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Impact of macromolecular crowding on DNA replication.

ABSTRACT: Enzymatic activities in vivo occur in a crowded environment composed of many macromolecules. This environment influences DNA replication by increasing the concentration of the constituents, desolvation, decreasing the degrees of freedom for diffusion and hopping of proteins onto DNA, and enhancing binding equilibria and catalysis. However, the effect of macromolecular crowding on protein structure is poorly understood. Here we examine macromolecular crowding using the replication system of bacteriophage T7 and we show that it affects several aspects of DNA replication; the activity of DNA helicase increases and the sensitivity of DNA polymerase to salt is reduced. We also demonstrate, using small-angle X-ray scattering analysis, that the complex between DNA helicase and DNA polymerase/trx is far more compact in a crowded environment. The highest enzymatic activity corresponds to the most compact structure. Better knowledge of the effect of crowding on structure and activity will enhance mechanistic insight beyond information obtained from NMR and X-ray structures.

SUBMITTER: Akabayov B

PROVIDER: S-EPMC3666333 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Impact of macromolecular crowding on DNA replication.

Akabayov Barak B Akabayov Sabine R SR Lee Seung-Joo SJ Wagner Gerhard G Richardson Charles C CC

Nature communications 20130101

Enzymatic activities in vivo occur in a crowded environment composed of many macromolecules. This environment influences DNA replication by increasing the concentration of the constituents, desolvation, decreasing the degrees of freedom for diffusion and hopping of proteins onto DNA, and enhancing binding equilibria and catalysis. However, the effect of macromolecular crowding on protein structure is poorly understood. Here we examine macromolecular crowding using the replication system of bacte ...[more]

PMID: 23511479

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Project description:Many protein functions can be directly linked to conformational changes. Inside cells, the equilibria and transition rates between different conformations may be affected by macromolecular crowding. We have recently developed a new approach for modeling crowding effects, which enables an atomistic representation of "test" proteins. Here this approach is applied to study how crowding affects the equilibria and transition rates between open and closed conformations of seven proteins: yeast protein disulfide isomerase (yPDI), adenylate kinase (AdK), orotidine phosphate decarboxylase (ODCase), Trp repressor (TrpR), hemoglobin, DNA beta-glucosyltransferase, and Ap(4)A hydrolase. For each protein, molecular dynamics simulations of the open and closed states are separately run. Representative open and closed conformations are then used to calculate the crowding-induced changes in chemical potential for the two states. The difference in chemical-potential change between the two states finally predicts the effects of crowding on the population ratio of the two states. Crowding is found to reduce the open population to various extents. In the presence of crowders with a 15 A radius and occupying 35% of volume, the open-to-closed population ratios of yPDI, AdK, ODCase and TrpR are reduced by 79%, 78%, 62% and 55%, respectively. The reductions for the remaining three proteins are 20-44%. As expected, the four proteins experiencing the stronger crowding effects are those with larger conformational changes between open and closed states (e.g., as measured by the change in radius of gyration). Larger proteins also tend to experience stronger crowding effects than smaller ones [e.g., comparing yPDI (480 residues) and TrpR (98 residues)]. The potentials of mean force along the open-closed reaction coordinate of apo and ligand-bound ODCase are altered by crowding, suggesting that transition rates are also affected. These quantitative results and qualitative trends will serve as valuable guides for expected crowding effects on protein conformation changes inside cells.

Dataset Information

Impact of macromolecular crowding on DNA replication.

Publications

Impact of macromolecular crowding on DNA replication.

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