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Organization and dynamics of tryptophan residues in erythroid spectrin: Novel structural features of denatured spectrin revealed by the wavelength-selective fluorescence approach

¹ Centre for Cellular and Molecular Biology, Hyderabad 500 007, India
² Biophysics Division, Saha Institute of Nuclear Physics, Kolkata, 700 037, India

Reprint requests to: Amitabha Chattopadhyay, Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India; e-mail: amit{at}ccmb.res.in; fax: +91-40-2716-0311.

We have investigated the organization and dynamics of the functionally important tryptophan residues of erythroid spectrin in native and denatured conditions utilizing the wavelength-selective fluorescence approach. We observed a red edge excitation shift (REES) of 4 nm for the tryptophans in the case of spectrin in its native state. This indicates that tryptophans in spectrin are localized in a microenvironment of restricted mobility, and that the regions surrounding the spectrin tryptophans offer considerable restriction to the reorientational motion of the water dipoles around the excited state tryptophans. Interestingly, spectrin exhibits a REES of 3 nm even when denatured in 8 M urea. This represents the first report of a denatured protein displaying REES. Observation of REES in the denatured state implies that some of the structural and dynamic features of this microenvironment around the spectrin tryptophans are retained even when the protein is denatured. Fluorescence quenching data of denatured spectrin support this conclusion. In addition, we have deduced the organization and dynamics of the hydrophobic binding site of the polarity-sensitive fluorescent probe PRODAN that binds erythroid spectrin with high affinity. When bound to spectrin, PRODAN exhibits a REES of 9 nm. Because PRODAN binds to a hydrophobic site in spectrin, such a result would directly imply that this region of spectrin offers considerable restriction to the reorientational motion of the solvent dipoles around the excited state fluorophore. The results of our study could provide vital insight into the role of tryptophans in the stability and folding of spectrin.

Keywords: Spectrin; REES; denaturation; tryptophan; PRODAN; wavelength-selective fluorescence

Abbreviations: DANCA, 2'-(N,N-dimethyl)amino-6-naphthoyl-4-trans-cyclohexanoic acid • DTT, dithiothreitol • EDTA, ethylenediaminetetraacetic acid • NATA, N-acetyl-L-tryptophanamide • NBD, 7-nitrobenz-2-oxa-1,3-diazol-4-yl • PMSF, phenylmethylsulfonyl fluoride • PRODAN, 6-propionyl-2-dimethylaminonaphthalene • REES, red edge excitation shift • SDS, sodium dodecyl sulfate • TNS, 2-(p-toluidinylnaphthalene)-6-sulfonate • TRIS, tris-(hydroxymethyl)aminomethane

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