Stationary phase EPR for exploring protein structure, conformation
Page 1 of 13
Supporting Information:
Stationary phase EPR for exploring protein structure, conformation, and dynamics in spin-labeled proteins Carlos J. López1, Mark R. Fleissner2, Evan K. Brooks1, and Wayne L. Hubbell1,*
1
Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles CA 90095 2
Present Address: Genzyme corporation, 500 Kendall st. Cambridge MA 02138
* To whom correspondence should be addressed: 100 Stein Plaza, Jules Stein Eye Institute BH973. University of California, Los Angeles. Los Angeles, California 90095. Phone number: (310) 206-8830. Email: [email protected]
Page 2 of 13
SI Methods Materials. Unnatural amino acids p-acetylphenlyalanine and p-azidophenylalanine were obtained from SynChem (Elk Grove Village, IL). N-(aminooxyacetyl)-N'-(D-biotinoyl) hydrazine, trifluoroacetic acid salt, was obtained from Invitrogen. CNBr-activated Sepharose 4B was obtained from GE Healthcare. Dibenzylcyclooctyne (DBCO)-biotin, DBCO-sulfo-NHS, and DBCO-PEG4-NHS were obtained from ClickChemistry Tools (Scottsdale, Az). High-capacity Streptavidin beads were obtained from Thermo Scientific Pierce. The nitroxide spin labeling reagents HO-225, HO-2242, and HO-1944 were a generous gift from Dr. Kálmán Hideg (University of Pécs, Hungary). These reagents react with cysteine residues to generate the nitroxide side chains designated R1,1 R8,2 and RX,3 respectively. Expression, purification, and spin labeling of T4L mutants bearing p-AcF or p-AzF and a single cysteine residue. The expression and purification of T4L mutants bearing p-AcF in BL21(DE3) cells was done as previously described.4 For expression of T4L mutants bearing pAzF, BL21(DE3) cells were co-transformed with pET11a vector containing T4L mutants and pEVOL-p-AzF,5 and then plated onto LB-agar plates containing ampicillin and chloramphenicol. Expression in the presence of 1mM of p-AzF was done in the dark (to minimize photoreduction of the azide) at 37°C for 3 hours or at 30 °C overnight. Cell lysis, protein purification, and spin labeling to generate the side chains designated R1, R8, or RX was done as previously described.2, 3
Tethering of T4L mutants on CNBr-activated Sepharose 4B. T4L mutants were covalently bound to commercially available CNBr-Sepharose as previously described.2
Page 3 of 13 Biotinylation of proteins bearing p-AcF. Purified and spin-labeled mutants bearing p-AcF in a buffer consisting of 50 mM sodium phosphate, 25 mM NaCl at pH 4.0 were incubated with 10fold molar excess of a freshly made solution of N-(aminooxyacetyl)-N'-(D-biotinoyl) hydrazine trifluoroacetic acid salt. The mixture was incubated overnight at 37 °C with nutation to yield the biotinylated protein. Excess reagent was removed by three 15-ml washes with PBS buffer (100 mM phosphate, 150 mM NaCl) at pH 7.2, using a 15-ml Amicon Ultra concentrator (10 kDa MWCO). Biotinylation of proteins bearing p-AzF. A stock solution of 10 mM of DBCO-biotin was prepared in DMF or DMSO and used immediately or stored at -20 °C. A 10-20-fold molar excess of DBCO-biotin was added to 50-100 μM of spin-labeled protein bearing p-AzF in PBS (pH 7.2) and the mixture was incubated at room temperature for 2-4 hours or at 4 °C overnight. Excess reagent was removed by repeated washes with PBS (pH 7.2) buffer using a 15-ml Amicon Ultra concentrator (10 kDa MWCO). It is noted that spin labeling of the cysteine residue should be done prior to incubation with DBCO-biotin to prevent thiol-yne addition.6 The final product was verified by mass spectrometry (Fig. S2). Tethering biotinylated proteins to high-capacity streptavidin beads. The desired quantity of Strepavidin beads was equilibrated in PBS buffer (pH 7.2) and washed 3X by centrifugation with a 5-fold excess of the same buffer. After the final wash and removal of the supernatant, the biotinylated protein was added to the beads in an amount equivalent to the stated capacity of the beads and the suspension was mixed at room temperature for at least 2 hours at room temperature or at 4 °C overnight. The supernatant was removed and the beads washed with buffer consisting of 50 mM MOPS, 25 mM NaCl at pH 6.8 prior to EPR measurements. The
