equiv), HBTU (1.5 equiv), and triethylamine (2.0 equiv) were added. The reaction completion was monitored by LCMS. Typically, after 1 h stirring, the mixture was concentrated, purified by RP-HPLC and lyophilized. Each substrate was analyzed by high-resolution mass spectrometry and UV absorption. Isolated yields are given in parentheses and are not optimized. The following substrates were purified using a water/acetonitrile gradient: SNAP-Surface 488 (70 %): ESI-TOFMS m/z
(calcd for C38
842.2021); UV (pH 7.5) λmax
=507 nm. CBG-488-DABCYL (32 %): ESI-TOFMS m/z
(calcd for C58
1207.3873); UV (pH 7.5) λmax
=505 nm. CBG-488-TQ2 (28 %): ESI-TOFMS m/z
(calcd for C63
1320.4172); UV (pH 7.5) λmax
=503 nm. SNAP-Surface 549 (76 %): ESI-TOFMS m/z
(calcd for C46
1069.2570); UV (H2
=555 nm. CBG-TF3 (51 %): ESI-TOFMS m/z
(calcd for C43
783.3249); UV (MeOH) λmax
=545 nm. CBG-TF3-DABCYL (21 %): ESI-TOFMS m/z
(calcd for C60
1076.4890); UV (MeOH) λmax
=556 nm. SNAP-Surface 647 (68 %): ESI-TOFMS m/z
(calcd for C45
895.3266); UV (EtOH) λmax
=652 nm. CBG-TF5 (63 %): ESI-TOFMS m/z
(calcd for C54
1203.2926); UV (MeOH) λmax
=655 nm. The following substrates were purified using a water/acetonitrile gradient with trifluoroacetic acid (0.1 %): CBG-549-TQ3 (13 %): ESI-TOFMS m/z
(calcd for C73
808.7328); UV (MeOH) λmax
=558 nm. CBG-549-QSY7 (54 %): ESI-TOFMS m/z
(calcd for C93
933.8127); UV (MeOH) λmax
=559 nm. CBG-TF3-TQ3 (17 %): ESI-TOFMS m/z
(calcd for C67
1260.5196); UV (MeOH) λmax
=556 nm. SNAP-Surface Alexa Fluor 647 (87 %): ESI-TOFMS m/z
(calcd for C49
1111.3028); UV (MeOH) λmax
=651 nm. CBG-TF5-QXL670 (47 %): ESI-TOFMS m/z
; UV (MeOH) λmax
=657 nm. CBG-TF5-QSY21 (67 %): ESI-TOFMS m/z
(calcd for C97
954.7887); UV (MeOH) λmax
=656 nm. The following substrates were purified using water/acetonitrile gradient with 1 m
triethylammonium bicarbonate buffer (0.1 %): CBG-549-QC1 (11 %): ESI-TOFMS m/z
(calcd for C101
1121.8057); UV (EtOH) λmax
=561 nm. CBG-AF647-QC1 (22 %): ESI-TOFMS m/z
(calcd for C103
1141.8150); UV (MeOH) λmax
=651 nm. CBG-647-QC1 (31 %): ESI-TOFMS m/z
(calcd for C99
1035.3376); UV (EtOH) λmax
=653 nm. Detailed experimental protocol and 1
H NMR spectrum for CBG-549-QSY7 (Scheme S1
) can be found in the Supporting Information. Substrates were further characterized by ESI-TOF mass spectrometry after their binding to the SNAPf
protein (Table S2
was constructed by insertion of the cDNA encoding SNAPf
, synthesized by IDT, between the restriction sites EcoRI and SbfI of pSNAP-tag(m) (New England Biolabs). This SNAP-tag variant, SNAPf
, contains 19 amino acid substitutions and an additional 24-residue deletion at the C-terminus compared to the wild-type AGT. Constitutive expression of the SNAPf
is under the control of a CMV promoter. The cDNA encoding the CLIPf
was introduced between the EcoRI and SbfI sites of pSNAPf
, resulting in pCLIPf
-tag(T7) and pCLIPf
-tag(T7) were constructed by replacing the SNAP-26 m coding region of pSNAP-tag(T7)-2 using the unique EcoRI and SbfI sites with the coding regions of SNAPf
The mouse EGF coding sequence was fused in-frame to the 5′-end of SNAPf and CLIPf, and a hexahistidine tag (His6) was fused to the 3′-end of SNAPf and CLIPf in pSNAPf-tag(T7) and pCLIPf-tag(T7), respectively. The resulting plasmids pEGF-SNAPf-His6 and pEGF-CLIPf-His6 were used for expression of EGF-SNAPf and EGF-CLIPf fusion proteins in E. coli and subsequent affinity purification by Ni-NTA agarose (Qiagen). A linker encoding the signal sequence of EGFR, formed by annealing 5′-CTAGC ATGCG ACCCT CCGGG ACGGC CGGGG CAGCG CTCCT GGCGC TGCTG GCTGC GCTCT GCCCG GCGAG TCGGG CTG-3′- and 5′-AATTC AGCCC GACTC GCCGG GCAGA GCGCA GCCAG CAGCG CCAGG AGCGC TGCCC CGGCC GTCCC GGAGG GTCGC ATG-3′, was inserted into the 5′-MCS of the pSNAPf vector using the unique NheI and EcoRI sites (underlined). Subsequently the coding sequence of mature EGFR (GeneCopoeia) was amplified by PCR and subcloned into the plasmid described above using the unique SbfI and NotI sites, creating pSNAPf-EGFR. SNAPf-β-tubulin was generated from the human β-tubulin coding sequence (Open Biosystems) which was amplified by PCR and fused in-frame to the 5′-end of SNAPf in the pSNAPf vector.
