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Kinetics and thermodynamics of salt-dependent T7 gene 2.5 protein binding to single- and double-stranded DNA.

Shokri L, Marintcheva B, Eldib M, Hanke A, Rouzina I, Williams MC - Nucleic Acids Res. (2008)

Bottom Line: We attribute the four orders of magnitude salt-independent differences between ssDNA and dsDNA binding to nonelectrostatic interactions involved only in ssDNA binding, in contrast to T4 gene 32 protein, which achieves preferential ssDNA binding primarily through cooperative interactions.The results support a model in which dimerization interactions must be broken for DNA binding, and gp2.5 monomers search dsDNA by 1D diffusion to bind ssDNA.We also quantitatively compare the salt-dependent ssDNA- and dsDNA-binding properties of the T4 and T7 ssDNA-binding proteins for the first time.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Northeastern University, 111 Dana Research Center, Boston, MA 02115, USA.

ABSTRACT
Bacteriophage T7 gene 2.5 protein (gp2.5) is a single-stranded DNA (ssDNA)-binding protein that has essential roles in DNA replication, recombination and repair. However, it differs from other ssDNA-binding proteins by its weaker binding to ssDNA and lack of cooperative ssDNA binding. By studying the rate-dependent DNA melting force in the presence of gp2.5 and its deletion mutant lacking 26 C-terminal residues, we probe the kinetics and thermodynamics of gp2.5 binding to ssDNA and double-stranded DNA (dsDNA). These force measurements allow us to determine the binding rate of both proteins to ssDNA, as well as their equilibrium association constants to dsDNA. The salt dependence of dsDNA binding parallels that of ssDNA binding. We attribute the four orders of magnitude salt-independent differences between ssDNA and dsDNA binding to nonelectrostatic interactions involved only in ssDNA binding, in contrast to T4 gene 32 protein, which achieves preferential ssDNA binding primarily through cooperative interactions. The results support a model in which dimerization interactions must be broken for DNA binding, and gp2.5 monomers search dsDNA by 1D diffusion to bind ssDNA. We also quantitatively compare the salt-dependent ssDNA- and dsDNA-binding properties of the T4 and T7 ssDNA-binding proteins for the first time.

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(a) Protein association rate (ka) as a function of protein concentration for gp2.5 in 5 mM salt (red diamond), 25 mM salt (green triangle) and 50 mM salt (blue circle). (b) Protein association rate (ka) as a function of protein concentration for gp2.5-Δ26C in 25 mM salt (green triangle), 50 mM salt (blue circle) and 100 mM salt (brown square). Lines are fit to the data using Equation (5). Dashed lines show the three-dimensional (3D) diffusion limit as discussed in the text (48).
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Figure 3: (a) Protein association rate (ka) as a function of protein concentration for gp2.5 in 5 mM salt (red diamond), 25 mM salt (green triangle) and 50 mM salt (blue circle). (b) Protein association rate (ka) as a function of protein concentration for gp2.5-Δ26C in 25 mM salt (green triangle), 50 mM salt (blue circle) and 100 mM salt (brown square). Lines are fit to the data using Equation (5). Dashed lines show the three-dimensional (3D) diffusion limit as discussed in the text (48).

Mentions: We determine ka for each Fk (v) versus ln (v) data set by extrapolating this dependence to the point , which according to Equation (2) is expected to correspond to the condition kν ≅ ka. These determinations of ka are shown in Figure 3a and b. Here, ka is a function of the protein concentration C, and exceeds the 3D diffusion limit for almost all conditions studied with gp2.5-Δ26C, given by kdiff = 4πDR = 2kBT/3η ≈109M−1s−1, where R is the protein size, estimated as 1 nm, D = kBT/6πηR is the 3D diffusion coefficient and η is the solution viscosity (27,28). The 3D diffusion limit is not exceeded for gp2.5 under these conditions.Figure 3.