Page 4 of 13 coupling efficiency was determined from the 280 nm absorbance of the supernatant and washes compared to that of the protein solution added to the beads. Functionalization of Sepharose beads with DBCO. Lyophilized CNBr-Sepharose 4B beads were resuspended in 10 mM HCl at pH 2, mixed for about 20 minutes, and then washed with a total volume of 100-200 ml of 10 mM HCl at pH 2 as suggested by the manufacturer. The beads were then suspended in coupling buffer (0.1 M NaHCO3, 0.5 M NaCl, at pH 8.3) and ethylenediamine was added to a final concentration of 1 M. The suspension was incubated for at least 2 hours at room temperature with mixing. Excess ethylenediamine was removed by repeated washes with at least 10 volumes of coupling buffer. To inactivate any remaining cyanate-ester group, the beads were incubated for 2 hours at room temperature with blocking buffer (0.1 M Tris-Cl at pH 8.0). The prepared amino-Sepharose was then washed with 3 cycles of high pH (0.1 M Tris-Cl, 0.5 M NaCl , pH 8.0) and low pH (0.1 M sodium acetate, 0.5 M NaCl, pH 4.0) buffers to remove any traces of ethylenediamine that may be nonspecifically bound to the beads. For long-term storage, the beads were stored in 20% ethanol. For functionalization with DBCO, the amino-Sepharose beads were pre-equilibrated in PBS (pH 7.2), then suspended in a freshly prepared 10 mM solution of DBCO-sulfo-NHS or DBCOPEG4-NHS and incubated overnight at room temperature with nutation. Unreacted DBCO-NHS was removed and inactivated via repeated washes with buffer consisting of 0.1 M Tris-Cl, 0.5 M NaCl at pH 8. The resin was washed 3 times with alternating high pH buffer (pH 8) – low pH buffer (pH 4) cycles to remove any nonspecifically bound reactant. The washed DBCOSepharose was used immediately or stored in 20% ethanol.
Page 5 of 13 Tethering spin-labeled proteins bearing p-AzF on DBCO beads. The desired quantity of DBCO-Sepharose beads were pre-equilibrated with PBS (pH 7.2), and protein bearing the pAzF was added and incubated overnight at room temperature or at 4 °C. The supernatant was removed and any uncoupled protein was removed by washing with several medium volumes of buffer consisting of 50 mM MOPS, 25 mM NaCl at pH 6.8. Binding efficiency was determined using A280 measurements of the supernatant and washes. DEER spectroscopy. Four-pulse DEER data at 80K were obtained on a Bruker ELEXSYS 580 operated at Q-band. The protein concentration was at or below 200 M. Samples of 20 L in spin labeling buffer containing 20 % v/v glycerol in a glass capillary (1.4 ID X 1.7 OD; VitroCom Inc., NJ) were flash-frozen in liquid nitrogen. A 36 ns π–pump pulse was set at the maximum absorption spectra and the observer π/2 (16 ns) and π (32 ns) pulses were positioned 50 MHz (17.8 Gauss) upfield, which corresponds to the absorption maxima of the center-field line. Distance distributions were obtained from the raw dipolar evolution data using the program “LongDistance” available at http://www.chemistry.ucla.edu/directory/hubbell-wayne-l. For samples immobilized on beads, the “VariableD” option was used for background correction. Halothane binding to immobilized T4L 128R1/121A/133A mutant. For the halothane titration experiments, a saturated solution of halothane in buffer (20.6 mM) was diluted to the appropriate concentration and added to the tethered protein. The EPR spectra were recorded immediately. Monitoring reduction of p-AzF by DTT and TCEP. Due to the practical importance of the pAzF reduction by DTT and TCEP, the kinetics of the reactions was determined. All experiments were done in the dark to minimize photolysis of the p-AzF. The UAA p-AzF in solution has an absorbance maximum at 251 nm, where the reduced para-aminophenylalanine (p-AmF) exhibits little absorbance 7; thus the conversion of p-AzF to p-AmF was followed in time at 251 nm. The
Page 6 of 13 absorbance at 251 nm as a function of time for 100 M p-AzF in the presence of 5 mM tris-(2carboxyethyl) phosphine (TCEP) is shown in Fig. S1A. Because there is a 50X excess of TCEP, the reaction is pseudo-first order in p-AzF; the pseudo first order rate constant is given in the Figure S1. The same experiment could not be done with the reducing agent DTT, because DTT and the product of p-AzF reduction by DTT (dithiolane)8 absorb in the same UV-Vis region that p-AzF absorbs. Reduction of p-AzF by DTT was instead followed for the amino acid in T4L as a change in the A253/A280 ratio. For T4L bearing p-AzF, A253/A280 ~ 0.9-1.0, while the same ratio for T4L containing p-AmF the ratio is ~ 0.60. Fig. S1B shows the absorption profile of T4L 65p-AzF as a function of time after 5 mM DTT was added. The data were obtained after excess DTT and dithiolane were