title: Hemoglobin interactions with αB crystallin: a direct test of sensitivity to protein instability.
abstract: As a small stress response protein, human αB crystallin, detects protein destabilization that can alter structure and function to cause self assembly of fibrils or aggregates in diseases of aging. The sensitivity of αB crystallin to protein instability was evaluated using wild-type hemoglobin (HbA) and hemoglobin S (HbS), the glutamate-6-valine mutant that forms elongated, filamentous aggregates in sickling red blood cells. The progressive thermal unfolding and aggregation of HbA and HbS in solution at 37°C, 50°C and 55°C was measured as increased light scattering. UV circular dichroism (UVCD) was used to evaluate conformational changes in HbA and HbS with time at the selected temperatures. The changes in interactions between αB crystallin and HbA or HbS with temperature were analyzed using differential centrifugation and SDS PAGE at 37°C, 50°C and 55°C. After only 5 minutes at the selected temperatures, differences in the aggregation or conformation of HbA and HbS were not observed, but αB crystallin bound approximately 6% and 25% more HbS than HbA at 37°C, and 50°C respectively. The results confirmed (a) the remarkable sensitivity of αB crystallin to structural instabilities at the very earliest stages of thermal unfolding and (b) an ability to distinguish the self assembling mutant form of HbS from the wild type HbA in solution.
authors: Department of Biological Structure, University of Washington School of Medicine, Seattle, Washington, United States of America.
authors: Tyler J W
authors: TJ
authors: Clark
authors: Scott A
authors: SA
authors: Houck
authors: John I
authors: JI
authors: Clark
mesh_terms: D006441:Hemoglobin A / Q000737:chemistry* / Q000378:metabolism*; D006451:Hemoglobin, Sickle / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D055503:Protein Multimerization; D055550:Protein Stability / Q000187:drug effects; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D058767:Protein Unfolding / Q000187:drug effects; D013696:Temperature; D038203:alpha-Crystallin B Chain / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D006451:Hemoglobin, Sickle; D038203:alpha-Crystallin B Chain; D006441:Hemoglobin A