Single-Molecule Studies of Unlbelled Full-Length p53 Protein Binding to DNA Philipp Nuttll, 1 Kidn Lee, 2 Pietro Ciccrell, 3 Mrco Crminti, 3 Giorgio Ferrri, 3 Ki- Bum Kim, 2 Tim Albrecht 1* 1 Imperil College London, Deprtment of Chemistry, Exhibition Rod, London SW7 2AZ, UK 2 Deprtment of Mterils Science nd Engineering, Seoul Ntionl University, Seoul 151-742, Kore 3 Politecnico di Milno, Diprtimento di Elettronic, Informzione e Bioingegneri, P.z Leonrdo d Vinci 32, Milno, Itly * t.lbrecht@imperil.c.uk Supporting Informtion S1
AFM results nd further nlysis Figure S1. Representtive AFM imges of. pet24, b. 26DNA, c. 36DNA nd d. 47DNA in ir. DNA ws deposited onto freshly cleved mic in 1 mm HEPES, 4 mm MgCl 2 nd dried under dry N 2. White nd blck rrows indicted prtilly nd completely folded molecules respectively. The finite size of the AFM probe gives rise to tip/smple convolution effects, which cn be corrected by modelling the tip s sphere of rdius nd DNA molecule s cylinder of S2
rdius. The mesured width of the DNA object from the AFM imge, w, cn therefore be clculted by: 4 eq 1 Tking the rdius of DNA to be 1. nm, nd using the men mesured DNA dimeter of 12. nm, eq. SI.1 gives the AFM tip rdius s 9. ±.7 nm. This is lrger thn the mnufcturers specifiction of less thn 5 nm, however, s mesurements were performed in tpping mode, the tip my become blunt due to driving the tip onto the smple surfce repetitively. The vlue of the AFM tip rdius obtined here ws used to clculte the dimeters of the p53 protein from mesured vlues, both s bre p53 protein nd when bound to DNA. Figure S2. Representtive AFM imges showing p53 protein dsorbed onto mic. It cn be seen from these imges tht the sizes of the protein vry significntly. Imging conditions: 124 points/line; scn speed:.3 Hz. Insets show individul p53 proteins, the scle br is 5 nm, the Z scle is - 1 nm. S3
.12 1.2 1 Reltive Frequency 8 4 2.15 5 2 2 4 6 8 1 12 14 16 18 2 4 6 8 p53 Height (nm) p53 Dimeter (nm) Figure S3. Histogrm of the mesured height nd dimeter of p53 proteins dsorbed onto mic. Two Gussin distributions re fitted to ech histogrm to obtin two popultions. Popultion 1 refers to the lrgest popultion, while popultion 2 refers to the smller distribution, s lbelled bove. Bin sizes re.25 nm nd 2 nm for p53 height nd dimeter histogrms respectively. The bin size ws chosen to be the sme s the DNA histogrms in figure 4.6. Chnging the bin size hs no effect on the vlues obtined from fitting. Popultion 1 for the height histogrm hs men pek vlue t 2.9 ±.8 nm, whilst the popultion 2 hs pek found t 5.5 ± 1.8 nm. The dimeter histogrm hs peks with men vlues of 16. ± 3.1 nd 3.2 ± 9.8 nm for popultions 1 nd 2 respectively. Reltive Frequency.4.3.2.1 47DNA-p53 complex 36DNA-p53 complex 26DNA-p53 complex pet24-p53 complex DNA Length (µm) 2. 1.8 1.6.4.2.4.8 1.2 1.6 2. 2.4 DNA Length (µm) pet24 26DNA 36DNA 47DNA DNA Smple Figure S4.. A histogrm of the mesured length of DNA with p53 protein bound for pet24-p53 (blue), 26DNA-p53 (blck), 36DNA-p53 (green) nd 47DNA-p53 (red). The histogrms re remrkbly similr, with Gussin fits yielding men vlues of 1.79 ± 5 µm, 1.65 ± 5 µm. 1.73 ± 8 µm, nd 1.75 ± 7 µm, respectively. b. A sctter plot of the DNA length for the DNA only smples (blck, circles) nd the DNA-p53 complex smples (red, tringles). The men length for ech DNA smple is within one stndrd devition of the length for the DNA with p53 bound. S4
