Research Mentor(s)

Spencer Anthony-Cahill

Affiliated Department

Chemistry

Sort Order

42

Start Date

14-5-2015 10:00 AM

End Date

14-5-2015 2:00 PM

Keywords

Hemoglobin, X-ray crystalography

Document Type

Event

Abstract

Our research is focused on the production of a hemoglobin-based oxygen carrier (HBOC) which can be used as a therapeutic in the event of acute blood loss. The administration of cell-free hemoglobin is associated with severe adverse effects due to dissociation of the tetrameric α₂β₂ complex into αβ heterodimers. Our approach to designing an effective HBOC is based on a recombinant circularly permuted human hemoglobin in which all of the subunits are linked in a single-chain fashion. This design would prevent the dissociation of the tetramer and allow for the biosynthesis of polymeric hemoglobins of defined mass. Preliminary ligand binding data with our permuted hemoglobins indicates that they prefer the high O₂-affinity R-state conformation over the low O₂-affinity T state. The βN108K and αV96W mutations were introduced to restore T state stability. Preliminary studies of the mutants have shown that while the βN108K mutation improved T-state stability, the αV96W mutation displays an unexpected destabilizing effect on the T state. We would like to understand the molecular basis for the surprising results. We intend to determine the X-ray crystal structure of the αV96W mutant as well as the βN108K mutant and the αV96W + βN108K double mutant to gain an atomic-level picture of protein structural differences that could explain these results.

Rights

Copying of this document in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this documentation for commercial purposes, or for financial gain, shall not be allowed without the author's written permission.

Language

English

Format

application/pdf

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May 14th, 10:00 AM May 14th, 2:00 PM

Structural Studies of a Circularly Permuted Human Hemoglobin Containing Low O₂-affinity Mutations

Chemistry

Our research is focused on the production of a hemoglobin-based oxygen carrier (HBOC) which can be used as a therapeutic in the event of acute blood loss. The administration of cell-free hemoglobin is associated with severe adverse effects due to dissociation of the tetrameric α₂β₂ complex into αβ heterodimers. Our approach to designing an effective HBOC is based on a recombinant circularly permuted human hemoglobin in which all of the subunits are linked in a single-chain fashion. This design would prevent the dissociation of the tetramer and allow for the biosynthesis of polymeric hemoglobins of defined mass. Preliminary ligand binding data with our permuted hemoglobins indicates that they prefer the high O₂-affinity R-state conformation over the low O₂-affinity T state. The βN108K and αV96W mutations were introduced to restore T state stability. Preliminary studies of the mutants have shown that while the βN108K mutation improved T-state stability, the αV96W mutation displays an unexpected destabilizing effect on the T state. We would like to understand the molecular basis for the surprising results. We intend to determine the X-ray crystal structure of the αV96W mutant as well as the βN108K mutant and the αV96W + βN108K double mutant to gain an atomic-level picture of protein structural differences that could explain these results.

 

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