washed out using an Amicon concentrator. A plot showing A253/A280 as a function of time and the pseudo-first order rate analysis is given in Fig. S1B, right panel, which is essentially identical to that for TCEP determined above. To show that the changes in the absorption profile were due to reduction of p-AzF, T4L 65p-AzF exposed to 5 mM DTT or with pure buffer (no DTT) was incubated with DBCO-biotin. The protein bearing 65p-AzF is biotinylated, while 65p-AmF is not. After overnight incubation with DBCO-biotin, the molecular weights of the samples were determined by MALDI-TOF. As shown in Fig. S1C, the mass of the major peak of the – DTT sample was 19345 Da corresponding to the biotinylated protein, while the same for the + DTT sample was 18664 Da. The difference in the mass of the main peaks between the two samples is 680 Da, corresponding to 654 Da from biotinylation and 26 Da from the reduction. Interestingly, the protein sample that was not incubated with DTT showed a small peak corresponding to reduced azide, which likely took place in vivo.9
Page 7 of 13 Minimizing reduction of p-AzF in T4L during purification. Based on the kinetics of reduction by DTT and TCEP, it is recommended that for proteins bearing p-AzF and a cysteine residue, that low concentrations of either reducing agent (1:1 molar ratio) are added for a short period of time (1 hour) just before spin labeling to minimize reduction of the azide. Previous studies have shown that azide reduction by mono-thiols (i.e. BME) or by DTT at pH < 6 are less efficient.7, 8 Thus, for SDSL studies equimolar amounts of DTT at pH ~ 6 should be used prior to spin labeling. For T4L mutants it was found that harvesting and purifying the protein at a low pH (pH=5) eliminated dimer formation from solvent-exposed cysteines in the absence of DTT or TCEP (data not shown), thus eliminating the need for addition of reducing cysteines prior to spin labeling. For harvesting and purification of T4L at low pH (pH 5), the lysis buffer consisted of 10 mM sodium citrate, 0.1 mM EDTA at pH 5. For ion exchange, a HiTrap CMFF column was used, and the protein was eluted with a linear gradient of buffer consisting of 10 mM sodium citrate, 1 M NaCl, 0.1 mM EDTA at pH 5. The protein eluted at a concentration of NaCl of ~ 270 mM. Non-reducing SDS-PAGE and gel filtration runs show no evidence of dimers even when using T4L mutants containing double cysteine mutations. For spin labeling at pH 5, 10fold molar excess MTSL was added and the mixture was incubated overnight. After overnight incubation, the pH of the protein solution was increased to 6, followed by an additional 2 hour incubation to ensure high labeling efficiency. Excess spin label was removed as described in the main text experimental procedure section.
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Figure S1: In vitro reduction of p-AzF to p-NH2F with TCEP and DTT. (A) UV-Vis spectra showing the time course of reduction of 100 μM p-AzF by 5 mM TCEP. Right panel: Change in p-AzF as a function of time monitored by the absorbance at 251nm (A251). The best fit of the data to a pseudo first-order process is shown in red along with the derived half-life (t1/2) and pseudo first order rate constant (k’). (B) UV-Vis spectra showing reduction of 20 μM T4L 65p-AzF with 5 mM DTT. The blue arrows indicate the change in the absorbance upon reduction of p-AzF in T4L. Right panel: Changes in A253/A280 as a function of time and the best fit of the data to a pseudo first order process are shown together with values for the rate constant and half-life. (C) MALDI-TOF data of T4L 65p-AzF after reaction with DBCO-biotin of samples incubated with no DTT (black) or with 5 mM DTT (red), demonstrating the lack of reaction following reduction.
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Figure S2: Efficiency of biotinylation and subsequent tethering of proteins bearing p-AzF. (A) Graph showing the binding efficiencies of the indicated T4L mutants to streptavidin and DBCO-functionalized beads. The horizontal dashed line represents the overall average (). (B) MALDI-TOF spectra of T4L S44p-AzF before and after incubation with excess of biotinylated DBCO for 1 hour. The T4L S44p-AzF mutant shown here contains a spin label at position 131, which is consistent with the mass of the protein prior to incubating with sulfo-DBCO biotin (18873 Da). The red circle in the diagram shown in the left panel represents a spin label side chain.
