Human Erythrocyte Spectrin Research Articles

Electron Microscopy and Persistence Length Analysis of Semi-Rigid Smooth Muscle Tropomyosin Strands

The structural mechanics of tropomyosin are essential determinants of its affinity and positioning on F-actin. Thus, tissue-specific differences among tropomyosin isoforms may influence both access of actin-binding proteins along the actin filaments and the cooperativity of actin-myosin interactions. Here, 40 nm long smooth and striated muscle tropomyosin molecules were rotary-shadowed and compared by means of electron microscopy. Electron microscopy shows that striated muscle tropomyosin primarily consists of single molecules or paired molecules linked end-to-end. In contrast, smooth muscle tropomyosin is more a mixture of varying-length chains of end-to-end polymers. Both isoforms are characterized by gradually bending molecular contours that lack obvious signs of kinking. The flexural stiffness of the tropomyosins was quantified and evaluated. The persistence lengths along the shaft of rotary-shadowed smooth and striated muscle tropomyosin molecules are equivalent to each other (approximately 100 nm) and to values obtained from molecular-dynamics simulations of the tropomyosins; however, the persistence length surrounding the end-to-end linkage is almost twofold higher for smooth compared to cardiac muscle tropomyosin. The tendency of smooth muscle tropomyosin to form semi-rigid polymers with continuous and undampened rigidity may compensate for the lack of troponin-based structural support in smooth muscles and ensure positional fidelity on smooth muscle thin filaments.

Spectrin functions upstream of ankyrin in a spectrin cytoskeleton assembly pathway

Prevailing models place spectrin downstream of ankyrin in a pathway of assembly and function in polarized cells. We used a transgene rescue strategy in Drosophila melanogaster to test contributions of four specific functional sites in β spectrin to its assembly and function. (1) Removal of the pleckstrin homology domain blocked polarized spectrin assembly in midgut epithelial cells and was usually lethal. (2) A point mutation in the tetramer formation site, modeled after a hereditary elliptocytosis mutation in human erythrocyte spectrin, had no detectable effect on function. (3) Replacement of repetitive segments 4–11 of β spectrin with repeats 2–9 of α spectrin abolished function but did not prevent polarized assembly. (4) Removal of the putative ankyrin-binding site had an unexpectedly mild phenotype with no detectable effect on spectrin targeting to the plasma membrane. The results suggest an alternate pathway in which spectrin directs ankyrin assembly and in which some important functions of spectrin are independent of ankyrin.

Safety of Total Therapy III for Newly Diagnosed Multiple Myeloma: Preliminary Analysis of 62 Consecutive Patients. Safety of Total Therapy III for Newly Diagnosed Multiple Myeloma: Preliminary Analysis of 62 Consecutive Patients.

Malaria is one of the world's major health problems, causing millions of deaths every year, primarily in Africa. The disease is caused by Plasmodium parasites, which invade and destroy human erythrocytes. Of the four species infecting humans, Plasmodium falciparum is responsible for the greatest morbidity and mortality burden. The erythrocyte membrane plays a vital role in all aspects of the pathogenic phase of the parasite's life cycle and the protein-protein interactions between host and parasite are a key focus of research. Spectrin is the main structural protein in the erythrocyte membrane skeleton and phage-display technology was used to probe the interaction between P falciparum peptide fragments and human erythrocyte spectrin. A phage-display library was constructed by isolating mRNA from P falciparum strain FCR-3, which was reverse transcribed using two-base anchored oligodT primers. Linkers facilitating directional cloning were added to the cDNA, followed by insertion into a gene encoding the 10B capsid protein of the T7 bacteriophage vector. The vector was packaged into viral particles and the library amplified using Escherichia coli as a host. The presence and size of inserts were determined by PCR amplification with T7 bacteriophage vector arm specific primers. Human erythrocyte membranes were prepared from whole blood by hypotonic lysis and spectrin was extracted with a low ionic strength buffer and purified by size exclusion chromatography. The protein was biotinylated, immobilized on streptavidin-coated magnetic beads and biopanned against the phage library. Bound phage were eluted and amplified in E coli for three additional rounds of biopanning to eliminate non-specific protein-protein interactions. The P falciparum cDNA inserts of interacting phage were sequenced and compared to the PlasmoDB database. One of the sequences was identified as a putative aminopeptidase (PFI1570c), which has a 30.7% homology to a human aspartyl aminopeptidase, an enzyme catalysing the release of N-terminal amino acids from a peptide. The parasite protein contains a putative transmembrane domain at the C terminal end and is larger than the human form, with an estimated molecular weight of 65 kD. Several features that are critical for enzyme activity are conserved in the P falciparum aminopeptidase. These include twelve amino acids (four histidine, three glutamic acid and five aspartic acid residues), which are involved in the binding of catalytic zinc ions in the active site, as well as a putative N-myristoylation site and phosphorylation sites for casein kinase II and protein kinase C. Interestingly, the peptide fragment that bound to spectrin in the initial phage display screening, corresponds to a 33 amino acid fragment that is not found in the human aspartyl aminopeptidase. This suggests an evolutionary development of the parasite that allows the protease to bind to human spectrin. Mass spectrometry and microarray data from the PlasmoDB database indicate that the protein is present at the erythrocyte membrane and is expressed in all the developmental stages of the parasite's erythrocytic life cycle. During the trophozoite stage the parasite modifies the erythrocyte membrane to allow transport of nutrients and waste products. The aminopeptidase could cleave spectrin and destabilise the membrane skeleton to facilitate the insertion of parasite protein channels during development. It may also play a role in proteolysis of the skeleton to enable the release of schizonts from infected erythrocytes.

