The dynamics of protein interactions in erythrocytes
Abstract
Much is known about the static nature of protein interactions in the erythrocyte membrane and spectrin skeleton; however, the dynamics of many protein interactions is not well understood. We performed structural and functional studies on erythrocyte proteins in order to understand these associations better. We evaluated the reasons why deletion of nine amino acids joining the membrane and cytoplasmic domains of band 3 from Southeast Asian Ovalocytes (SAO) renders erythrocytes rigid by comparing the structural and functional properties of SAO and normal band 3. We found that SAO erythrocytes had more band 3 nonspecifically entrapped in the spectrin skeleton; thus, we propose that this entrapment may be responsible for the rigidity of the SAO erythrocyte. Using differential scanning calorimetry and the anion transport inhibitor, DIDS, we determined that one membrane-spanning domain of band 3 could allosterically communicate with another. We also found that we could perturb ankyrin's association with band 3 by shifting the subunit equilibrium to favor the dimer, a form of band 3 that ankyrin cannot bind. This allowed us to clarify previous data on the association of ankyrin and band 3. We then disrupted half of the band 3-ankyrin linkages by converting half of the band 3 tetramers to dimers and determined the behavior of the erythrocytes under shear stress. This disruption did not change the cell stability or deformability. These results suggest that either breakage of 50% of the ankyrin-band 3 linkages is not critical for the normal deformability/stability of red cells or that other compensating interactions offset the DIDS-induced defect. We hypothesize that the anticipated weakening of the membrane due to the loss of membrane-to-skeleton tethers is largely compensated by increased interactions between band 3 and lipid molecules.
Degree
Ph.D.
Advisors
Low, Purdue University.
Subject Area
Biochemistry|Biophysics
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