Tble S1 Mesured heights nd dimeters of p53 bound to DNA smples, nd clculted dimensions Mesured Clculted Smple Height Dimeter 1 Dimeter 2 Dimeter 1 Dimeter 2 Height (nm) (nm) (nm) (nm) (nm) (nm) pet24 3.5 ± 1.1 24.9 ± 7. 17.2 ± 1.9 7.9 ± 2.5 8.6 ± 1.8 4.1 ±.4 8.3 ±.5 44.3 ± 2.6 26.2 ± 1.6 18.8 ± 1.2 27.4 ± 1.9 9.6 ±.7 26DNA 36DNA 2.6 ±.3 21.6 ± 4.6 18.4 ± 4.5 5.8 ±.8 6.5 ± 1.1 4.7 ±.8 4.7 ± 2. 1.8 ± 4.6 2.4 ±.3 17.4 ± 3.8 15.8 ± 3.6 5.4 ±.7 4.2 ±.7 3.5 ±.6 3.9 ± 1.3 3.2 ± 9.9 28.3 ± 4.7 8.9 ± 2.9 12.7 ± 3. 11.2 ± 1.4 47DNA 2.6 ±.6 16.4 ± 2.2 13.5 ± 1.3 5.9 ± 1.3 3.7 ±.4 2.5 ±.2 5.5 ± 2.8 26.1 ± 1 21.6 ± 7.4 12.5 ± 6.3 9.5 ± 2.6 6.5 ± 1.6 The first nd second rows for ech DNA smples correspond to popultion 1 nd 2 respectively. Dimeters re clculted using eqution SI.1, whilst the height is estimted s hving reduction fctor of.44, s clculted for DNA, see min text. Tble S2. Concentrtion of DNA, p53 nd percentge of DNA with bound p53 protein DNA smple pet24 26DNA 36DNA 47DNA Percentge of DNA with p53 bound p53 concentrtion (in dimers) 5.8 % 2.5 % 15.6 % 6.5 % 6.1 nm 1.4 nm 9.8 nm 32.1 nm DNA concentrtion.25 nm.55 nm.47 nm 3.4 nm Rtio of p53 monomers to DNA 8:1 8:1 8:1 8:1 Simultion of "rndom" protein binding to DNA S5
Mtlb script: close ll cler ll k=1; % number of repets x=:25:.5; = ; b = 1; ABV=; ABVend=; numbers = 28; % number of observtions cutoff =.14286; rng(,'twister'); m2=; m3=1; % This clcultes the verge probbility of finding protein binding % The higher 'cutoff' in comprison to 'vprob', the less likely % histogrm will contin vlues bove 'cutoff' [~,xc]=size(x); vprob=x(end)/(xc); for c1= 1:k c1 % optionl output counter rng('shuffle'); c = (b-).*rnd(numbers,1); i = c>.5; j = c<.5; cbove = +i.*c; cbelow = +j.*c; cdjust = 1.-cbove; cdjust2 = +i.*cdjust; cfinl= cbelow + cdjust2; n_elements=histc(cfinl,x); norm_elements=n_elements./numbers; dt(:,c1) = norm_elements; r(:,c1) = (norm_elements > cutoff); if isempty(find(r(:,c1),1))==; [rf,~]=size(find(r(:,c1))); if rf==2; % simultes two mxim t ny pos. ABVend=ABVend+1; histdat(:,m3)=dt(:,c1); m3=m3+1; end ABV=ABV+1; m2=m2+1; EVL(m2,1:5)=[c1,ABV,ABV/c1,ABVend,ABVend/c1]; end end figure(1) semilogx(evl(:,1),evl(:,3)); title('rel. no. trils bove cutoff vs. no. trils'); figure(2) semilogx(evl(:,1),evl(:,5)); title('rel. no. trils bove cutoff AT DNA EDGE vs. no. trils'); Astr=['cutoff rel. to v. prob.:',' ',num2str(cutoff/vprob)]; disp(astr); S6