Kinetics and thermodynamics of salt-dependent T7 gene 2.5 protein binding to single- and double-stranded DNA.

Shokri L, Marintcheva B, Eldib M, Hanke A, Rouzina I, Williams MC - Nucleic Acids Res. (2008)

(a) Protein association rate (ka) as a function of protein concentration for gp2.5 in 5 mM salt (red diamond), 25 mM salt (green triangle) and 50 mM salt (blue circle). (b) Protein association rate (ka) as a function of protein concentration for gp2.5-Δ26C in 25 mM salt (green triangle), 50 mM salt (blue circle) and 100 mM salt (brown square). Lines are fit to the data using Equation (5). Dashed lines show the three-dimensional (3D) diffusion limit as discussed in the text (48).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553585&req=5

Figure 3: (a) Protein association rate (ka) as a function of protein concentration for gp2.5 in 5 mM salt (red diamond), 25 mM salt (green triangle) and 50 mM salt (blue circle). (b) Protein association rate (ka) as a function of protein concentration for gp2.5-Δ26C in 25 mM salt (green triangle), 50 mM salt (blue circle) and 100 mM salt (brown square). Lines are fit to the data using Equation (5). Dashed lines show the three-dimensional (3D) diffusion limit as discussed in the text (48).
Mentions: We determine ka for each Fk (v) versus ln (v) data set by extrapolating this dependence to the point , which according to Equation (2) is expected to correspond to the condition kν ≅ ka. These determinations of ka are shown in Figure 3a and b. Here, ka is a function of the protein concentration C, and exceeds the 3D diffusion limit for almost all conditions studied with gp2.5-Δ26C, given by kdiff = 4πDR = 2kBT/3η ≈109M−1s−1, where R is the protein size, estimated as 1 nm, D = kBT/6πηR is the 3D diffusion coefficient and η is the solution viscosity (27,28). The 3D diffusion limit is not exceeded for gp2.5 under these conditions.Figure 3.

Bottom Line: We attribute the four orders of magnitude salt-independent differences between ssDNA and dsDNA binding to nonelectrostatic interactions involved only in ssDNA binding, in contrast to T4 gene 32 protein, which achieves preferential ssDNA binding primarily through cooperative interactions.The results support a model in which dimerization interactions must be broken for DNA binding, and gp2.5 monomers search dsDNA by 1D diffusion to bind ssDNA.We also quantitatively compare the salt-dependent ssDNA- and dsDNA-binding properties of the T4 and T7 ssDNA-binding proteins for the first time.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Northeastern University, 111 Dana Research Center, Boston, MA 02115, USA.

ABSTRACT
Bacteriophage T7 gene 2.5 protein (gp2.5) is a single-stranded DNA (ssDNA)-binding protein that has essential roles in DNA replication, recombination and repair. However, it differs from other ssDNA-binding proteins by its weaker binding to ssDNA and lack of cooperative ssDNA binding. By studying the rate-dependent DNA melting force in the presence of gp2.5 and its deletion mutant lacking 26 C-terminal residues, we probe the kinetics and thermodynamics of gp2.5 binding to ssDNA and double-stranded DNA (dsDNA). These force measurements allow us to determine the binding rate of both proteins to ssDNA, as well as their equilibrium association constants to dsDNA. The salt dependence of dsDNA binding parallels that of ssDNA binding. We attribute the four orders of magnitude salt-independent differences between ssDNA and dsDNA binding to nonelectrostatic interactions involved only in ssDNA binding, in contrast to T4 gene 32 protein, which achieves preferential ssDNA binding primarily through cooperative interactions. The results support a model in which dimerization interactions must be broken for DNA binding, and gp2.5 monomers search dsDNA by 1D diffusion to bind ssDNA. We also quantitatively compare the salt-dependent ssDNA- and dsDNA-binding properties of the T4 and T7 ssDNA-binding proteins for the first time.

Show MeSH
Related in: MedlinePlus