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Figure S3: EPR spectra of spin-labeled T4L mutants. (A) EPR spectra of 5RX9 tethered to streptavidin beads via site 131p-AzF or 44p-AzF are shown in black and red, respectively. The spectra are identical and overlap completely, illustrating the equivalence of the attachment sites for the immobilization. (B) Top panel: Ribbon diagram of T4L showing the location of lysine as red spheres. Bottom panel: EPR spectra of 131R1 attached to CNBr-Sepharose containing all native lysines (red) or with lysine at sites 83, 85, 124, 135, and 147 mutated to alanine or arginine (cyan). The arrow indicates a component corresponding to an immobilized state of the spin label which is reduced in intensity in the mutant with replaced lysine residues. (C) EPR spectra of R1 at the indicated sites in 30% w/w sucrose solution (black) and of T4L tethered to streptavidin beads (green) via 44p-AzF or 72p-AzF. (D) Comparison of EPR spectra of 86R1 and 131R1 tethered using schemes 3 and 4 (see main text) via site 44, showing the similarity of the lineshapes.
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Figure S4: Monitoring contribution of overall rotational diffusion with T4L 72R8. Top panel: Structure of the R8 side chain. Bottom panel: EPR spectra of 72R8 under the indicated conditions. The hyperfine splitting values for each condition are shown.
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Figure S5: Interspin distance measurements of doubly-R1 labeled T4L tethered to DBCO-beads (Scheme 1) and compared with the protein tethered via streptavidin (Scheme 3). The DEF and corresponding distance distribution are shown in the left and right panels, respectively. The fits of the DEF are shown as dashed gray traces. The green and cyan traces in the distance distribution correspond to T4L tethered to streptavidin and DBCO beads, respectively.
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Figure S6: Evaluating ligand binding to T4 lysozyme mutant tethered site-specifically to streptavidin beads. Left panel: Ribbon diagram showing the structure of the T4L mutant 121A/133A (PDB code: 251L). The blue sphere at the Cα indicates the site where R1 was introduced. The surface of the engineered binding site for halothane is shown in green. Right panel: EPR spectra of 128R1/121A/133A tethered via scheme 3 at site 44 in the presence of increasing amounts of halothane from 0-20 mM showing the continuous change with concentration.
Supporting Information References: [1] Mchaourab, H. S., Lietzow, M. A., Hideg, K., and Hubbell, W. L. (1996) Motion of Spin-Labeled Side Chains in T4 Lysozyme. Correlation with Protein Structure and Dynamics†, Biochemistry 35, 7692-7704. [2] López, C. J., Fleissner, M. R., Guo, Z., Kusnetzow, A. K., and Hubbell, W. L. (2009) Osmolyte perturbation reveals conformational equilibria in spin-labeled proteins, Protein science : a publication of the Protein Society 18, 1637-1652. [3] Fleissner, M. R., Bridges, M. D., Brooks, E. K., Cascio, D., Kalai, T., Hideg, K., and Hubbell, W. L. (2011) Structure and dynamics of a conformationally constrained nitroxide side chain and applications in EPR spectroscopy, Proceedings of the National Academy of Sciences of the United States of America 108, 16241-16246. [4] Fleissner, M. R., Brustad, E. M., Kalai, T., Altenbach, C., Cascio, D., Peters, F. B., Hideg, K., Peuker, S., Schultz, P. G., and Hubbell, W. L. (2009) Site-directed spin labeling of a genetically encoded unnatural amino acid, Proceedings of the National Academy of Sciences of the United States of America 106, 21637-21642. [5] Kálai, T., Fleissner, M. R., Jekő, J., Hubbell, W. L., and Hideg, K. (2011) Synthesis of new spin labels for Cu-free click conjugation, Tetrahedron Letters 52, 2747-2749. [6] van Geel, R., Pruijn, G. J. M., van Delft, F. L., and Boelens, W. C. (2012) Preventing Thiol-Yne Addition Improves the Specificity of Strain-Promoted Azide–Alkyne Cycloaddition, Bioconjugate Chemistry 23, 392-398. [7] Staros, J. V., Bayley, H., Standring, D. N., and Knowles, J. R. (1978) Reduction of aryl azides by thiols: Implications for the use of photoaffinity reagents, Biochemical and Biophysical Research Communications 80, 568-572. [8] Cartwright IL, H. D., and Armstrong VW. (1976) The reaction between thiols and 8-azidoadenosine derivatives, Nucleic Acid Research 3, 2331-2340. [9] Chin, J. W., Santoro, S. W., Martin, A. B., King, D. S., Wang, L., and Schultz, P. G. (2002) Addition of p-azido-Lphenylalanine to the genetic code of Escherichia coli, Journal of the American Chemical Society 124, 9026-9027.
