Table 1. One-dimensional diffusion constants (D 1), equilibrium dissociation constants (K d) and size of binding site on DNA.
| Protein (bp DNA) MW (aa) | D1±s.e. (bp)2 s−1 × 10−6 | s.d. D1 (bp)2 × 10−6 | Kd(app.)nM (pH) | DNA footprint (bp) | Notes |
|---|---|---|---|---|---|
| pVIc (12) 1,350 (11) | ND | ND | 693±84 (pH 8) | 7 | 1, 2, 3 |
| pVIc(low salt) | 26.0±1.8 | 11 | 264±25 (pH 7) | 7 | 1, 4, 5 |
| pVIc(high salt) | 17.9±3.5 | 10.7 | ND | ND | 1, 4 |
| AVP (12) 23,087 (204) | (0.02±0.07) | 63.1±5.8 (pH 7) | ND | 1, 3, 6, 7 | |
| AVP–pVIc (36) 24,435 (215) | 21.0±1.9 | 15.6 | 4.6±2.2 (pH 7.5) | 6 | 1, 3, 5, 8 |
| AVP–pVIc (high salt) | 17.1±3.5 | 16.2 | ND | ND | 1, 4, 5 |
| (pVIc-biotin: streptavidin) (18) ∼70,000 (651) | 2.21±0.21 | 1.99 | 35±5.0 (pH 7.5) | ND | 1, 8 |
| pVI (33) 27,014 (250) | 1.45±0.13 | 1.61 | 46±1.6 (pH 8) | 8 | 1, 2, 7, 9 |
| Protein VI (33) 22,118 (206) | ND | ND | 307±38 (pH 8) | ND | 1, 2, 9 |
| 8-Actin-C (12) 988 (8) | 5.45 | 3.63 | 5.0±0.8 (pH 7) | ND | 1 |
| 11-Actin-C (12) 1,230 (11) | 6.40 | 3.29 | ND | ND | 1 |
| 13-p53-C (30) 1,593 (13) | 11.2±0.8 | 3.5 | 780±96 (pH 7.4) | ND | 1, 10 |
AVP, Adenovirus proteinase; ND, not determined; pVIc, peptide derived from C terminus of pVI, the precursor to protein VI; pVI, Precursor to adenovirus protein VI; Protein VI, Adenovirus protein VI.
Notes: (1) to convert from bp to nm: 106 (bp)2 s−1=102,400 (nm)2 s−1. For Kd(app.) determinations, at pH 7.5 or 8, the dye was fluorescein, and the label was on the DNA; at pH 7 the dye was Cy3B and the label was on the protein. For pVIc-biotin: streptavidin experiments at pH 7.5, the dye used was Alexa Fluor 546, and there were two dye molecules per streptavidin. pVIc was labelled with Cy3B at Cys10′. AVP and AVP–pVIc were labelled with Cy3B at Cys199. The actin C-terminal peptides were labelled on their cysteine residue with Cy3B. pVI was labelled at Cys249 with Cy3B. The p53 CTD segment (aa 376–388) was labelled at its N terminus with tetramethyl rhodamine; (2) assay buffer was 20 mM Tris-HCl, pH 8, 0.025% DDM, 0.1 mM DTT; (3) ref. 10; (4) Binding assay buffer was 20 mM HEPES, pH 7, 0.025% DDM, 10 mM NaCl and 0.1 mM DTT. The NaCl concentration in the sliding assay buffer was 2–6 mM NaCl; in the high salt-sliding assay buffer, 20–25 mM NaCl was present (see Supplementary Text); (5) ref. 14; (6) whole population-mean D1 calculated from one population (99–96% of the molecules bound to DNA) having a D1 of zero and another population (1–4% of the molecules bound to DNA) having a D1 of 1.7 × 106 (bp)2 s−1, with s.d. of 1.9 × 106 (bp)2 s−1; (7) ref. 11; (8) assay buffer was 20 mM Na phosphate, pH 7.5, 0.05% DDM; (9) ref. 12, (10) Assay in 10 mM sodium phosphate buffered at pH 6.5 or 7.4.