Theory predicts that the net charge (
- Award ID(s):
- 2203441
- PAR ID:
- 10451773
- Date Published:
- Journal Name:
- Protein Science
- Volume:
- 31
- Issue:
- 9
- ISSN:
- 0961-8368
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Z ) of a protein can be altered by the net charge of a neighboring protein as the two approach one another below the Debye length. This type of charge regulation suggests that a protein's charge and perhaps function might be affected by neighboring proteins without direct binding. Charge regulation during protein crowding has never been directly measured due to analytical challenges. Here, we show that lysine specific protein crosslinkers (NHS ester‐Staudinger pairs) can be used to mimic crowding by linking two non‐interacting proteins at a maximal distance of ~7.9 Å. The net charge of the regioisomeric dimers and preceding monomers can then be determined with lysine‐acyl “protein charge ladders” and capillary electrophoresis. As a proof of concept, we covalently linked myoglobin (Z monomer = −0.43 ± 0.01) and α‐lactalbumin (Z monomer = −4.63 ± 0.05). Amide hydrogen/deuterium exchange and circular dichroism spectroscopy demonstrated that crosslinking did not significantly alter the structure of either protein or result in direct binding (thus mimicking crowding). Ultimately, capillary electrophoretic analysis of the dimeric charge ladder detected a change in charge of ΔZ = −0.04 ± 0.09 upon crowding by this pair (Z dimer = −5.10 ± 0.07). These small values of ΔZ are not necessarily general to protein crowding (qualitatively or quantitatively) but will vary per protein size, charge, and solvent conditions. -
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Abstract Protein enzymes are the main catalysts in the crowded and complex cellular interior, but their activity is almost always studied in dilute buffered solutions. Studies that attempt to recreate the cellular interior
in vitro often utilize synthetic polymers as crowding agents. Here, we report the effects of the synthetic polymer cosolutes Ficoll, dextran, and polyvinylpyrrolidone, and their respective monomers, sucrose, glucose, and 1‐ethyl‐2‐pyrrolidone, on the activity of the 18‐kDa monomeric enzyme,Escherichia coli dihydrofolate reductase. At low concentrations, reductase activity increases relative to buffer and monomers, suggesting a macromolecular effect. However, the effect decreases at higher concentrations, approaching, and, in some cases, falling below buffer values. We also assessed activity in terms of volume occupancy, viscosity, and the overlap concentration (where polymers form an interwoven mesh). The trends vary with polymer family, but changes in activity are within threefold of buffer values. We also compiled and analyzed results from previous studies and conclude that alterations of steady‐state enzyme kinetics in solutions crowded with synthetic polymers are idiosyncratic with respect to the crowding agent and enzyme. -
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Abstract The degree by which metalloproteins partially regulate net charge (
Z ) upon electron transfer (ET) was recently measured for the first time using “protein charge ladders” of azurin, cytochrome c, and myoglobin [Angew. Chem. Int. Ed .2018 ,57 (19), 5364–5368;Angew. Chem .2018 ,130 , 5462–5466Z ET(SOD1)=0.05±0.08 per electron, compared to ΔZ ET(Cyt‐c)=1.19±0.02). This total regulation of net charge by SOD1 is attributed to the protonation of the bridging histidine upon copper reduction, yielding redox centers that are isoelectric at both copper oxidation states. Charge regulation by SOD1 would prevent long range coulombic perturbations to residue pK a’s upon ET at copper, allowing SOD1’s “electrostatic loop” to attract superoxide with equal affinity (at both redox states of copper) during diffusion‐limited reduction and oxidation of superoxide. -
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Abstract The degree by which metalloproteins partially regulate net charge (
Z ) upon electron transfer (ET) was recently measured for the first time using “protein charge ladders” of azurin, cytochrome c, and myoglobin [Angew. Chem. Int. Ed .2018 ,57 (19), 5364–5368;Angew. Chem .2018 ,130 , 5462–5466Z ET(SOD1)=0.05±0.08 per electron, compared to ΔZ ET(Cyt‐c)=1.19±0.02). This total regulation of net charge by SOD1 is attributed to the protonation of the bridging histidine upon copper reduction, yielding redox centers that are isoelectric at both copper oxidation states. Charge regulation by SOD1 would prevent long range coulombic perturbations to residue pK a’s upon ET at copper, allowing SOD1’s “electrostatic loop” to attract superoxide with equal affinity (at both redox states of copper) during diffusion‐limited reduction and oxidation of superoxide. -
The rotational diffusion of a protein in the presence of protein crowder molecules was analyzed via computer simulations. Cluster formation as a result of transient intermolecular contacts was identified as the dominant effect for reduced rotational diffusion upon crowding. The slow-down in diffusion was primarily correlated with direct protein–protein contacts rather than indirect interactions via shared hydration layers. But increased solvent viscosity due to crowding contributed to a lesser extent. Key protein–protein contacts correlated with a slow-down in diffusion involve largely interactions between charged and polar groups suggesting that the surface composition of a given protein and the resulting propensity for forming interactions with surrounding proteins in a crowded cellular environment may be the major determinant of its diffusive properties.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1002/pro.4384