In vivo phosphorylation of human erythrocyte spectrin occurs in a sequential manner.

Spectrin is the major component of the erythrocyte membrane skeleton and exists as a 526 kDa alphabeta heterodimer. The 246 kDa beta-chain of human spectrin is phosphorylated near the C-terminus, but the exact phosphorylation sites are unknown and the role of this phosphorylation is not fully characterized. In this study, we produced a monoclonal antibody, Sp316, capable of recognizing the C-terminal region of beta-spectrin regardless of its phosphorylation state and used it to purify the phosphorylated region after 2-nitro-5-thiocyanobenzoic acid cleavage of spectrin. Two-dimensional gels, mass spectrometry, and reversed-phase high-performance liquid chromatography were used to characterize these phosphorylation states. Only about 1.5% of spectrin isolated from fresh blood is unphosphorylated, about 9% has more than four phosphates per molecule, and the majority of the protein has one to four phosphates per molecule. A total of six phosphorylation sites were identified by tandem mass spectrometry. Quantitative analysis of the phosphorylation states by reversed-phase high-performance liquid chromatography revealed that phosphorylation of beta-spectrin occurs in a sequential manner where each specific site is completely phosphorylated before the next site is modified. The first phosphorylation event occurs on Ser-2114, followed by Ser-2125, Ser-2123, Ser-2128, Ser-2117, and Thr-2110. The identification of the specific phosphorylated beta-spectrin residues and the ordered sequence of phosphorylation events in vivo should provide an invaluable basis for further studies of the role of these posttranslational modifications in spectrin function in situ.

Construction and use of Plasmodium falciparum phage display libraries to identify host parasite interactions

BackgroundThe development of Plasmodium falciparum within human erythrocytes induces a wide array of changes in the ultrastructure, function and antigenic properties of the host cell. Numerous proteins encoded by the parasite have been shown to interact with the erythrocyte membrane. The identification of new interactions between human erythrocyte and P. falciparum proteins has formed a key area of malaria research. To circumvent the difficulties provided by conventional protein techniques, a novel application of the phage display technology was utilised.MethodsP. falciparum phage display libraries were created and biopanned against purified erythrocyte membrane proteins. The identification of interacting and in-frame amino acid sequences was achieved by sequencing parasite cDNA inserts and performing bioinformatic analyses in the PlasmoDB database.ResultsFollowing four rounds of biopanning, sequencing and bioinformatic investigations, seven P. falciparum proteins with significant binding specificity toward human erythrocyte spectrin and protein 4.1 were identified. The specificity of these P. falciparum proteins were demonstrated by the marked enrichment of the respective in-frame binding sequences from a fourth round phage display library.ConclusionThe construction and biopanning of P. falciparum phage display expression libraries provide a novel approach for the identification of new interactions between the parasite and the erythrocyte membrane.

Important region in the beta-spectrin C-terminus for spectrin tetramer formation.