Fluorescence in-gel detection: SNAPf protein was labeled at 37 °C for 30 min in the presence of SNAPf (5 μm), BG conjugate (10 μm) and DTT (1 mm) in PBS. The samples were submitted to electrophoresis on a 10–20 % Tris-glycine gel under denaturing conditions. The gels were scanned using a Typhoon 9400 imager at 300 V PMT with a 488/526 nm (, 488 in green), 532/580 nm (, 549 in orange) or 633/670 nm excitation/emission filter set (, TF5 and Alexa Fluor 647 in red).
Assay of quenching efficiency: Fluorescence signals of the SNAPf proteins labeled with a fluorophore from a quenched or non-quenched substrate were analyzed with a FLEXstation scanning fluorometer (Molecular Devices). The reactions were performed in 96-well plates (Costar) and the fluorescence was measured at the appropriate wavelength. Reactions were carried out with dye (5 μm) and DTT (1 mm) in PBS in the presence or absence of SNAPf protein (10 μm). SNAP-Surface 488 and its fluorogenic derivatives were excited at 488 nm and measured at the maximum emission wavelength of 526 nm. SNAP-Surface 549, CBG-TF3 and their fluorogenic derivatives were excited at 546 nm and measured at the maximum emission wavelength of 580 nm. Fluorescence of SNAP-Surface 647, SNAP-Alexa Fluor 647, CBG-TF5 and their fluorogenic derivatives was read at 636 nm with maximum emission of 670 nm. Fluorescence was followed in 5 min intervals over 2 h at 25 °C. Quenching efficiencies were calculated by the equation E=1−(IFD/ISNAPf), where IFD indicates fluorescence intensity of free dyes and ISNAPf indicates fluorescence intensity of labeled SNAPf protein at the end of the 2 h reaction.
Kinetic study: Labeling reactions were carried out at 22 °C in the presence of dye (5 μm), SNAPf protein (1 μm) and DTT (1 mm) in PBS. At each of the following time points: 0, 15, 30 or 45 s, 1, 2, 4, 8, 16, 32 or 64 min, 18 μL of the labeling reaction was removed and added to a microfuge tube containing 18 μL of 3×Red SDS-PAGE loading buffer (New England Biolabs). After boiling the samples for 5 min, each sample (7.5 μL) was loaded on a 10–20 % Tris-glycine gel (Invitrogen). Following separation of proteins and free dyes on SDS-PAGE, the labeled SNAPf protein was detected with fluorescence imager Typhoon 9400 (GE Healthcare). Gel scanning was performed with appropriate filter sets: excitation at 488 nm and emission at 526 nm for SNAP-Surface 488 and its fluorogenic derivatives; excitation at 533 nm and emission at 580 nm for SNAP-Surface 549, CBG-TF3 and their fluorogenic derivatives; excitation at 633 nm and emission at 670 nm for SNAP-Surface 647, SNAP-Alexa Fluor 647, CBG-TF5 and their fluorogenic derivatives. The imaging data were quantified with ImageQuant TL software (GE Healthcare). The data were fitted to an exponential rise model using the KaleidaGraph 4.0 software (Synergy Software) to get the pseudo-first-order rate constants. Second-order rate constants were then obtained by dividing the pseudo first-order constant by the concentration of substrate.
