Peptides with More than One 106-amino Acid Sequence Motif Are Needed to Mimic the Structural Stability of Spectrin

doi:10.1074/jbc.271.48.30410

Peptides with More than One 106-amino Acid Sequence Motif Are Needed to Mimic the Structural Stability of Spectrin*

From the Department of Chemistry, Loyola University of Chicago, Chicago, Illinois 60626

^§ To whom correspondence should be addressed:
Dept. of Chemistry, Loyola University of Chicago, 6525 N. Sheridan Rd., Chicago, IL 60626.
Tel.: 773-508-3128; Fax: 773-508-3086.

Abstract

The primary sequence of human erythrocyte spectrin contains repetitive homologous sequence motifs of approximately 106 amino acids with 22 such motifs in the α-subunit and 17 in the β-subunit. These homologous sequence motifs have been proposed to form domains with a triple-helical bundle type structure (Speicher, D. W., and Marchesi, V. T. (1984) Nature 311, 177-180; Parry, D. A. D., Dixon, T. W., and Cohen, C. (1992) Biophys. J. 61, 858-867). In this study, we show that these sequence motifs, while they do form compact proteolytically resistant units, are not completely independent. Peptides composed of two or three such motifs in tandem are substantially more stable than peptides composed of a single motif, as measured by proteolysis or by fluorescence or circular dichroism studies of urea or thermal denaturation. Circular dichroism and infrared spectroscopy measurements also indicate that these larger, more stable peptides exhibit greater secondary structure. In these respects, the peptides with tandem sequence motifs are more similar to intact spectrin than the peptide with a single sequence motif. Thus, we conclude that peptides with more than one sequence motif model spectrin more adequately than the peptides with one sequence motif, and that these sequence motifs are not completely independent domains.

Footnotes

↵^‡ Supported in part by fellowships from the Graduate Assistance in Areas of National Need Program.
↵* This work was supported in part by the National Science Foundation and Loyola University of Chicago. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

FTIR

Fourier transform infrared

Spαi-j

human erythroid α-spectrin recombinant peptide composed of residues i-j

δG

free energy change in unfolding

Φ₂₂₂

CD ellipticity at 222 nm

I_λ

fluorescence intensity at wavelength λ

λ_mean

mean emission wavelength

λ_max

wavelength with maximum intensity

PBS

phosphate-buffered saline

τ_f1

time constant for the fast phase in urea unfolding (from fluorescence results)

τ_f2

time constant for the slow phase in urea unfolding (from fluorescence results)

τ_g

time constant for the disappearance of peptide by elastase digestion (from gel data)

U

urea concentration

[urea]_mid

mean urea concentrations for transition from native to denatured state

T_m

temperature of maximum value of δΦ₂₂₂/δT

PAGE

polyacrylamide gel electrophoresis.
↵² ProtParam can be obtained from the following molecular biology server on the World Wide Web (http://expasy.hcuge.ch/www/expasy-top.html).
- Received May 21, 1996.
- Revision received September 11, 1996.