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Quantitative experimental assessment of macromolecular crowding effects at membrane surfaces.

ABSTRACT: We examined how crowding of the surfaces of lipid vesicles with either grafted polyethyleneglycol (PEG) chains or bilayer-anchored protein molecules affects the binding of soluble proteins to the vesicle surface. Escherichia coli dihydrofolate reductase (DHFR, 18 kDa) or a larger fusion protein, NusA-DHFR (72 kDa), binds reversibly but with high affinity to a methotrexate-modified lipid (MTX-PE) incorporated into large unilamellar vesicles. Incorporation of phosphatidylethanolamine-PEG5000 into the vesicles strongly decreases the affinity of binding of both proteins, to a degree that varies roughly exponentially with the lateral density of the PEG chains. Covalently coupling maltose-binding protein (MBP) to the vesicle surfaces also strongly decreases the affinity of binding of NusDHFR or DHFR, to a degree that likewise varies roughly exponentially with the surface density of anchored MBP. Surface-coupled MBP strongly decreases the rate of binding of NusDHFR to MTX-PE-incorporating vesicles but does not affect the rate of NusDHFR dissociation. The large magnitudes of these effects (easily exceeding an order of magnitude for moderate degrees of surface crowding) support previous theoretical analyses and suggest that surface-crowding effects can markedly influence a variety of important aspects of protein behavior in membranes.

SUBMITTER: Leventis R

PROVIDER: S-EPMC3042566 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Quantitative experimental assessment of macromolecular crowding effects at membrane surfaces.

Leventis Rania R Silvius John R JR

Biophysical journal 20101001 7

We examined how crowding of the surfaces of lipid vesicles with either grafted polyethyleneglycol (PEG) chains or bilayer-anchored protein molecules affects the binding of soluble proteins to the vesicle surface. Escherichia coli dihydrofolate reductase (DHFR, 18 kDa) or a larger fusion protein, NusA-DHFR (72 kDa), binds reversibly but with high affinity to a methotrexate-modified lipid (MTX-PE) incorporated into large unilamellar vesicles. Incorporation of phosphatidylethanolamine-PEG5000 into ...[more]

PMID: 20923646

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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.

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Quantitative experimental assessment of macromolecular crowding effects at membrane surfaces.

Publications

Quantitative experimental assessment of macromolecular crowding effects at membrane surfaces.

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