Many hereditary hemolytic anemias are due to spectrin mutations at the C-terminal region of beta-spectrin (the betaC region) that destabilize spectrin tetramer formation. However, little is known about the betaC region of spectrin. We have prepared four recombinant beta-peptides of different lengths from human erythrocyte spectrin, all starting at position 1898 of the C-terminal region, but terminating at position 2070, 2071, 2072 or 2073. Native polyacrylamide gel electrophoresis showed that the two peptides terminating at positions 2070 and 2071 did not associate with an N-terminal region alpha-peptide (Spalpha1-156) in the micromolar range. However, the peptides that terminated at positions 2072 and 2073 associated with the alpha-peptide. Circular dichroism results showed that the unassociated helices in both alpha- and beta-peptides became associated, presumably to form a helical bundle, for those beta-peptides that formed an alphabeta complex, but not for those beta-peptides that did not form an alphabeta complex. In addition, upon association, an increase in the alpha-helical content was observed. These results showed that the beta-peptides ending prior to residue 2072 (Thr) would not associate with alpha-peptide, and that no helical bundling of the partial domains was observed. Thus, we suggest that the C-terminal segment of beta-spectrin, starting from residue 2073 (Thr), is not critical to spectrin tetramer formation. However, the C-terminal region ending with residue 2072 is important for its association with alpha-spectrin in forming tetramers.

Dynamic molecular modeling of pathogenic mutations in the spectrin self-association domain

AbstractDisruption of spectrin self-association underlies many inherited hemolytic disorders. Using dynamic modeling and energy minimization, the 3-dimensional structure of the self-association domain has been estimated in human erythrocyte spectrin and the structural consequences of 17 elliptogenic mutations determined. The predicted structure of the normal self-association domain was remarkably similar to the crystal structure of the Drosophila α-spectrin 14th repeat unit, despite replacement in the human sequence of over 70% of the amino acids relative to fly spectrin, including 2 prolines in the human sequence that appear in helical regions of the fly structure. The predicted structure placed all hydrophilic residues at the surface and identified 4 salt bridges, 9 hydrophobic interactions, and 4 H-bonds that stabilize the native self-association unit. Remarkably, every pathologic point mutation, including seemingly conservative substitutions such as G for A, A for V, or K for R (single-letter amino acid codes), led to conformational rearrangements in the predicted structure. The degree of structural disruption, as measured by root-mean-square deviation of the predicted backbone structure from theDrosophila structure, correlated strongly with the severity of clinical disease associated with each mutation. This approach thus enables an accurate prediction, from the primary sequence, of the clinical consequences of specific point mutations in spectrin. The 3-dimensional structure of the self-association domain derived here is likely to be accurate. It provides a powerful heuristic model for understanding how point mutations disrupt cytoskeletal function in a variety of hemolytic disorders.

Role of terminal nonhomologous domains in initiation of human red cell spectrin dimerization.

Human erythrocyte spectrin is an antiparallel heterodimer comprised of a 280 kDa alpha subunit and a 246 kDa beta subunit which further associates into tetramers in the red cell membrane cytoskeleton. Lateral association of the flexible rodlike monomers involves a multiple-step process that is initiated by a high affinity association near the actin-binding end of the molecule (dimer nucleation site). In this study, recombinant alpha and beta proteins comprising two or four "spectrin type" motifs with and without adjacent, terminal nonhomologous domains were evaluated for their relative contributions to dimer initiation, and the thermodynamic properties of these heterodimer complexes were measured. Sedimentation equilibrium studies showed that in the absence of the heterologous subunit, individual recombinant proteins formed weak homodimers (K(d) > 0.3 mM). When 2-motif (alpha20-21 and beta1-2) and 4-motif (alpha18-21 and beta1-4) recombinants lacking the terminal nonhomologous domains were paired with the complementary protein, high affinity heterodimers were formed in sedimentation equilibrium analysis. Both the alpha20-21/beta1-2 complex and the alpha20-21EF/betaABD1-2 complex showed stoichiometric binding with similar binding affinities (K(d) approximately 10 nM) using isothermal titration calorimetry. The alpha20-21/beta1-2 complex showed an enthalpy of -10 kcal/mol, while the alpha20-21EF/betaABD1-2 complex showed an enthalpy of -13 kcal/mol. Pull-down assays using alpha spectrin GST fusion proteins showed strong associations between all heterodimer complexes in physiological buffer, but all heterodimer complexes were destabilized by the presence of Triton X-100 and other detergents. Complexes lacking the nonhomologous domains were destabilized to a greater extent than complexes that included the nonhomologous domains. The detergent effect appears to be responsible for the apparent essential role of the nonhomologous domains in prior reports. Taken together, our results indicate that the terminal nonhomologous domains do not contribute to dimer initiation nor are they required for formation of high affinity spectrin heterodimers in physiological buffers.

Laboratory method to study mutational effects on human erythrocyte spectrin tetramerization.