Supporting Information:
Stationary phase EPR for exploring protein structure, conformation, and dynamics in spin-labeled proteins Carlos J. López1, Mark R. Fleissner2, Evan K. Brooks1, and Wayne L. Hubbell1,*
1
Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles CA 90095 2
Present Address: Genzyme corporation, 500 Kendall st. Cambridge MA 02138
* To whom correspondence should be addressed: 100 Stein Plaza, Jules Stein Eye Institute BH973. University of California, Los Angeles. Los Angeles, California 90095. Phone number: (310) 206-8830. Email: [email protected]
Page 2 of 13
SI Methods Materials. Unnatural amino acids p-acetylphenlyalanine and p-azidophenylalanine were obtained from SynChem (Elk Grove Village, IL). N-(aminooxyacetyl)-N'-(D-biotinoyl) hydrazine, trifluoroacetic acid salt, was obtained from Invitrogen. CNBr-activated Sepharose 4B was obtained from GE Healthcare. Dibenzylcyclooctyne (DBCO)-biotin, DBCO-sulfo-NHS, and DBCO-PEG4-NHS were obtained from ClickChemistry Tools (Scottsdale, Az). High-capacity Streptavidin beads were obtained from Thermo Scientific Pierce. The nitroxide spin labeling reagents HO-225, HO-2242, and HO-1944 were a generous gift from Dr. Kálmán Hideg (University of Pécs, Hungary). These reagents react with cysteine residues to generate the nitroxide side chains designated R1,1 R8,2 and RX,3 respectively. Expression, purification, and spin labeling of T4L mutants bearing p-AcF or p-AzF and a single cysteine residue. The expression and purification of T4L mutants bearing p-AcF in BL21(DE3) cells was done as previously described.4 For expression of T4L mutants bearing pAzF, BL21(DE3) cells were co-transformed with pET11a vector containing T4L mutants and pEVOL-p-AzF,5 and then plated onto LB-agar plates containing ampicillin and chloramphenicol. Expression in the presence of 1mM of p-AzF was done in the dark (to minimize photoreduction of the azide) at 37°C for 3 hours or at 30 °C overnight. Cell lysis, protein purification, and spin labeling to generate the side chains designated R1, R8, or RX was done as previously described.2, 3
Tethering of T4L mutants on CNBr-activated Sepharose 4B. T4L mutants were covalently bound to commercially available CNBr-Sepharose as previously described.2
Page 3 of 13 Biotinylation of proteins bearing p-AcF. Purified and spin-labeled mutants bearing p-AcF in a buffer consisting of 50 mM sodium phosphate, 25 mM NaCl at pH 4.0 were incubated with 10fold molar excess of a freshly made solution of N-(aminooxyacetyl)-N'-(D-biotinoyl) hydrazine trifluoroacetic acid salt. The mixture was incubated overnight at 37 °C with nutation to yield the biotinylated protein. Excess reagent was removed by three 15-ml washes with PBS buffer (100 mM phosphate, 150 mM NaCl) at pH 7.2, using a 15-ml Amicon Ultra concentrator (10 kDa MWCO). Biotinylation of proteins bearing p-AzF. A stock solution of 10 mM of DBCO-biotin was prepared in DMF or DMSO and used immediately or stored at -20 °C. A 10-20-fold molar excess of DBCO-biotin was added to 50-100 μM of spin-labeled protein bearing p-AzF in PBS (pH 7.2) and the mixture was incubated at room temperature for 2-4 hours or at 4 °C overnight. Excess reagent was removed by repeated washes with PBS (pH 7.2) buffer using a 15-ml Amicon Ultra concentrator (10 kDa MWCO). It is noted that spin labeling of the cysteine residue should be done prior to incubation with DBCO-biotin to prevent thiol-yne addition.6 The final product was verified by mass spectrometry (Fig. S2). Tethering biotinylated proteins to high-capacity streptavidin beads. The desired quantity of Strepavidin beads was equilibrated in PBS buffer (pH 7.2) and washed 3X by centrifugation with a 5-fold excess of the same buffer. After the final wash and removal of the supernatant, the biotinylated protein was added to the beads in an amount equivalent to the stated capacity of the beads and the suspension was mixed at room temperature for at least 2 hours at room temperature or at 4 °C overnight. The supernatant was removed and the beads washed with buffer consisting of 50 mM MOPS, 25 mM NaCl at pH 6.8 prior to EPR measurements. The
