Nick Menhart

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

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.

Mammaglobin Complexes with BU101 in Breast Tissue

We queried the Lifeseq EST database (Incyte Genomics) with the known human uteroglobin sequences to investigate their tissue specificity. The database comprises over 1100 libraries prepared from well-characterized tissues, both diseased and normal. The libraries have significant depth, averaging 4000 ESTs of greater than 200 bases each. The database provides an electronic link between the tissue, its pathology, and the profile of expressed sequences generated from that tissue. One can query the database with a sequence and retrieve the tissues in which the sequence was found. The human sequences queried included mammaglobin,1 mammaglobin B,2 BU101,3 ESSBF I,4 and lipophilin A.5 TABLE 1 presents the abundance (number of ESTs) found in the tissue that encodes the sequence of interest. As other investigators have observed,1,5 mammaglobin is very specific for mammary tissue (included under exocrine tissue). Mammaglobin B does not present the same profile. It is more specific for tissues of the female reproductive system, specifically the uterus. BU101 is present in both tissue types, exocrine (breast) and reproductive (uterus), whereas ESSBF I is present in only the reproductive tissues. Lipophilin A was not found in the database, which may indicate its lower expression level or a difficulty in generating ESTs from the message. For comparison, CC10 (Clara cell 10-kDa protein) was present only in respiratory tissue. This tissue distribution profile of the uteroglobin member sequences provides a platform for proposing potential partners in protein complex formation. In breast tissue, Mam and BU101 are expressed at measurable levels, unlike MamB, which is barely detected. The presence of MamB in breast tissue is suspect until abundance levels rise to include multiple specimens of the same tissue type. Based on known uteroglobin protein structures, the potential is there for Mam and BU101 to be involved in complex formation in breast tissue. In uterine tissue, BU101 may partner with MamB and/or ESSBF I. The rat prostatein complex provides an interesting model6 for these human sequences. BU101, ESSBF I, and lipophilin A are closely related to the C1 and C2 chains, which appear only once in the rat heterotetramer. Mam and MamB, however, are more closely related to the C3 chain, which appears twice in the heterotetramer.

Spin label EPR structural studies of the N-terminus of α-spectrin

Spectrin, a vital component in human erythrocyte, is composed of α‐ and β‐subunits, which associate to form (αβ)2 tetramers. The tetramerization site is believed to involve the α‐spectrin N‐terminus and the β‐spectrin C‐terminus. Abnormal interactions in this region may lead to blood disorders. It has been proposed that both termini consist of partial structural domains and that tetramerization involves the association of these partial domains. We have studied the N‐terminal region of a model peptide for α‐spectrin by making a series of double spin‐labeled peptides and studying their dipolar interaction by electron paramagnetic resonance methods. Our results indicate that residues 21–42 of the N‐terminus region exhibit an α‐helical conformation, even in the absence of β‐spectrin.

Flexibility of the α-Spectrin N-Terminus by EPR and Fluorescence Polarization

The structure and flexibility of the biologically important alpha-spectrin amino terminal region was examined by the use of fluorescence and EPR spectroscopy. The region studied has been previously demonstrated to be essential for the alpha-spectrin:beta-spectrin association of the tetramerization site. Appropriate spectroscopic probe moieties were coupled to this region in a recombinant fragment of human erythroid alpha-spectrin. There was good agreement between the EPR and fluorescence techniques in most of this region. Mobility determinations indicated that a portion of the region was relatively immobilized. This is significant, since although predictive methods have indicated that this region should be alpha-helical, previous experimental evidence obtained on smaller synthetic peptides had indicated that this region was disordered. Observed rigidity appears to be incompatible with such a disordered state, and has important ramifications for the flexibility of this molecule that is so integral to its role in stabilizing erythrocyte membranes.