A Reduction in Structural Specificity by Polar-to-Hydrophobic Surface Substitutions in the Arc Repressor Protein: A Romance of Three Folds

Persistent Link:
http://hdl.handle.net/10150/311174
Title:
A Reduction in Structural Specificity by Polar-to-Hydrophobic Surface Substitutions in the Arc Repressor Protein: A Romance of Three Folds
Author:
Stewart, Katie Lynn
Issue Date:
2013
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Embargo:
Release 14-Apr-2014
Abstract:
Most amino acid sequences are predicted to specify a single three-dimensional protein structure. However, the identification of "metamorphic" proteins, which can adopt two folds from a single amino acid sequence, has challenged the one sequence/one structure paradigm. Polar-to-hydrophobic substitutions have been suggested computationally as one mechanism to decrease structural specificity, allowing the population of novel folds. Here, we experimentally investigate the role of polar-to-hydrophobic substitutions on structural specificity in the homodimeric ribbon-helix-helix protein Arc repressor. Previous work showed that a single polar-to-hydrophobic surface substitution in the strand region of Arc repressor (Arc-N11L) populates the wild-type fold and a novel dimeric "switch" fold. In this work, we investigate an Arc repressor variant with the N11L substitution plus two additional polar-to-hydrophobic surface substitutions (Arc-S-VLV). We determine that this sequence folds into at least three structures: both dimer forms present in Arc-N11L, and a novel octamer structure containing higher stability and less helicity than the dimer folds. We are able to isolate and stabilize a core of the S-VLV octamer by limited trypsinolysis and deletion mutagenesis (Arc-VLV 4-44). The shortened construct contains only the octameric structure by removing disordered C-terminal segments nonessential for this fold. A two-dimensional NMR spectrum of VLV 4-44 and subsequent trypsinolysis of this construct suggests that at least two types of subunits comprise the S-VLV octamer: subunits structured from residues 4 to 44 and subunits structured from residues 4 to 31. Crystal trials of trypsinolyzed Arc-VLV 4-44 yielded several leads, suggesting that obtaining a high resolution structure of the S-VLV octamer is possible. Relatedly, we determine that the proline residues flanking the Arc repressor strand act in concert as "gatekeepers" to prevent aggregation in the S-VLV sequence. We also find that three highly hydrophobic surface substitutions in the Arc repressor strand region are necessary and sufficient to promote higher-order oligomer formation. In summation, this work reveals in an experimental context that progressive increases in polar-to-hydrophobic surface substitutions populate increasingly diverse, structurally degenerate folds. These results suggest that "metamorphic" as well as "polymetamorphic" proteins, which adopt numerous folds, are possible outcomes for a single protein sequence.
Type:
text; Electronic Dissertation
Keywords:
fold switching; polar-to-hydrophobic substitutions; polymetamorphic protein; structural degeneracy; structural specificity; Biochemistry; Arc repressor
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Biochemistry
Degree Grantor:
University of Arizona
Advisor:
Cordes, Matthew H. J.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleA Reduction in Structural Specificity by Polar-to-Hydrophobic Surface Substitutions in the Arc Repressor Protein: A Romance of Three Foldsen_US
dc.creatorStewart, Katie Lynnen_US
dc.contributor.authorStewart, Katie Lynnen_US
dc.date.issued2013-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.releaseRelease 14-Apr-2014en_US
dc.description.abstractMost amino acid sequences are predicted to specify a single three-dimensional protein structure. However, the identification of "metamorphic" proteins, which can adopt two folds from a single amino acid sequence, has challenged the one sequence/one structure paradigm. Polar-to-hydrophobic substitutions have been suggested computationally as one mechanism to decrease structural specificity, allowing the population of novel folds. Here, we experimentally investigate the role of polar-to-hydrophobic substitutions on structural specificity in the homodimeric ribbon-helix-helix protein Arc repressor. Previous work showed that a single polar-to-hydrophobic surface substitution in the strand region of Arc repressor (Arc-N11L) populates the wild-type fold and a novel dimeric "switch" fold. In this work, we investigate an Arc repressor variant with the N11L substitution plus two additional polar-to-hydrophobic surface substitutions (Arc-S-VLV). We determine that this sequence folds into at least three structures: both dimer forms present in Arc-N11L, and a novel octamer structure containing higher stability and less helicity than the dimer folds. We are able to isolate and stabilize a core of the S-VLV octamer by limited trypsinolysis and deletion mutagenesis (Arc-VLV 4-44). The shortened construct contains only the octameric structure by removing disordered C-terminal segments nonessential for this fold. A two-dimensional NMR spectrum of VLV 4-44 and subsequent trypsinolysis of this construct suggests that at least two types of subunits comprise the S-VLV octamer: subunits structured from residues 4 to 44 and subunits structured from residues 4 to 31. Crystal trials of trypsinolyzed Arc-VLV 4-44 yielded several leads, suggesting that obtaining a high resolution structure of the S-VLV octamer is possible. Relatedly, we determine that the proline residues flanking the Arc repressor strand act in concert as "gatekeepers" to prevent aggregation in the S-VLV sequence. We also find that three highly hydrophobic surface substitutions in the Arc repressor strand region are necessary and sufficient to promote higher-order oligomer formation. In summation, this work reveals in an experimental context that progressive increases in polar-to-hydrophobic surface substitutions populate increasingly diverse, structurally degenerate folds. These results suggest that "metamorphic" as well as "polymetamorphic" proteins, which adopt numerous folds, are possible outcomes for a single protein sequence.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectfold switchingen_US
dc.subjectpolar-to-hydrophobic substitutionsen_US
dc.subjectpolymetamorphic proteinen_US
dc.subjectstructural degeneracyen_US
dc.subjectstructural specificityen_US
dc.subjectBiochemistryen_US
dc.subjectArc repressoren_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineBiochemistryen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorCordes, Matthew H. J.en_US
dc.contributor.committeememberCordes, Matthew H. J.en_US
dc.contributor.committeememberBandarian, Vaheen_US
dc.contributor.committeememberFane, Bentleyen_US
dc.contributor.committeememberGhosh, Indraneelen_US
dc.contributor.committeememberMcEvoy, Meganen_US
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