Page 4 of 13 coupling efficiency was determined from the 280 nm absorbance of the supernatant and washes compared to that of the protein solution added to the beads. Functionalization of Sepharose beads with DBCO. Lyophilized CNBr-Sepharose 4B beads were resuspended in 10 mM HCl at pH 2, mixed for about 20 minutes, and then washed with a total volume of 100-200 ml of 10 mM HCl at pH 2 as suggested by the manufacturer. The beads were then suspended in coupling buffer (0.1 M NaHCO3, 0.5 M NaCl, at pH 8.3) and ethylenediamine was added to a final concentration of 1 M. The suspension was incubated for at least 2 hours at room temperature with mixing. Excess ethylenediamine was removed by repeated washes with at least 10 volumes of coupling buffer. To inactivate any remaining cyanate-ester group, the beads were incubated for 2 hours at room temperature with blocking buffer (0.1 M Tris-Cl at pH 8.0). The prepared amino-Sepharose was then washed with 3 cycles of high pH (0.1 M Tris-Cl, 0.5 M NaCl , pH 8.0) and low pH (0.1 M sodium acetate, 0.5 M NaCl, pH 4.0) buffers to remove any traces of ethylenediamine that may be nonspecifically bound to the beads. For long-term storage, the beads were stored in 20% ethanol. For functionalization with DBCO, the amino-Sepharose beads were pre-equilibrated in PBS (pH 7.2), then suspended in a freshly prepared 10 mM solution of DBCO-sulfo-NHS or DBCOPEG4-NHS and incubated overnight at room temperature with nutation. Unreacted DBCO-NHS was removed and inactivated via repeated washes with buffer consisting of 0.1 M Tris-Cl, 0.5 M NaCl at pH 8. The resin was washed 3 times with alternating high pH buffer (pH 8) – low pH buffer (pH 4) cycles to remove any nonspecifically bound reactant. The washed DBCOSepharose was used immediately or stored in 20% ethanol.
Page 5 of 13 Tethering spin-labeled proteins bearing p-AzF on DBCO beads. The desired quantity of DBCO-Sepharose beads were pre-equilibrated with PBS (pH 7.2), and protein bearing the pAzF was added and incubated overnight at room temperature or at 4 °C. The supernatant was removed and any uncoupled protein was removed by washing with several medium volumes of buffer consisting of 50 mM MOPS, 25 mM NaCl at pH 6.8. Binding efficiency was determined using A280 measurements of the supernatant and washes. DEER spectroscopy. Four-pulse DEER data at 80K were obtained on a Bruker ELEXSYS 580 operated at Q-band. The protein concentration was at or below 200 M. Samples of 20 L in spin labeling buffer containing 20 % v/v glycerol in a glass capillary (1.4 ID X 1.7 OD; VitroCom Inc., NJ) were flash-frozen in liquid nitrogen. A 36 ns π–pump pulse was set at the maximum absorption spectra and the observer π/2 (16 ns) and π (32 ns) pulses were positioned 50 MHz (17.8 Gauss) upfield, which corresponds to the absorption maxima of the center-field line. Distance distributions were obtained from the raw dipolar evolution data using the program “LongDistance” available at http://www.chemistry.ucla.edu/directory/hubbell-wayne-l. For samples immobilized on beads, the “VariableD” option was used for background correction. Halothane binding to immobilized T4L 128R1/121A/133A mutant. For the halothane titration experiments, a saturated solution of halothane in buffer (20.6 mM) was diluted to the appropriate concentration and added to the tethered protein. The EPR spectra were recorded immediately. Monitoring reduction of p-AzF by DTT and TCEP. Due to the practical importance of the pAzF reduction by DTT and TCEP, the kinetics of the reactions was determined. All experiments were done in the dark to minimize photolysis of the p-AzF. The UAA p-AzF in solution has an absorbance maximum at 251 nm, where the reduced para-aminophenylalanine (p-AmF) exhibits little absorbance 7; thus the conversion of p-AzF to p-AmF was followed in time at 251 nm. The
Page 6 of 13 absorbance at 251 nm as a function of time for 100 M p-AzF in the presence of 5 mM tris-(2carboxyethyl) phosphine (TCEP) is shown in Fig. S1A. Because there is a 50X excess of TCEP, the reaction is pseudo-first order in p-AzF; the pseudo first order rate constant is given in the Figure S1. The same experiment could not be done with the reducing agent DTT, because DTT and the product of p-AzF reduction by DTT (dithiolane)8 absorb in the same UV-Vis region that p-AzF absorbs. Reduction of p-AzF by DTT was instead followed for the amino acid in T4L as a change in the A253/A280 ratio. For T4L bearing p-AzF, A253/A280 ~ 0.9-1.0, while the same ratio for T4L containing p-AmF the ratio is ~ 0.60. Fig. S1B shows the absorption profile of T4L 65p-AzF as a function of time after 5 mM DTT was added. The data were obtained after excess DTT and dithiolane were