Molecular sled is an eleven-amino acid vehicle facilitating biochemical interactions via sliding components along DNA

Recently, we showed the adenovirus proteinase interacts productively with its protein substrates in vitro and in vivo in nascent virus particles via one-dimensional diffusion along the viral DNA. The mechanism by which this occurs has heretofore been unknown. We show sliding of these proteins along DNA occurs on a new vehicle in molecular biology, a ‘molecular sled' named pVIc. This 11-amino acid viral peptide binds to DNA independent of sequence. pVIc slides on DNA, exhibiting the fastest one-dimensional diffusion constant, 26±1.8 × 106 (bp)2 s−1. pVIc is a ‘molecular sled,' because it can slide heterologous cargos along DNA, for example, a streptavidin tetramer. Similar peptides, for example, from the C terminus of β-actin or NLSIII of the p53 protein, slide along DNA. Characteristics of the ‘molecular sled' in its milieu (virion, nucleus) have implications for how proteins in the nucleus of cells interact and imply a new form of biochemistry, one-dimensional biochemistry.


Supplementary
1c. This implied that one molecule of pVIc occluded 7 base pairs of DNA, Table 1.

Supplementary Note 3 Ι Hopping and sliding on DNA
The mean one-dimensional diffusion constant for pVIc sliding on DNA was extremely large. Such a large diffusion constant caused us to consider the possibility that processes other than persistent 'sliding' along DNA might be occurring 9  <D 1 > was found to be 17.9 ± 3.5 x 10 6 (bp) 2 s -1 , Table 1. Since this is not a higher value than the 26.0 ± 1.8 x 10 6 (bp) 2 s -1 we observed in low salt, ~2 mM NaCl., Table 1, it appeared that the observed transport we observed in the sliding assays was dominated by sliding of pVIc in contact with the DNA and not by hopping.

Supplementary Note 4 Ι Rotational friction is rate limiting for AVP-pVIc sliding
Recently, we showed that molecules sliding along DNA, including AVP-pVIc complexes, diffuse along a helical path defined by DNA, rotating in order to keep the DNA-binding face of the protein in contact with DNA 10,14,15 . Based on this analysis, we estimate the free energy barriers to translocation average approximately 1 k B T. That the peptide slides only slightly more rapidly than the AVP-pVIc complex (despite being much smaller and having lower friction with the solvent as a result), suggests that the peptide's configuration in the AVP-pVIc complex, Fig. 3 and Supplementary Fig. 1, is optimized by AVP to reduce free energy barriers to diffusion along DNA.

Supplementary Discussion
Many proteins with functions relevant to specific loci or features in the genome have been found capable of one-dimensional diffusion along DNA, an activity that can help to maintain biologically-relevant bimolecular association rates in the face of high concentrations of non-target DNA 16 .
But what about proteins that must interact with one another in a similar setting, e.g. inside a virus particle or the nucleus of a cell, with functions independent of specific loci or features on the genome? Would there be advantages to these proteins if they were to form bimolecular interactions via one-dimensional diffusion along DNA? Recently, we showed that the active form of the adenovirus proteinase and one of its substrates slide along DNA during activation and processing reactions, reactions that take place inside the virus particle but do not involve DNA metabolism 6,8,17,18 .
The sliding assays were done under nonphysiological conditions to ensure accurate measurements of one-dimensional diffusion constants for sliding along DNA. The protein or peptide and DNA concentrations were low as was the ionic strength. The proteins or peptides were labeled with a fluorophore. To keep the fluorescence background low, the protein or peptide concentrations had to be kept low. The DNA concentration was well below the concentration it is in a virus particle. In the assay, the DNA is attached to a glass surface via a biotin-streptavidin linkage. High concentrations of DNA would result in their interaction thereby making sliding events difficult to interpret; for example jumping from one DNA strand to another. The ionic strength was kept low because increasing ionic strength decreases the association rate constant for a protein binding to DNA 19,20 ; it also decreases the residence time for a protein being bound to DNA (the time a protein slides along DNA) 13,21 . These conditions in the assay were required in order for there to be enough binding events to observe and quantitatively measure sliding without ambiguity.
Sequence-nonspecific DNA affinity can lead to widely disparate 3D (in solution) and 1D (on DNA) reactivities, creating an opportunity for the regulation of protein-protein interactions by targeting proteins to DNA, analogous to regulatory mechanisms that target interacting partners to membranes or molecular scaffolding 22,23   reaction.
An exception for the approximate equivalence of self-reaction (A + A -> P) and distinguishable interaction partners (B + C -> P) is the case for the 3D solution to solution reaction with one binding partner bound to DNA, where the B + C -> P rate will be significantly slower due to the low concentration of one binding partner in solution. Using parameters estimated for adenovirus, t 1/2 for this reaction is ≥ 3300 seconds when formulated as B + C -> P rather than self-reaction when using the initial rate of reaction presuming the DNA binding equilibrium is fast with respect to the in-solution reaction.