Structure‐based engineering starting from the antiparallel coiled coil domain of the Bcr‐Abl oncoprotein (BCR30‐65) afforded a new homodimeric antiparallel coiled coil whose folding is responsive to solution pH. The BCR30‐65homodimer contains two glutamic acids (Glu52) buried in its hydrophobic core which one would expect to impart pH responsive folding behavior. Surprisingly, BCR30‐65is resistant to thermal denaturation over a wide pH range. This behavior arises from stabilizing salt bridges formed between solvent‐exposed Arg55 and Glu52 that passivates the glutamates’ negative charge within the protein’s core. Replacing Arg55 with alanine imparts pH‐responsive folding behavior, but also results in the loss of directional specificity between the coiled coil’s helices. We find that an Ile42Glu mutation preserves the salt bridges and imparts the desired pH responsive folding behavior. The Ile42Glu protein forms an antiparallel homodimeric coiled coil under acidic conditions but unfolds under basic conditions and is only marginally less thermally stable compared to BCR30‐65.
Charge regulation mechanism in end-tethered weak polyampholytes
Weak polyampholytes, containing oppositely charged dissociable groups, are expected to be responsive to changes in ionic conditions. Here, we determine structural and thermodynamic properties, including the charged groups’ degrees of dissociation, of end-tethered weak polyampholyte layers as a function of salt concentration, pH, and the solvent quality. For diblock weak polyampholytes grafted by their acidic blocks, we find that the acidic monomers increase their charge while the basic monomers decrease their charge with decreasing salt concentration for pH values less than the p K a value of both monomers and vice versa when the pH > p K a . This complex charge regulation occurs because the electrostatic attraction between oppositely charged blocks is stronger than the repulsion between monomers with the same charge in both good and poor solvents when the screening by salt ions is weak. This is evidenced by the retraction of the top block into the bottom layer. In the case of poor solvent conditions to the basic block (the top block), we find lateral segregation of basic monomers into micelles, forming a two-dimensional hexagonal pattern on the surface at intermediate and high pH values for monovalent salt concentrations from 0.01 to 0.1 M. When the solvent is poor to both blocks, we find lateral segregation of the grafted acidic block into lamellae with longitudinal undulations of low and high acidic monomer density. By exploiting weak block polyampholytes, our work expands the parameter space for creating responsive surfaces stable over a wide range of pH and salt concentration.
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- Award ID(s):
- 1833214
- NSF-PAR ID:
- 10289117
- Date Published:
- Journal Name:
- Soft Matter
- Volume:
- 16
- Issue:
- 38
- ISSN:
- 1744-683X
- Page Range / eLocation ID:
- 8832 to 8847
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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null (Ed.)When oppositely charged polyelectrolytes mix in an aqueous solution, associative phase separation gives rise to coacervates. Experiments reveal the phase diagram for such coacervates, and determine the impact of charge density, chain length and added salt. Simulations often use hybrid MC-MD methods to produce such phase diagrams, in support of experimental observations. We propose an idealized model and a simple simulation technique to investigate coacervate phase behavior. We model coacervate systems by charged bead-spring chains and counterions with short-range repulsions, of size equal to the Bjerrum length. We determine phase behavior by equilibrating a slab of concentrated coacervate with respect to swelling into a dilute phase of counterions. At salt concentrations below the critical point, the counterion concentration in the coacervate and dilute phases are nearly the same. At high salt concentrations, we find a one-phase region. Along the phase boundary, the total concentration of beads in the coacervate phase is nearly constant, corresponding to a “Bjerrum liquid''. This result can be extended to experimental phase diagrams by assigning appropriate volumes to monomers and salts.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/d0sm01323d
