The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy

Scott Blackman, Charles E. Cobb, Albert H. Beth, David W. Piston

Research output: Contribution to journalArticle

Abstract

The dominant motional mode for membrane proteins is uniaxial rotational diffusion about the membrane normal axis, and investigations of their rotational dynamics can yield insight into both the oligomeric state of the protein and its interactions with other proteins such as the cytoskeleton. However, results from the spectroscopic methods used to study these dynamics are dependent on the orientation of the probe relative to the axis of motion. We have employed polarized fluorescence confocal microscopy to measure the orientation of eosin-5-maleimide covalently reacted with Lys-430 of human erythrocyte band 3. Steady-state polarized fluorescence images showed distinct intensity patterns, which were fit to an orientation distribution of the eosin absorption and emission dipoles relative to the membrane normal axis. This orientation was found to be unchanged by trypsin treatment, which cleaves band 3 between the integral membrane domain and the cytoskeleton- attached domain. This result suggests that phosphorescence anisotropy changes observed after trypsin treatment are due to a rotational constraint change rather than a reorientation of eosin. By coupling time-resolved prompt fluorescence anisotropy with confocal microscopy, we calculated the expected amplitudes of the e(-Dt) and e(-4Dt) terms from the uniaxial rotational diffusion model and found that the e(-4Dt) term should dominate the anisotropy decay. Delayed fluorescence and phosphorescence anisotropy decays of control and trypsin-treated band 3 in ghosts, analyzed as multiple uniaxially rotating populations using the amplitudes predicted by confocal microscopy, were consistent with three motional species with uniaxial correlation times ranging from 7 μs to 1.4 ms.

Original languageEnglish (US)
Pages (from-to)194-208
Number of pages15
JournalBiophysical Journal
Volume71
Issue number1
StatePublished - Jul 1996
Externally publishedYes

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Polarization Microscopy
Fluorescence Polarization
Fluorescence Microscopy
Confocal Microscopy
Trypsin
Erythrocytes
Anisotropy
Eosine Yellowish-(YS)
Cytoskeleton
Membranes
Membrane Proteins
Proteins
Fluorescence
Population
eosin maleimide

ASJC Scopus subject areas

  • Biophysics

Cite this

The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy. / Blackman, Scott; Cobb, Charles E.; Beth, Albert H.; Piston, David W.

In: Biophysical Journal, Vol. 71, No. 1, 07.1996, p. 194-208.

Research output: Contribution to journalArticle

Blackman, Scott ; Cobb, Charles E. ; Beth, Albert H. ; Piston, David W. / The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy. In: Biophysical Journal. 1996 ; Vol. 71, No. 1. pp. 194-208.
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