washed out using an Amicon concentrator. A plot showing A253/A280 as a function of time and the pseudo-first order rate analysis is given in Fig. S1B, right panel, which is essentially identical to that for TCEP determined above. To show that the changes in the absorption profile were due to reduction of p-AzF, T4L 65p-AzF exposed to 5 mM DTT or with pure buffer (no DTT) was incubated with DBCO-biotin. The protein bearing 65p-AzF is biotinylated, while 65p-AmF is not. After overnight incubation with DBCO-biotin, the molecular weights of the samples were determined by MALDI-TOF. As shown in Fig. S1C, the mass of the major peak of the – DTT sample was 19345 Da corresponding to the biotinylated protein, while the same for the + DTT sample was 18664 Da. The difference in the mass of the main peaks between the two samples is 680 Da, corresponding to 654 Da from biotinylation and 26 Da from the reduction. Interestingly, the protein sample that was not incubated with DTT showed a small peak corresponding to reduced azide, which likely took place in vivo.9
Page 7 of 13 Minimizing reduction of p-AzF in T4L during purification. Based on the kinetics of reduction by DTT and TCEP, it is recommended that for proteins bearing p-AzF and a cysteine residue, that low concentrations of either reducing agent (1:1 molar ratio) are added for a short period of time (1 hour) just before spin labeling to minimize reduction of the azide. Previous studies have shown that azide reduction by mono-thiols (i.e. BME) or by DTT at pH < 6 are less efficient.7, 8 Thus, for SDSL studies equimolar amounts of DTT at pH ~ 6 should be used prior to spin labeling. For T4L mutants it was found that harvesting and purifying the protein at a low pH (pH=5) eliminated dimer formation from solvent-exposed cysteines in the absence of DTT or TCEP (data not shown), thus eliminating the need for addition of reducing cysteines prior to spin labeling. For harvesting and purification of T4L at low pH (pH 5), the lysis buffer consisted of 10 mM sodium citrate, 0.1 mM EDTA at pH 5. For ion exchange, a HiTrap CMFF column was used, and the protein was eluted with a linear gradient of buffer consisting of 10 mM sodium citrate, 1 M NaCl, 0.1 mM EDTA at pH 5. The protein eluted at a concentration of NaCl of ~ 270 mM. Non-reducing SDS-PAGE and gel filtration runs show no evidence of dimers even when using T4L mutants containing double cysteine mutations. For spin labeling at pH 5, 10fold molar excess MTSL was added and the mixture was incubated overnight. After overnight incubation, the pH of the protein solution was increased to 6, followed by an additional 2 hour incubation to ensure high labeling efficiency. Excess spin label was removed as described in the main text experimental procedure section.
Page 8 of 13
Figure S1: In vitro reduction of p-AzF to p-NH2F with TCEP and DTT. (A) UV-Vis spectra showing the time course of reduction of 100 μM p-AzF by 5 mM TCEP. Right panel: Change in p-AzF as a function of time monitored by the absorbance at 251nm (A251). The best fit of the data to a pseudo first-order process is shown in red along with the derived half-life (t1/2) and pseudo first order rate constant (k’). (B) UV-Vis spectra showing reduction of 20 μM T4L 65p-AzF with 5 mM DTT. The blue arrows indicate the change in the absorbance upon reduction of p-AzF in T4L. Right panel: Changes in A253/A280 as a function of time and the best fit of the data to a pseudo first order process are shown together with values for the rate constant and half-life. (C) MALDI-TOF data of T4L 65p-AzF after reaction with DBCO-biotin of samples incubated with no DTT (black) or with 5 mM DTT (red), demonstrating the lack of reaction following reduction.
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Figure S2: Efficiency of biotinylation and subsequent tethering of proteins bearing p-AzF. (A) Graph showing the binding efficiencies of the indicated T4L mutants to streptavidin and DBCO-functionalized beads. The horizontal dashed line represents the overall average (). (B) MALDI-TOF spectra of T4L S44p-AzF before and after incubation with excess of biotinylated DBCO for 1 hour. The T4L S44p-AzF mutant shown here contains a spin label at position 131, which is consistent with the mass of the protein prior to incubating with sulfo-DBCO biotin (18873 Da). The red circle in the diagram shown in the left panel represents a spin label side chain.