Quantification of SNAPf-β-tubulin in cell lysates: To generate a standard curve of fluorescence intensity versus SNAPf protein concentration, purified SNAPf protein (25 μL) at a final concentration of 0.025, 0.05, 0.075, 0.1, 0.125, and 0.25 μm were incubated with CBG-488-TQ2 (2 μm, 25 μL, final concentration 0.5 μm) and of cell lysate (50 μL) from nontransfected U2OS cells at room temperature for 4.5 h. The reaction was performed in triplicate in a 96-well plate (Costar). The fluorescence intensity was recorded at 526 nm emission maximum upon excitation at 488 nm and plotted against SNAPf protein concentration. The curve was fitted to a linear equation.
The concentration of SNAPf-β-tubulin was measured from cell lysates of U2OS cells stably expressing SNAPf-β-tubulin. Cells grown at 37 °C in phenol red-free DMEM medium supplemented with 10 % fetal bovine serum (FBS), L-glutamine (2 mm), penicillin (100 units per mL), streptomycin (100 μg mL−1) and G418 (200 μg mL−1) were harvested from a 75 cm2; cell culture flask (BD Falcon) with 0.25 % trypsin treatment, then washed and spun down. The cell pellet was lysed in 500 μL of CelLytic M cell lysis reagent (Sigma–Aldrich) for 15 min at room temperature. Total protein concentration was determined by the Bradford assay. The cell lysate was serially diluted with PBS buffer (1:1, 1:2, 1:4, 1:8, and 1:16) to generate cell lysate samples with various total protein concentrations. 50 μL of each dilution was mixed with 1 μm CBG-488-TQ2 (50 μL, final concentration 0.5 μm) and incubated at room temperature for 4.5 h. The reaction was performed in triplicate in a 96-well plate and the fluorescence intensity was recorded at 526 nm upon excitation at 488 nm. The fluorescence intensities were converted to SNAPf-β-tubulin protein concentrations by using the standard curve generated for SNAPf. The total protein concentration (mg mL−1) was plotted against the concentration of SNAPf-β-tubulin in the cell lysate (μm). The signal-to-noise (S/N) ratios were determined as S/N=(IF−IB)/SD, where IF is the average fluorescence intensity, IB is the average background intensity, and SD is the standard deviation of background. The signal-to-background (S/B) ratios were determined as S/B=IFT/IFNT, where IFT is the average fluorescence intensity of transfected U2OS cells and IFNT is the average fluorescence intensity of nontransfected U2OS cells.
Live cell labeling and imaging: Human embryonic kidney (HEK 293) cells stably transfected with pSNAPf-EGFR were maintained at 37 °C in phenol red-free DMEM medium supplemented with 10 % fetal bovine serum (FBS), penicillin (100 units per mL), streptomycin (100 μg mL−1) and G418 (200 μg mL−1). Cells were seeded in Lab Tek II chambered coverglasses (Nalge Nunc Int). At 24 h post-seeding, cell membrane-localized SNAPf-EGFR was labeled by incubation of live HEK 293 cells stably expressing SNAPf-EGFR with SNAP-tag substrate (1 μm) for 30 min at 37 °C. Then SNAP-Surface Block (New England Biolabs) was added to the cells (final concentration 20 μm) to inhibit further labeling. Images were taken on a wide-field Axiovert 200 m Zeiss microscope using a 63× objective and fixed exposure setting. Cell nuclei were counterstained with Hoechst 33342. For imaging with medium removal, labeling was carried out as above, except that labeling medium was replaced with complete growth medium containing SNAP-Surface Block (20 μm). Images were processed using AxioVision 4.7 software.
EGF-CLIPf isolation and labeling: Expression of recombinant EGF-CLIPf-His6 was performed in SHuffle T7 E. coli (New England Biolabs). EGF-CLIPf-His6 was purified from E. coli cell lysate using Ni-NTA Agarose (Qiagen). Analysis of protein expression and purification was done with Coomassie Blue-stained SDS-PAGE. Labeling of EGF-CLIPf-His6 was carried out with EGF-CLIPf-His6 (40 μm), CLIP-Surface 488 (15 μm) and DTT (1 mm) in PBS on ice for 4 h.
Colocalization of SNAPf-EGFR and EGF-CLIPf: HEK293 cells stably expressing SNAPf-EGFR were labeled with 5 μm CBG-549-QSY7 (red) at 25 °C for 5 min. Cells were then incubated for 2 min with EGF-CLIPf labeled with CLIP-Surface 488 (green) at 500 ng mL−1 prior to imaging by confocal fluorescence microscopy. Cells were counterstained with Hoechst 33342 for nucleus (blue). Images were acquired on a Zeiss LSM 510 laser scanning confocal microscope using a 63X objective. Images were processed using LSM 510 Meta software.