We have developed a laboratory method combining a random mutagenesis method and a yeast two-hybrid system to study effects of mutation on human erythrocyte spectrin tetramerization. A PCR-based procedure was used to generate random mutations in DNA fragments of the first 55 residues of alpha-spectrin. Each of the DNA fragments from random mutagenesis was fused with a DNA fragment of native spectrin consisting of residues 56 to 368 to give a DNA fragment of the first 368 residues in alpha-spectrin. The alpha-spectrin DNA fragment and a DNA fragment containing the last 449 residues in beta-spectrin were introduced into the yeast two-hybrid system for rapid screening of alpha- and beta-spectrin interaction. Yeast colonies with interacting alpha- and beta-peptides were blue, and those with non-interacting alpha- and beta-peptides were white. Six single amino acid mutations (R27G, Y35N, F38S, L49H, Y53N, and Y53C) and a double amino acid mutation (K16M, I24N) were identified from 8 white colonies, but no mutations were found in the DNA fragments of 14 blue colonies. Thus this simple laboratory method allows us to study effects of mutation on interactions of alpha- and beta-spectrin at the tetramerization site.

High-Performance Liquid Chromatography Analysis of Spectrin Oligomerization

Gel filtration chromatography has been used to analyze the oligomerization of human erythrocyte spectrin. By applying an exponentially modified Gaussian function we have been able to resolve overlapping elution peaks. From these peaks it was possible to calculate the equilibrium composition of each spectrin concentration and thus also the dissociation constants describing the oligomeric process. The determined dissociation constants for tetramer formation (1.3 μM) and for hexamer formation (24 μM) agree well with other measurements.

SPECTRIN-LIKE PROTEINS IN PLANT NUCLEI

We analysed the presence and localization of spectrin-like proteins in nuclei of various plant tissues, using several anti-erythrocyte spectrin antibodies on isolated pea nuclei and nuclei in cells. Western blots of extracted purified pea nuclei show a cross-reactive pair of bands at 220–240kDa, typical for human erythrocyte spectrin, and a prominent 60kDa band. Immunolocalization by means of confocal laser scanning microscopy reveals spectrin-like proteins in distinct spots equally distributed in the nucleoplasm and over the nuclear periphery, independent of the origin of the anti-spectrin antibodies used. In some nuclei tracks of spectrin-like proteins are also observed. No signal is present in nucleoli. The amount and intensity of signal increases when nuclei were extracted, successively, with detergents, DNase I and RNase A, and high salt, indicating that the spectrin-like protein is associated with the nuclear matrix. The labelling is similar in nuclei of various plant tissues. These data are the first that show the presence and localization of spectrin-like epitopes in plant nuclei, where they may stabilize specific interchromatin domains.

Investigations of spectrin–lipid interactions using fluoresceinphosphatidylethanolamine as a membrane probe

The binding of human erythrocyte spectrin to large unilamellar vesicles (LUVET) formed by the extrusion technique has been studied using fluoresceinphosphatidylethanolamine (FPE) as a reporter of electrostatic membrane potential. Spectrin aliquots were added to a suspension of FPE-labelled LUVETs to elucidate both the type of charge involved and the dissociation constants for spectrin binding to various lipids. All binding experiments showed serial increases in FPE fluorescence intensity upon serial additions of spectrin, indicative of increasing positive charge at the membrane surface. This proves for the first time that although exhibiting an overall net negative charge, spectrin binds to lipid surfaces by presenting positive charges to the lipid surface. Binding curves were obtained from the change in fluorescence intensity upon each spectrin addition and analysed to determine dissociation constants. A K d of 0.14±0.12 μM was found for spectrin binding to FPE-labelled phosphatidylcholine/phosphatidylserine (PC/PS) LUVETs at 22°C in high salt conditions. A similar K d of 0.17±0.11 μM was obtained for spectrin binding to neutral LUVETs composed of PC. However, binding was found to be much weaker for PC/PS LUVETs under low salt conditions with a K d of 1.22±0.48 μM.

