Sequestered Water and Binding Energy are Coupled in Complexes of λ Cro Repressor with Non-consensus Binding Sequences

Rau, Donald C.

doi:10.1016/j.jmb.2006.06.036

Cited by 16 publications

(23 citation statements)

References 62 publications

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“…The dissociation rate is insensitive to oligonucleotide/fragment concentration ratios between ~100–1000. The reaction was stopped at various times by adding 10 μl triethylene glycol as described in Rau 27.…”

Section: Methodsmentioning

confidence: 99%

“…Sliding is the base pair by base pair diffusion along DNA; the protein remains in contact with the helix. Several crystal and NMR structures of nonspecific DNA-protein complexes (e.g., 22; 23; 24) and our own measurements of the difference in sensitivity to water activity between specific and nonspecific binding modes for several DNA-protein complexes 25; 26; 27; 28 show that substantial water is present at the DNA-protein interface of nonspecific complexes that can `lubricate' movement along the helix 22. Hopping is the transient dissociation and rebinding of the protein to the same helix 29; 30.…”

Section: Introductionmentioning

confidence: 97%

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Diffusion of the Restriction Nuclease EcoRI along DNA

Rau

Sidorova

2010

Journal of Molecular Biology

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Many specific sequence DNA binding proteins locate their target sequence by first binding to DNA nonspecifically, then linearly diffusing or hopping along DNA until either the protein dissociates from the DNA or it finds the recognition sequence. We have devised a method for measuring 1-dimensional diffusion along DNA based on the ratio of the dissociation rates of EcoRI from DNA fragments containing one and two specific binding sites. Our extensive measurements of dissociation rates and specific-nonspecific relative binding constants of the restriction nuclease EcoRI enable us to determine the diffusion rate of nonspecifically bound protein along the DNA. By varying the distance between the two binding sites we confirm a linear diffusion mechanism. The sliding rate is relatively insensitive to salt concentration and osmotic pressure indicating the protein moves smoothly along the DNA probably following the helical phosphate-sugar backbone of DNA. We calculate a diffusion coefficient for EcoRI of 3 × 10 4 bp 2 sec −1 . EcoRI is able to diffuse ~150 base pairs on average along DNA in 1 second. This diffusion rate is about 2000-fold slower than the diffusion of the free protein in solution. A factor of 40-50 can be accounted for by a rotational friction resulting from following the helical path of the DNA backbone. Two possibilities could account for remaining activation energy: the salt bridges between the DNA and protein are transiently broken or the water structure at the protein-DNA interface is disrupted as the two surfaces move past one another.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 97%

Diffusion of the Restriction Nuclease EcoRI along DNA

Rau

Sidorova

2010

Journal of Molecular Biology

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“…In this case,

normalΔ N_{w}^{†}

represents changes both in sterically sequestered water and in the preferential hydration of solvent exposed surface area between the initial and transition states of each kinetic step (19, 25, 26). …”

Section: Resultsmentioning

confidence: 99%

“…Dissociation reactions typically require binding extra water molecules. This can be made energetically even less favorable by applying osmotic pressure, thus slowing dissociation considerably (15, 19, 25, 28). In our view, ideal single-turnover experiments should bracket as few kinetic steps as possible.…”

Section: Discussionmentioning

confidence: 99%

“…We have previously employed the osmotic stress technique (15, 16, 19, 24–27, 42, 43) to measure changes in hydration coupled to conformational differences from the dependence of equilibrium constants or kinetic rates on osmotic pressure (water activity). As with

normalΔ N_{H^{+}}^{†}

and

normalΔ N_{{M g}^{2 +}}^{†}

, values of

normalΔ N_{w}^{†}

linked to k 1 and k 2 (Table 3) are quite similar, averaging ~28–35 extra water molecules for each kinetic step, and are fairly insensitive to pH and Mg 2+ concentration.…”

Section: Discussionmentioning

confidence: 99%

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Using Single-Turnover Kinetics with Osmotic Stress To Characterize the EcoRV Cleavage Reaction

2013

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Type II restriction endonucleases require metal ions to specifically cleave DNA at canonical sites. Despite the wealth of structural and biochemical information, the number of Mg2+ ions used for cleavage by EcoRV, in particular, at physiological divalent ion concentrations is still not established. In this work we employ a single-turnover technique that uses osmotic stress in order to probe reaction kinetics between an initial specific EcoRV-DNA complex formed in the absence of Mg2+ and the final cleavage step. With osmotic stress, complex dissociation before cleavage is minimized and the reaction rates are slowed to a convenient timescale of minutes to hours. We find that cleavage occurs by a two-step mechanism that can be characterized by two rate constants. The dependence of these rate constants on Mg2+ concentration and osmotic pressure gives the number of Mg2+ ions and water molecules coupled to each kinetic step of the EcoRV cleavage reaction. Each kinetic step is coupled to the binding 1.5 – 2.5 Mg2+ ions, the uptake of ~30 water molecules, and the cleavage of a DNA single strand. We suggest that each kinetic step reflects an independent, rate limiting conformational change of each monomer of the dimeric enzyme that allows Mg2+ ion binding. This modified single turnover protocol has general applicability for metalloenzymes.

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Hydration Forces

Todd¹,

Sidorova²,

Rau³

et al. 2008

Wiley Encyclopedia of Chemical Biology

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Force measurements on a wide variety of biologic macromolecules indicate that the perturbation of water structuring as hydrated surfaces closely approach can dominate interaction energetics. Forces with remarkably similar characteristics are observed for the repulsive interactions of charged polymer surfaces, of uncharged polymer surfaces, of charged solutes with uncharged surfaces, and of uncharged solutes with charged surfaces stressing the universality of the hydration force in aqueous solution. Repulsive interactions are caused by a disruption of the hydrogen bonding network of the intervening water between surfaces. The available measurements of forces between macromolecules that spontaneously assemble are consistent with an attractive hydration interaction. A water‐order parameter theory has been developed that is successful for predicting force curve behaviors. The direct involvement of water structuring in the interaction between macromolecules has important consequences for recognition, assembly, and conformational transitions that underlie biologic function.

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Sequestered Water and Binding Energy are Coupled in Complexes of λ Cro Repressor with Non-consensus Binding Sequences

Cited by 16 publications

References 62 publications

Diffusion of the Restriction Nuclease EcoRI along DNA

Diffusion of the Restriction Nuclease EcoRI along DNA

Using Single-Turnover Kinetics with Osmotic Stress To Characterize the EcoRV Cleavage Reaction

Hydration Forces

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