menevl=double(men(evl((end):end,3))); Bstr=['verge of rel. no. trils over lst 1 dt pts.',' ',num2str(menevl)]; disp(bstr); menevlend=double(men(evl((end):end,5))); Cstr=['verge of rel. no. trils over lst 1 dt pts, end of chin.',' ',num2str(menevlend)]; disp(cstr); Exemplry results from the script: Reltive Frequency.14.12 8 6 4 2.1.2.3.4.5 Distnce from DNA terminus Distnce from DNA terminus Figure S5. Rndomly generted histogrms to investigte the probbility of the significnt peks t the end of the DNA in figure 4.1 being rndom rtefct. Here. illustrtes histogrm generted rndomly which does not exhibit significnt pek t the end of the DNA. b. shows n exmple of significnt pek t the end of the DNA from rndomly generted numbers. It cn be seen tht this histogrm looks similr to those shown in the min text. b.14.12 8 6 4 2.1.2.3.4.5 Further nnopore trnsloction results nd nlysis S7
.2 36DNA-p53 Group 1 36DNA-p53 Group 2.2 pet24-p53 Group 1 pet24 p53 Group 2 Reltive Frequency c Reltive Frequency.15 5-8 -6-4 8 6 4 2 36DNA mv ecd (fas) -8-6 -4 ecd (fas) Most Probble ecd (fas) -8-6 -4 ecd (fas) Smple Figure S6. Histogrms of ecd for Group 1 trnsloction events (red) nd Group 2 events (blck) for. 36DNA-p53 complex, b. pet24-p53 complex nd c. bre 36DNA, which only exhibits Group 1 chrcteristics. Gussin distributions were fitted to the histogrms to obtin the most probble ecd vlues of 4.27 ± 2.52 fas nd 3.2 ± 7.26 fas for Group 1 nd Group 2 events respectively for 36DNA-p53 complex, 4.33 ± 3.4 fas nd 8.3 ± 6.91 fas for Group 1 nd Group 2 events respectively for pet24-p53 complex nd 8.22 ± 4.31 fas for 36DNA. d. shows the most probble ecd vlues for ech smple nd ech group type. It cn be clerly seen tht the ecd vlues obtined for Group 1 nd Group 2 events for both 36DNA-p53 complex nd pet24-p53 complex re the sme within experimentl error, lso compred to the vlue obtined for bre 36DNA. This suggests tht binding of ( smll number of) p53 protein to DNA does not significntly lter the chrge on the complex..15 5-5 5 5 Group 1 Group 2 36DNA 36DNA-p53 pet24-p53 Exmple trnsloction events for vrious nlytes used in this study S8
G/G (%) 36DNA -4 5 1-4 5 1 36DNA-p53 complex b G/G (%) -.5 -.5 5 1.. 5 1 1 2 3 4 1 2 3 4 pet24-p53 complex b G/G (%) -4-4 -.5-5 -5.. 5 1 5 1 1 2 3 4 1 2 3 4 Time (µs) Time (µs) Time (µs) Time (µs) Figure S7. Plots of G/G versus time disply typicl events for popultions of. Group 1 type trnsloction events nd b. Group 2 type trnsloction events for 36DNA, 36DNA-p53 complex nd pet24-p53 complex. It cn be seen for Group 1 events tht the reltive conductnce blockde is lrger thn 1.7 %, whilst the dwell time is short (< 1 µs). The DNA-p53 complex type events exhibit smller conductnce blockdes but significntly longer dwell times, with individul peks in some events reching lrger conductnce vlues. -.5 Exmple of trnsloction time τ vs. V bis plot, for 36DNA-flp53 (Group 1, DNA-only) S9
2 15 τ [s] 1 5 5 1 15 2 V bis [V ] Figure S8. Plot of t vs. V bis for Group 1 of the 36 DNA-flp53 smple (conditions described in the min text). Bsed on our dt, we cnnot exclude liner correltion between τ nd the inverse of V bis, but devitions become more pprent t low V bis. S1