Rapid diffusion of spectrin bound to a lipid surface

Human erythrocyte spectrin was labelled with the probe 5,5′-disulfato-1-(6-hexanoic acid N-hydroxysuccinimide ester)-1′-ethyl-3,3,3′,3′-tetramethylindocarbocyanine (Cy3). Cy3-spectrin was bound to the outer surface of dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles and its diffusion measured by fluorescence recovery after photobleaching (FRAP). It was found that at 30°C, above the lipid gel to liquid-crystalline phase transition of the lipids, Cy3-spectrin had an unexpectedly high diffusion coefficient D=(2.1±0.6)×10 −7 cm 2/s. At the phase transition, diffusion of Cy3-spectrin was only slightly lower; D=(1.3±0.3)×10 −7 cm 2/s, whereas at 14°C, well below the lipid phase transition, diffusion was found to be much slower with D=(3.1±0.12)×10 −9 cm 2/s. The fast diffusion of Cy3-spectrin on the lipid surface implies that the individual bonds which bind spectrin to the lipid surface must rapidly be made and broken. In the light of these results, spectrin–lipid interactions alone appear unlikely to have any significant role in supporting the cell membrane. Probably, the interactions serve only to localise the spectrin at the inner lipid surface in order to facilitate formation of the cytoskeleton.

SPECTRIN-LIKE PROTEINS IN GREEN ALGAE (DESMIDIACEAE)

Immunochemical detection of actin as well as spectrin-like proteins have been carried out in the green algae Micrasterias denticulata, Closterium lunula, and Euastrum oblongum. In these algae, actin is detected on Western blots at 43kDa with antibodies to actin from higher plant and animal origin. By use of antibodies to human and chicken erythrocyte spectrin a cross-reactivity with desmid proteins is found at about the molecular mass of 220kDa, where also human erythrocyte spectrin is detected. Additional bands are present at 120kDa and 70kDa, which are probably breakdown products. An antibody against chicken α-actinin, a small protein of the spectrin superfamily, recognizes bands at 90kDa, where it is expected, and 70kDa, probably the same breakdown product as mentioned for spectrin. Isoelectric focusing provides staining at pI4.6 with antibodies against spectrin. Immunogold labelling of spectrin and α-actinin antigens on high-pressure frozen, freeze-substituted Micrasterias denticulata cells with the same antibodies exhibits staining, especially at membranes of different populations of secretory vesicles, at dictyosomes, and the plasma membrane. However, no clear correlation to the growth pattern of the cell could be observed. Taken together, our results demonstrate the presence of spectrin-like proteins in desmid cells which are probably functional in exocytosis.

Ionic strength effect on the thermal unfolding of alpha-spectrin peptides.

In previous work, we have shown that the ionic strength-mediated differences found for the hydrodynamic dimensions of the human erythrocyte spectrin are not caused by secondary structural changes, but are caused more probably by subtle changes in tertiary interactions (LaBrake, C. C., Wang, L., Keiderling, T. A., and Fung, L. W.-M. (1993) Biochemistry 32, 10296-10302.). The substructure of spectrin has been suggested to be composed largely of triple alpha-helical bundle structural domains in tandem. In the present study, we used fluorescence and circular dichroism methods to study ionic strength effects on intact spectrin dimers and on recombinant peptides of spectrin domains of different lengths. We observed little ionic strength effect on the thermal unfolding temperature, Tm, values in these systems. However, we found that ionic strength-induced cooperativity in the unfolding processes was similar for the spectrin dimer and for peptides with two or three domains, as measured by entropy changes (DeltaSm). Although single-domain peptides exhibited rather variable DeltaSm values, depending on the specific domain, they showed little salt effects on the DeltaSm values themselves. This suggests that spectrin undergoes subtle ionic strength-induced conformational changes, probably near the interdomain regions of the molecule. These conformational changes may be responsible for the observed hydrodynamic and unfolding properties in intact spectrin under different ionic strength conditions. We suggest that recombinant peptides of various lengths may serve as models for studying the structural flexibility in spectrin.

Detection of dimers of dimers of human leukocyte antigen (HLA)-DR on the surface of living cells by single-particle fluorescence imaging.

The technique of single-particle fluorescence imaging was used to investigate the oligomeric state of MHC class II molecules on the surface of living cells. Cells transfected with human leukocyte antigen (HLA)-DR A and B genes were labeled at saturation with a univalent probe consisting of Fab coupled to R-phycoerythrin. Analysis of the intensities of fluorescent spots on the cell surface revealed the presence of single and double particles consistent with the simultaneous presence of HLA-DR heterodimers and dimers of dimers. The proportion of double particles was lower at 37 degrees C than at 22 degrees C, suggesting that the heterodimers and dimers of dimers exist in a temperature-dependent equilibrium. These results are discussed in the context of a possible role for HLA-DR dimers of dimers in T cell receptor-MHC interactions. The technique is validated by demonstrating that fluorescence imaging can distinguish between dimers and tetramers of human erythrocyte spectrin deposited from solution onto a solid substrate. The methodology will have broad applicability to investigation of the oligomeric state of immunological and other membrane-bound receptors in living cells.

