Physicochemical characterization of bovine retinal arrestin

Susan Kotake, Patricia Hey, Raghu Mirmira, Robert A. Copeland

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

The native conformation of bovine retinal arrestin has been characterized by a variety of spectroscopic methods. The purified protein gives rise to a near uv absorption band centered at 279 nm which results from the absorbance of its 14 tyrosine and one tryptophan residue. The extinction coefficient for this absorption band was determined to be 38.64 mm-1, cm-1 using the tyrosinate-tyrosine difference spectrum method; this extinction coefficient is ca. 17% lower than the previously reported value, and provides estimates of protein concentration which are in good agreement with estimates from the Bradford colorimetric assay. When native arrestin is purified to homogeneity, it displays a fluorescence spectrum which is dominated by tyrosine emission with no discernible contribution from tryptophan. Observation of the tyrosine-like fluorescence is dependent on the purity and structural integrity of the protein. Denaturation of arrestin by guanidine hydrochloride results in a diminution of tyrosine fluorescence and the concomitant appearance of a second fluorescence maximum at ca. 340 nm, presumably due to the single tryptophan residue. Thermal denaturation of arrestin leads to a conformation characterized by a broad fluorescence band centered at ca. 325 nm. Study of the arrestin fluorescence spectrum as a function of temperature indicates that the thermal denaturation is well modeled as a two-state transition with a transition midpoint of 60 °C. Temperature-dependent far uv circular dichroism studies indicate that changes in secondary structure occur coincident with the change in fluorescence. Studies of the temperature dependence of arrestin binding to light-adapted phosphorylated rhodopsin shows a strong correlation between the fluorescence spectral features of arrestin and its ability to bind rhodopsin. These data suggest that the relative intensities of tyrosine and tryptophan fluorescence are sensitive to the structural integrity of the native (i.e., rhodopsin binding) state of arrestin, and can thus serve as useful markers of conformational transitions of this protein. The lack of tryptophan fluorescence for native arrestin suggests an unusual environment for this residue. Possible mechanisms for this tryptophan fluorescence quenching are discussed.

Original languageEnglish (US)
Pages (from-to)126-133
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume285
Issue number1
DOIs
StatePublished - Feb 15 1991
Externally publishedYes

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Arrestin
Fluorescence
Tryptophan
Tyrosine
Denaturation
Rhodopsin
Structural integrity
Temperature
Conformations
Absorption spectra
Proteins
Hot Temperature
Guanidine
Circular Dichroism
Quenching
Assays

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Physicochemical characterization of bovine retinal arrestin. / Kotake, Susan; Hey, Patricia; Mirmira, Raghu; Copeland, Robert A.

In: Archives of Biochemistry and Biophysics, Vol. 285, No. 1, 15.02.1991, p. 126-133.

Research output: Contribution to journalArticle

Kotake, Susan ; Hey, Patricia ; Mirmira, Raghu ; Copeland, Robert A. / Physicochemical characterization of bovine retinal arrestin. In: Archives of Biochemistry and Biophysics. 1991 ; Vol. 285, No. 1. pp. 126-133.
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abstract = "The native conformation of bovine retinal arrestin has been characterized by a variety of spectroscopic methods. The purified protein gives rise to a near uv absorption band centered at 279 nm which results from the absorbance of its 14 tyrosine and one tryptophan residue. The extinction coefficient for this absorption band was determined to be 38.64 mm-1, cm-1 using the tyrosinate-tyrosine difference spectrum method; this extinction coefficient is ca. 17{\%} lower than the previously reported value, and provides estimates of protein concentration which are in good agreement with estimates from the Bradford colorimetric assay. When native arrestin is purified to homogeneity, it displays a fluorescence spectrum which is dominated by tyrosine emission with no discernible contribution from tryptophan. Observation of the tyrosine-like fluorescence is dependent on the purity and structural integrity of the protein. Denaturation of arrestin by guanidine hydrochloride results in a diminution of tyrosine fluorescence and the concomitant appearance of a second fluorescence maximum at ca. 340 nm, presumably due to the single tryptophan residue. Thermal denaturation of arrestin leads to a conformation characterized by a broad fluorescence band centered at ca. 325 nm. Study of the arrestin fluorescence spectrum as a function of temperature indicates that the thermal denaturation is well modeled as a two-state transition with a transition midpoint of 60 °C. Temperature-dependent far uv circular dichroism studies indicate that changes in secondary structure occur coincident with the change in fluorescence. Studies of the temperature dependence of arrestin binding to light-adapted phosphorylated rhodopsin shows a strong correlation between the fluorescence spectral features of arrestin and its ability to bind rhodopsin. These data suggest that the relative intensities of tyrosine and tryptophan fluorescence are sensitive to the structural integrity of the native (i.e., rhodopsin binding) state of arrestin, and can thus serve as useful markers of conformational transitions of this protein. The lack of tryptophan fluorescence for native arrestin suggests an unusual environment for this residue. Possible mechanisms for this tryptophan fluorescence quenching are discussed.",
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