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Figure S3: EPR spectra of spin-labeled T4L mutants. (A) EPR spectra of 5RX9 tethered to streptavidin beads via site 131p-AzF or 44p-AzF are shown in black and red, respectively. The spectra are identical and overlap completely, illustrating the equivalence of the attachment sites for the immobilization. (B) Top panel: Ribbon diagram of T4L showing the location of lysine as red spheres. Bottom panel: EPR spectra of 131R1 attached to CNBr-Sepharose containing all native lysines (red) or with lysine at sites 83, 85, 124, 135, and 147 mutated to alanine or arginine (cyan). The arrow indicates a component corresponding to an immobilized state of the spin label which is reduced in intensity in the mutant with replaced lysine residues. (C) EPR spectra of R1 at the indicated sites in 30% w/w sucrose solution (black) and of T4L tethered to streptavidin beads (green) via 44p-AzF or 72p-AzF. (D) Comparison of EPR spectra of 86R1 and 131R1 tethered using schemes 3 and 4 (see main text) via site 44, showing the similarity of the lineshapes.
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Figure S4: Monitoring contribution of overall rotational diffusion with T4L 72R8. Top panel: Structure of the R8 side chain. Bottom panel: EPR spectra of 72R8 under the indicated conditions. The hyperfine splitting values for each condition are shown.
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Figure S5: Interspin distance measurements of doubly-R1 labeled T4L tethered to DBCO-beads (Scheme 1) and compared with the protein tethered via streptavidin (Scheme 3). The DEF and corresponding distance distribution are shown in the left and right panels, respectively. The fits of the DEF are shown as dashed gray traces. The green and cyan traces in the distance distribution correspond to T4L tethered to streptavidin and DBCO beads, respectively.
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Figure S6: Evaluating ligand binding to T4 lysozyme mutant tethered site-specifically to streptavidin beads. Left panel: Ribbon diagram showing the structure of the T4L mutant 121A/133A (PDB code: 251L). The blue sphere at the Cα indicates the site where R1 was introduced. The surface of the engineered binding site for halothane is shown in green. Right panel: EPR spectra of 128R1/121A/133A tethered via scheme 3 at site 44 in the presence of increasing amounts of halothane from 0-20 mM showing the continuous change with concentration.
Supporting Information References: [1] Mchaourab, H. S., Lietzow, M. A., Hideg, K., and Hubbell, W. L. (1996) Motion of Spin-Labeled Side Chains in T4 Lysozyme. Correlation with Protein Structure and Dynamics†, Biochemistry 35, 7692-7704. [2] López, C. J., Fleissner, M. R., Guo, Z., Kusnetzow, A. K., and Hubbell, W. L. (2009) Osmolyte perturbation reveals conformational equilibria in spin-labeled proteins, Protein science : a publication of the Protein Society 18, 1637-1652. [3] Fleissner, M. R., Bridges, M. D., Brooks, E. K., Cascio, D., Kalai, T., Hideg, K., and Hubbell, W. L. (2011) Structure and dynamics of a conformationally constrained nitroxide side chain and applications in EPR spectroscopy, Proceedings of the National Academy of Sciences of the United States of America 108, 16241-16246. [4] Fleissner, M. R., Brustad, E. M., Kalai, T., Altenbach, C., Cascio, D., Peters, F. B., Hideg, K., Peuker, S., Schultz, P. G., and Hubbell, W. L. (2009) Site-directed spin labeling of a genetically encoded unnatural amino acid, Proceedings of the National Academy of Sciences of the United States of America 106, 21637-21642. [5] Kálai, T., Fleissner, M. R., Jekő, J., Hubbell, W. L., and Hideg, K. (2011) Synthesis of new spin labels for Cu-free click conjugation, Tetrahedron Letters 52, 2747-2749. [6] van Geel, R., Pruijn, G. J. M., van Delft, F. L., and Boelens, W. C. (2012) Preventing Thiol-Yne Addition Improves the Specificity of Strain-Promoted Azide–Alkyne Cycloaddition, Bioconjugate Chemistry 23, 392-398. [7] Staros, J. V., Bayley, H., Standring, D. N., and Knowles, J. R. (1978) Reduction of aryl azides by thiols: Implications for the use of photoaffinity reagents, Biochemical and Biophysical Research Communications 80, 568-572. [8] Cartwright IL, H. D., and Armstrong VW. (1976) The reaction between thiols and 8-azidoadenosine derivatives, Nucleic Acid Research 3, 2331-2340. [9] Chin, J. W., Santoro, S. W., Martin, A. B., King, D. S., Wang, L., and Schultz, P. G. (2002) Addition of p-azido-Lphenylalanine to the genetic code of Escherichia coli, Journal of the American Chemical Society 124, 9026-9027.
