TY - JOUR
T1 - UV resonance Raman spectra of ligand binding intermediates of sol-gel encapsulated hemoglobin
AU - Juszczak, Laura J.
AU - Friedman, Joel M.
PY - 1999/10/22
Y1 - 1999/10/22
N2 - We report for the first time specific conformational changes for a homogeneous population of ligand-bound adult deoxy human hemoglobin A (HbA) generated by introducing CO into a sample of deoxy-HbA with the effector, inositol hexaphosphate, encapsulated in a porous sol-gel. The preparation of ligand-bound deoxy-HbA results from the speed of ligand diffusion relative to globin conformational dynamics within the sol-gel (1). The ultraviolet resonance Raman (UVRR) difference spectra obtained reveal that E helix motion is initiated upon ligand binding, as signaled by the appearance of an α14β15 Trp W3 band difference at 1559 cm-1. The subsequent appearance of Tyr (Y8a and Y9a) and W3 (1549 cm-1) UVRR difference bands suggest conformational shifts for the penultimate Tyrα140 on the F helix, the 'switch' region Tyrα42, and the 'hinge' region Trpβ37. The UVRR results expose a sequence of conformational steps leading up to the ligation-induced T to R quaternary structure transition as opposed to a single, concerted switch. More generally, this report demonstrates that sol-gel encapsulation of proteins can be used to study a sequence of specific conformational events triggered by substrate binding because the traditional limitation of substrate diffusion times is overcome.
AB - We report for the first time specific conformational changes for a homogeneous population of ligand-bound adult deoxy human hemoglobin A (HbA) generated by introducing CO into a sample of deoxy-HbA with the effector, inositol hexaphosphate, encapsulated in a porous sol-gel. The preparation of ligand-bound deoxy-HbA results from the speed of ligand diffusion relative to globin conformational dynamics within the sol-gel (1). The ultraviolet resonance Raman (UVRR) difference spectra obtained reveal that E helix motion is initiated upon ligand binding, as signaled by the appearance of an α14β15 Trp W3 band difference at 1559 cm-1. The subsequent appearance of Tyr (Y8a and Y9a) and W3 (1549 cm-1) UVRR difference bands suggest conformational shifts for the penultimate Tyrα140 on the F helix, the 'switch' region Tyrα42, and the 'hinge' region Trpβ37. The UVRR results expose a sequence of conformational steps leading up to the ligation-induced T to R quaternary structure transition as opposed to a single, concerted switch. More generally, this report demonstrates that sol-gel encapsulation of proteins can be used to study a sequence of specific conformational events triggered by substrate binding because the traditional limitation of substrate diffusion times is overcome.
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U2 - 10.1074/jbc.274.43.30357
DO - 10.1074/jbc.274.43.30357
M3 - Article
C2 - 10521410
AN - SCOPUS:0032746488
SN - 0021-9258
VL - 274
SP - 30357
EP - 30360
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 43
ER -