Supplementary Material
Interactions of Histone Acetyltransferase p300 with the Nuclear Proteins Histone and HMGB1, as revealed by Single Molecule Atomic Force Spectroscopy
S. Banerjee, T. Rakshit, S. Sett and R. Mukhopadhyay*
Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata-700 032, India
Cantilever SNL-10 Resonance frequency
before modification (kHz)
Resonance frequency after modification (kHz)
Resonance frequency after the
experiment (kHz) With APTES, glutaraldehyde With p300 FL 1 56.738 55.898 -- 55.898 2 54.283 53.397 52.762 52.762 3 56.847 55.997 55.411 55.410 4 68.950 68.166 67.506 67.506
Protein/complex Condition Unbinding force value
mean±standard error of mean (pN)
Sample size P value
p300 FL-histone octamer non-acetylated 95±0.8 516 < 0.0001 p300 FL-Histone H3 non-acetylated 117±0.8 567 p300 FL-histone octamer non-acetylated 95±0.8 516 < 0.0001 acetylating condition 66±0.8 461 p300 FL-Histone H3 non-acetylated 117±0.8 567 < 0.0001 acetylating condition 97±0.7 516 p300 FL-HMGB1 non-acetylated 122±0.7 517 < 0.0001 acetylating condition 102±0.8 490
Table S2: Different force values measured in acetylating and non-acetylating condition with their
Full length p300 (p300 FL) purification method (see ref. [1]):
His6-tagged recombinant full-length p300 was expressed in Sf21 insect cells by using the baculovirus expression system and purified by nickel-nitrilotriacetic acid affinity purification. Briefly, Sf21 cells were infected with the recombinant baculovirus for 72 h, following which they were harvested and homogenized in a homogenization buffer (10 mM Tris-HCl, pH 7.5, containing 10% glycerol, 0.1% Nonidet P-40, 2 mM β-mercaptoethanol, 2 mM phenylmethylsulfonyl fluoride, 50 μg/ml leupeptin, 50 μg/ml aprotinin, 500 mM NaCl, and 15 mM imidazole). The cleared lysate was bound to the nickel-nitrilotriacetic acid-agarose beads (Qiagen) and washed in the same buffer with 300 mM NaCl and 15 mM imidazole. The protein was eluted in the homogenization buffer containing 200 mM NaCl and 250 mM imidazole and stored at -80°C.
AFM sample preparation for imaging p300 FL-histone (or p300FL-HMGB1) complexes in fluid: For fluid phase imaging of the complexes, gold(111) on mica substrate was cleaned by flame-annealing and then immersed into 0.5 mM ethanolic solution of 3-mercaptopropionic acid (3-MPA) for 1 h followed by washing with 2 ml (500 µl×4) filtered ethanol and 2 ml (500 µl×4) milli-Q water, and drying with a stream of nitrogen gas. Then 3-MPA modified gold substrate was immersed into freshly prepared aqueous solution of 75 mM 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide hydrochloride (EDC) and 15 mM N-hydroxysuccinimide (NHS), kept for 30 min, followed by washing with 2 ml (500 µl×4) Milli-Q water. Then the 3-MPA-NHS-EDC modified gold substrate was dried with a stream of nitrogen gas. 2 µl of the protein complex solution was deposited onto modified gold surface for 10 min. The sample surface was gently washed with the buffer and then with milli-q water and then placed under the fluid cell.
AFM imaging and force measurement experiments:
For ambient imaging, rectangular silicon cantilever of force constant 7.5 N/m and oscillation frequency of 207 kHz was used. SNL-10 cantilevers of oscillation frequency 56-69 kHz were used for fluid phase imaging. The probes were cleaned in a UV-ozone cleaner (Bioforce, Nanosciences) before imaging. Amplitude set point was 85–90% of the free oscillation amplitude (7.5–8.0 V in ambient and 4.0–6.0 V in fluid). Scan speed was 0.5–2.0 lines/s.
In all the force measurements, the retrace curves were considered because the unbinding event took place during cantilever retraction. The effective spring constants of the cantilevers were calibrated by thermal noise method [2, 3]. For measurement of the unbinding force in acetylating condition, AFM tip was stopped near the baseline, and a certain pause time was allowed in the presence of acetylating buffer. After the pause, the AFM tip was retracted and the unbinding force was recorded [4]. The unbinding force value was determined as a product of the cantilever deflection (nm) and the calibrated spring constant (nN/nm). From the set of unbinding force values, the most probable unbinding force was determined by fitting a Gaussian function to the histogram of the force distribution.Standard error of mean (σM) has been calculated by dividing
the standard deviation (σ) by square root of sample size (N). Hence, σM = (σ/√N). P values have
been obtained by Microsoft Excel 2007 software. Dynamic light scattering experiment:
The measurements were performed on a Malvern Particle Size Analyser (Model No: ZEN 3690 Zetasizer Nano ZS 90) having a He–Ne laser (beam wavelength: 632.8 nm) utilizing 4 mW power as the light source. 0.45 μM octameric histone solution in buffer containing 10 mM Tris-HCl (pH 7.5), 2 M NaCl, 1 mM EDTA, and 5 mM 2-mercaptoethanol was used.
(a) (b) (c) (d) (e) (f) 2 0 p N 50 nm
Figure S1: Force-distance curve of different control experiments between (a) bare tip and bare gold
surface, (b) bare tip and 3-MPA-NHS-EDC modified gold, (c) APTES-glutaraldehyde modified tip and 3-MPA-NHS-EDC modified gold, (d) APTES-glutaraldehyde modified tip and 13 nM histone octamer-3-MPA-NHS-EDC modified gold surface, (e) p300-APTES-glutaraldehyde modified tip and 3-MPA-NHS-EDC modified gold surface, (f) p300 buffer coated tip and 13 nM histone octamer on 3-MPA-NHS-EDC modified gold surface. All the force curves were taken in buffered condition [10 mM HEPES (pH 7.6), 200 mM NaCl, 10 mM KCl, 1.5 mM MgCl2, 10 % glycerol, 0.05 % NP-40, 2 mM PMSF, 1 mM DTT]. All the force curves shown here are the retrace curves.
(a)
Figure S2: Distribution of hydrodynamic diameter of histone
octamer in buffer containing 10 mM Tris-HCl (pH 7.5), 2 M NaCl, 1 mM EDTA, and 5 mM 2-mercaptoethanol.
1 10 100 1000 10000 0 10 In te n si ty(% ) Diameter (nm)
2 0 p N 50 nm (a) (b)
Figure S3: Force-distance curve of control experiments between (a) APTES-glutaraldehyde
modified tip and 10 nM HMGB1-3-MPA-NHS-EDC modified gold surface, (b) p300 FL buffer coated tip and 10 nM HMGB1 on 3-MPA, NHS, EDC modified gold surface. All the force curves shown here are the retrace curves.
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