Steric exclusion is the principal source of the preferential hydration of proteins in the presence of polyethylene glycols

Bhat, Rajiv; Timasheff, Serge N.

doi:10.1002/pro.5560010907

Cited by 342 publications

(339 citation statements)

References 45 publications

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“…Can a random coiled polymer be modeled as an effective hard sphere of fixed diameter? The measured exclusion of polyethylene glycol of different molecular weights from proteins is arguably (23,24) consistent with crowding by hard spheres (whose radii are approximated by the radii of gyration of the polyethylene glycol polymers). Molecular Coulter Counter measurements (25,26), however, show that polyethylene glycol polymers of different sizes are not always excluded from welldefined cavities, as a hard-sphere model would require [e.g., figure 4 of Bezrukov et al (25)].…”

Section: Crowdingmentioning

confidence: 62%

Osmotic stress, crowding, preferential hydration, and binding: A comparison of perspectives

Parsegian

Rand

Rau

2000

Proc. Natl. Acad. Sci. U.S.A.

469

606

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There has been much confusion recently about the relative merits of different approaches, osmotic stress, preferential interaction, and crowding, to describe the indirect effect of solutes on macromolecular conformations and reactions. To strengthen all interpretations of measurements and to forestall further unnecessary conceptual or linguistic confusion, we show here how the different perspectives all can be reconciled. Our approach is through the Gibbs-Duhem relation, the universal constraint on the number of ways it is possible to change the temperature, pressure, and chemical potentials of the several components in any thermodynamically defined system. From this general Gibbs-Duhem equation, it is possible to see the equivalence of the different perspectives and even to show the precise identity of the more specialized equations that the different approaches use.

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Section: Crowdingmentioning

confidence: 62%

Osmotic stress, crowding, preferential hydration, and binding: A comparison of perspectives

Parsegian

Rand

Rau

2000

Proc. Natl. Acad. Sci. U.S.A.

469

606

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“…PEGs of varying sizes were used to analyze this possibility. The average radius of gyration for PEG400 is 8.1 Å; however, its radius increases because of hydration; the radii probed vary from 9 to 15 Å, depending on the macromolecule studied (84). This range of values contrasts with a maximal pore radius in R67 DHFR of 12 Å (5).…”

Section: Discussionmentioning

confidence: 99%

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

Chopra

Dooling

Horner

et al. 2008

Journal of Biological Chemistry

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R67 dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate using NADPH as a cofactor. This enzyme is a homotetramer possessing 222 symmetry, and a single active site pore traverses the length of the protein. A promiscuous binding surface can accommodate either DHF or NADPH, thus two nonproductive complexes can form (2NADPH or 2DHF) as well as a productive complex (NADPH⅐DHF). The role of water in binding was monitored using a number of different osmolytes. From isothermal titration calorimetry (ITC) studies, binding of NADPH is accompanied by the net release of 38 water molecules. In contrast, from both steady state kinetics and ITC studies, binding of DHF is accompanied by the net uptake of water. Although different osmolytes have similar effects on NADPH binding, variable results are observed when DHF binding is probed. Sensitivity to water activity can also be probed by an in vivo selection using the antibacterial drug, trimethoprim, where the water content of the media is decreased by increasing concentrations of sorbitol. The ability of wild type and mutant clones of R67 DHFR to allow host Escherichia coli to grow in the presence of trimethoprim plus added sorbitol parallels the catalytic efficiency of the DHFR clones, indicating water content strongly correlates with the in vivo function of R67 DHFR.R67 dihydrofolate reductase, a type II DHFR, 2 catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate. The gene for this enzyme is carried by an R-plasmid, and its presence confers resistance to the antibiotic drug, trimethoprim (TMP). Trimethoprim is a competitive inhibitor of chromosomal DHFR with a picomolar K i (1). Although R67 DHFR is not a good catalyst, it is not inhibited by TMP very well, and thus it allows cell growth in the presence of this antibacterial drug (2, 3). R67 DHFR shares no homology in sequence or structure with chromosomal DHFR and has been proposed to be a good model of a primitive enzyme (4).R67 DHFR is a homotetramer with a single active site pore; the overall structure possesses 222 symmetry as seen in Fig. 1 (5). The symmetry of the active site results in overlapping binding sites for substrate, DHF, and cofactor, NADPH. This can be observed as R67 DHFR binds a total of two ligands as follows: either two NADPH molecules or two folate/DHF molecules or one NADPH plus one folate/DHF molecule (6). The first two complexes are dead-end (binary) complexes, whereas the third is the productive ternary complex. Because of the 222 symmetry, binding to either ligand is unlikely to be optimal.The active site pore of R67 DHFR is unusual in its hourglass shape as well as its large size (2938 Å 3 for apoR67 DHFR with hydrogens added, calculated by CASTp (4, 7)). Because of this large volume, DHF and NADPH cannot occupy all the space in the pore and must use water to mediate some contacts with the protein.How do substrate and cofactor bind to R67 DHFR? From NMR, crystallography, and docking studies, the pteridine ring of DHF/fo...

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“…Importantly, polymer molecular weight plays a critical role on the effectiveness of PEG as precipitating agent, since its main action on protein solutions can be attributed to an increase in protein-protein net attraction due to depletion interactions or macromolecular crowding. 15,34,[37][38][39][40] Thus, further precision studies with PEG of higher molecular weight are needed.…”

Section: Introductionmentioning

confidence: 99%

Quaternary Diffusion Coefficients in a Protein−Polymer−Salt−Water System Determined by Rayleigh Interferometry

et al. 2009

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We have experimentally investigated multicomponent diffusion in a protein-polymer-salt-water quaternary system. Specifically, we have measured the nine multicomponent diffusion coefficients, D ij , for the lysozyme-poly(ethylene glycol)-NaCl-water system at pH 4.5 and 25°C using precision Rayleigh interferometry. Lysozyme is a model protein for protein-crystallization and enzymology studies. We find that the protein diffusion coefficient, D 11 , decreases as polymer concentration increases at a given salt concentration. This behavior can be quantitatively related to the corresponding increase in fluid viscosity only at low polymer concentration. However, at high polymer concentration (250 g/L), protein diffusion is enhanced compared to the corresponding viscosity prediction. We also find that a protein concentration gradient induces salt diffusion from high to low protein concentration. This effect increases in the presence of poly(ethylene glycol). Finally, we have evaluated systematic errors associated with measurements of protein diffusion coefficients by dynamic light scattering. This work overall helps characterize protein diffusion in crowded environments and may provide guidance for further theoretical developments in the field of protein crystallization and protein diffusion in such crowded systems, such as the cytoplasm of living cells.

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Steric exclusion is the principal source of the preferential hydration of proteins in the presence of polyethylene glycols

Cited by 342 publications

References 45 publications

Osmotic stress, crowding, preferential hydration, and binding: A comparison of perspectives

Osmotic stress, crowding, preferential hydration, and binding: A comparison of perspectives

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

Quaternary Diffusion Coefficients in a Protein−Polymer−Salt−Water System Determined by Rayleigh Interferometry

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