Presentation Title

Sequence based conformational bias in a 3-helix bundle

Authors' Names

Moses J. Leavens

Presentation Type

Oral Presentation

Abstract

It is well established that proteins spontaneously refold into their native structure, and this structure is important for function. The denatured (i.e. unfolded) state of a protein has been found to contain pockets of non-random structure, and this has provided clues to deciphering the protein folding code. Previous work with the four-helix bundle protein cytochrome c’ in Rhodopseudomonas palustris using histidine-heme loop formation thermodynamic methods revealed fold-specific deviations from random coil character in its denatured state ensemble. To examine the generality of this finding, we extend this work to a three-helix bundle polypeptide, the human DNA excision repair protein’s second ubiquitin-associated (UBA) domain, UBA(2). We use yeast iso-1-cytochrome c as a scaffold, fusing the UBA(2) domain to the N-terminus of iso-1-cytochrome c. Using site-directed mutagenesis, we have engineered the amino acid histidine into solvent accessible surface amino acid positions within UBA(2), creating eight single histidine variants. Isothermal equilibrium denaturation studies reveal that the fusion protein unfolds in a 3-state process, commencing with iso-1-cytochrome c followed by UBA(2). Thermodynamic stability experiments also demonstrate that the histidine positions in the UBA(2) domain strongly destabilize iso-1-cytochrome c. Furthermore, histidine-heme loop formation equilibria in the denatured state show lower apparent pKa’s compared to the pseudo-wild type variant, indicating significant interactions in the denatured state. Comparing the degree of deviation of loop stability versus loop size using the Jacobson-Stockmayer relationship, we observe significant deviation from random coil behavior in reverse turn sequences within the UBA(2) domain, in agreement with observations discovered in cytochrome c’. Kinetic experiments indicate that these reverse turns in UBA(2) are persisting in the denatured state. Therefore, reverse turn sequences are biasing the conformational search for these helix bundles, are important for setting up the overall structure of these proteins, and are critical in understanding the nature of the protein folding code. The significance of this work would be applied to treating protein misfolding diseases, such as Alzheimer’s, Parkinson’s, and Cystic fibrosis.

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Apr 27th, 10:00 AM Apr 27th, 10:15 AM

Sequence based conformational bias in a 3-helix bundle

UC Ballroom, Pod #3

It is well established that proteins spontaneously refold into their native structure, and this structure is important for function. The denatured (i.e. unfolded) state of a protein has been found to contain pockets of non-random structure, and this has provided clues to deciphering the protein folding code. Previous work with the four-helix bundle protein cytochrome c’ in Rhodopseudomonas palustris using histidine-heme loop formation thermodynamic methods revealed fold-specific deviations from random coil character in its denatured state ensemble. To examine the generality of this finding, we extend this work to a three-helix bundle polypeptide, the human DNA excision repair protein’s second ubiquitin-associated (UBA) domain, UBA(2). We use yeast iso-1-cytochrome c as a scaffold, fusing the UBA(2) domain to the N-terminus of iso-1-cytochrome c. Using site-directed mutagenesis, we have engineered the amino acid histidine into solvent accessible surface amino acid positions within UBA(2), creating eight single histidine variants. Isothermal equilibrium denaturation studies reveal that the fusion protein unfolds in a 3-state process, commencing with iso-1-cytochrome c followed by UBA(2). Thermodynamic stability experiments also demonstrate that the histidine positions in the UBA(2) domain strongly destabilize iso-1-cytochrome c. Furthermore, histidine-heme loop formation equilibria in the denatured state show lower apparent pKa’s compared to the pseudo-wild type variant, indicating significant interactions in the denatured state. Comparing the degree of deviation of loop stability versus loop size using the Jacobson-Stockmayer relationship, we observe significant deviation from random coil behavior in reverse turn sequences within the UBA(2) domain, in agreement with observations discovered in cytochrome c’. Kinetic experiments indicate that these reverse turns in UBA(2) are persisting in the denatured state. Therefore, reverse turn sequences are biasing the conformational search for these helix bundles, are important for setting up the overall structure of these proteins, and are critical in understanding the nature of the protein folding code. The significance of this work would be applied to treating protein misfolding diseases, such as Alzheimer’s, Parkinson’s, and Cystic fibrosis.