Purification of erythrocyte spectrin alpha- and beta-subunits at alkaline pH and structural and hydrodynamic properties of the isolated subunits.

A new method for the isolation of the alpha- and beta-subunits of human erythrocyte spectrin was developed, and structural properties and association behavior of the isolated subunits were studied by means of CD, nondenaturing gel electrophoresis, and analytical ultracentrifugation. The alpha- and beta-subunits were isolated using ion-exchange FPLC (pH 11) followed by size-exclusion FPLC (pH 7.5), having shown that alkaline pH dissociates spectrin polymers to their monomers [see Fujita et al. (1998) Biochemistry 37, 264-271]. The isolated subunits had alpha-helical content and thermal stability almost equivalent to those of native spectrin and reassembled to form heterodimers and tetramers which were indistinguishable from native spectrin with respect to secondary structure content, thermal stability, migration pattern on nondenaturing gels, and sedimentation coefficients. Thus, our data show that the increase in the structural stability of a heterodimer by association of the two monomers is very small. Sedimentation coefficients for the isolated alpha- and beta-subunits were 6.3 and 5.7 S, respectively. The similar frictional ratios (f/f0) of the isolated alpha-subunit (2.42) and the beta-subunit (2.45) indicate that the flexibility of both these wormlike chains and the range of shapes they can adopt in solution are very similar. The f/f0 value for spectrin dimer (2.41) indicates that its flexibility is somewhat, but not grossly, reduced compared to that of the individual subunits. Consequently, the folded repeat units of the subunits and the flexible connections between them are probably "in register" along the length of the dimer.

Solution structure of the spectrin repeat: a left-handed antiparallel triple-helical coiled-coil

Cytoskeletal proteins belonging to the spectrin family have an elongated structure composed of repetitive units. The three-dimensional solution structure of the 16th repeat from chicken brain α-spectrin (R16) has been determined by NMR spectroscopy and distance geometry-simulated annealing calculations. We used a total of 1035 distance restraints, which included 719 NOE-based values obtained by applying the ambiguous restraints for iterative assignment (ARIA) method. In addition, we performed a direct refinement against 1H-chemical shifts. The final ensemble of 20 structures shows an average RMSD of 1.52 Å from the mean for the backbone atoms, excluding loops and N and C termini. R16 is made up of three antiparallel α-helices separated by two loops, and folds into a left-handed coiled-coil.The basic unit of spectrin is an antiparallel heterodimer composed of two homologous chains, β and α. These assemble a tetramer via a mechanism that relies on the completion of a single repeat by association of the partial repeats located at the C terminus of the β-chain (two helices) and at the N terminus of the α-chain (one helix). This tetramer is the assemblage able to cross-link actin filaments. Model building by homology of the “tetramerization” repeat from human erythrocyte spectrin illuminates the possible role of point mutations which cause hemolytic anemias.

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 alpha-subunit and 17 in the beta-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.

Assessment of the validity of the Adams and Fujita approximation for the higher oligomers of human spectrin

Analysing the self-association behaviour of human erythrocyte spectrin is complicated by a large degree of nonideality. Adams and Fujita [1] proposed that, as a first order approximation, the logarithm of the activity coefficient of the protomer of a self-associating system can be considered to be linearly dependent on the total concentration of the protein, and that the same second virial coefficient could be considered to apply to all species. As a consequence of the Adams and Fujita approximation, the apparent equilibrium constant is equal to the thermodynamic equilibrium constant. The equilibrium concentrations at 30°C of each oligomer spectrin species up to the 14-mer were determined after electrophoresis at low temperature. An apparent equilibrium constant for forming tetramer ( K 2,4) of (1.2 ± 0.1) × 10 6 l/mol was obtained, a value of (9.4 ± 0.7) × 10 4 l/mol was obtained for K 4,6 and for all reactions forming oligomers higher than the hexamer an average approximate value of (2.7 ± 0.4) × 10 5 l/mol was obtained. The apparent equilibrium constants for the formation of all oligomer species of spectrin up to the tetrakaidecamer (14-mer) remain relatively independent of total spectrin concentration, and indicate that within the precision of the measurements a single virial coefficient is sufficient to account for the nonideality of spectrin self-association over the range 2–42 g/l, thus further justifying the use of the Adams and Fujita approximation for this protein over this concentration range.