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

Abstract

The primary sequence of human erythrocyte spectrin contains repetitive homologous sequence motifs of approximately 106 amino acids with 22 such motifs in the ␣-subunit and 17 in the ␤-subunit. These homologous sequence motifs have been proposed to form domains with a triple-helical bundle type structure (Speicher, 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.DHuman erythrocytes contain a dense, two-dimensional network of spectrin and other proteins that provides support to the lipid bilayer and maintains erythrocyte deformability (1). Spectrin, comprising ␣-and ␤-subunits, plays a critical role in maintaining the architecture and therefore the integrity of the red cell membrane. Many hereditary hemolytic anemias involve spectrin mutations (2-4). Thus, it is important to understand the structural properties of spectrin. The bulk (about 90%) of the primary structure of spectrin comprises repetitive homologous units of approximately 106 amino acids in length. Several other proteins, including brain spectrin (fodrin), dystrophin, and ␣-actinin, also have similar repetitive amino acid units in their sequences and are known as the spectrin superfamily (5).A triple-helical bundle model has been suggested for the 106-amino acid sequence motif in which the three helices are aligned side by side, with the first and third parallel and the intervening second helix antiparallel (6 -8). X-ray diffraction studies of one such sequence motif unit from a non-erythroid spectrin support this model (9). In these x-ray studies, the peptide used was found to form a homodimer containing two triple-helical structures, in which two of the three helices are contributed by one monomer and the remaining helix by the other monomer. It is thought that this peculiar arrangement may be an artifact of crystallization, and the true structure of a single unit may be similar to the earlier suggested triplehelical bundle with a zigzag arrangement of the helices (9, 10). This arrangement aligns the amino-and carboxyl-terminal residues at opposite ends of this triple-helical bundle, and thus sequential motifs are thought to be linked in tandem in intact spectrin, producing a very long rod-shaped molecule, approximately 100 nm in l...

Interactions of the α-Spectrin N-terminal Region with β-Spectrin

“…Purification procedures were as previously discussed (25,32), except that the buffers for the FPLC MonoQ purification were changed to 25 mM bis-Tris at pH 6.0 and the NaCl concentration was changed to 500 mM. Peptide identity was checked by SDS-polyacrylamide gel electrophoresis and amino-terminal sequencing (Biocore Facility, University of Notre Dame, South Bend, IN).…”

“…These peptides were expressed as glutathione S-transferase fusion proteins in Escherichia coli and released by thrombin cleavage with soluble thrombin (32). Purification procedures were as previously discussed (25,32), except that the buffers for the FPLC MonoQ purification were changed to 25 mM bis-Tris at pH 6.0 and the NaCl concentration was changed to 500 mM.…”

“…Preparation and Analysis of ␤-Spectrin and of ␣-Spectrin PeptidesSpectrin dimer was obtained by low ionic strength extraction of erythrocyte ghosts at 37°C, followed by molecular exclusion chromatography, as described previously (32). ␤-spectrin was obtained from spectrin dimer as before (21), except that ␤-spectrin was purified on the FPLC system using MonoQ column (Amersham Pharmacia Biotech) and NaCl gradient.…”

“…Fidelity of the polymerase-mediated reactions was subsequently confirmed by direct DNA sequencing. Genes encoding for the peptides Sp␣-(1-368), Sp␣-(1-262), Sp␣-(1-156), and Sp␣-(52-368) were prepared as described previously (25,32). The genes encoding for the peptides Sp␣-(12-368), Sp␣-(22-368), and Sp␣-(37-368) were derived from the gene for Sp␣-(1-368).…”

“…The molecular masses of these peptides were determined by mass spectrometry using electrospray ionization techniques (Washington University, St. Louis, MO). Protein concentrations were determined with absorbance values at 280 nm, using extinction coefficients determined from the primary sequence (32). In order to determine whether the peptides formed aggregates in buffer solution (5 mM phosphate buffer with 150 mM NaCl at pH 7.4), the solution molecular masses of the peptides at 1-2 mg/ml were determined at 4°C with an absolute molecular mass determination instrument based on multiangle (15 and 90°) light scattering (PD2000; Precision Detectors, Inc., Amherst, MA) as described previously (32).…”