Biophysical properties of human erythrocyte spectrin at alkaline pH: implications for spectrin structure, function, and association.

The effects of pH 6-13 on the conformation and assembly of spectrin were studied by means of analytical ultracentrifugation, circular dichroism (CD), 1H NMR, and UV spectrophotometry. Sedimentation velocity analysis showed that spectrin oligomers dissociate cooperatively into component alpha- and beta-subunits above pH 9.5, and that spectrin tetramers, heterodimers, and monomers adopt more extended and/or expanded shapes above this pH. The dissociation to monomers is mostly completed by pH 10.5 and is used as the basis for purifying the subunits [see Fujita et al. (1998) Biochemistry 37, 272-280]. Along with the dissociation, biphasic unfolding of spectrin was observed above pH 9.5 as detected by CD. The first phase of the transition occurred between pH 9.5 and 11, and the second phase between pH 11 and 13. A similar biphasic dependence was observed for the upfield shift of lysine epsilon-CH2 resonances detected by spin-echo 1H NMR and the spectrophotometric titration of the absorbance at 294 nm. These data indicate that deprotonation of tyrosine and lysine residues is closely correlated with (i) the dissociation of spectrin oligomers into heterodimers, (ii) the dissociation of heterodimers into monomers, and (iii) the unfolding of spectrin. Taken together, our data suggest that hydrophobic and electrostatic interactions involving tyrosine and lysine residues play a critical role in the formation of the alpha-helix of spectrin and assembly of physiologically relevant spectrin oligomers from the two component subunits.

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.

Comparison of the salt-dependent self-association of brain and erythroid spectrin.

The self-association of ovine brain spectrin in 0.1-1.5 M NaCl (pH 7.5) was studied using sedimentation velocity and sedimentation equilibrium techniques. Brain spectrin is tetrameric at sedimentation equilibrium at a 0.13 M ionic strength at 18-37 degrees C and at ionic strengths of up to 0.33 M at 20 degrees C. At ionic strengths greater than 0.33 M at 20 degrees C, the brain spectrin tetramer is destabilized, resulting in both dissociation to dimers and indefinite association to higher oligomers, in a manner similar to that seen with erythroid spectrin. The equilibrium constants describing all steps in the association involving the addition of dimers are around 15-fold higher for brain spectrin than for erythroid spectrin, at ionic strengths of > or = 0.43 M. We propose that the stronger association of brain spectrin compared to that of erythroid spectrin is due to a relative inability of brain spectrin to form closed dimers. Sedimentation velocity analysis confirms that brain spectrin readily forms open dimers and that the association of open dimers is not kinetically trapped even at 2 degrees C. Our results suggest that the destabilization of spectrin tetramers in high-ionic strength conditions is due to increased independent movement of the alpha and beta subunits resulting from disruption of electrostatic interactions. The greater stability of brain spectrin oligomers relative to those of erythroid spectrin is due to stronger nonelectrostatic interactions which stabilize the rigidity of the individual subunits and thereby increase the conformational strain associated with dimer closure.

Macromolecular crowding: effects on actin polymerisation.

Dextran has been found to enhance the polymerisation of actin. This enhancement increases exponentially with increasing mass concentrations of dextran, in a manner that is consistent with excluded volume theory. Mathematical prediction of experimental results is difficult due to the fact that all participating species, namely F-actin, G-actin and dextran are best represented by differently shaped hard particles. Modelling dextran as a sphere of radius defined by an effective thermodynamic radius (Reff), we have predicted our experimental results to an acceptable degree, given the relative crudity of the model. The results imply that the highly crowded cellular environment may help to stabilise the filamentous actin network in vivo.

Reorganization of terminator DNA upon binding replication terminator protein: implications for the functional replication fork arrest complex.

Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the replication terminator protein (RTP) and DNA terminator sites. While determination of the crystal structure of RTP has facilitated our understanding of how a single RTP dimer interacts with terminator DNA, additional information is required in order to understand the assembly of a functional fork arrest complex, which requires an interaction between two RTP dimers and the terminator site. In this study, we show that the conformation of the major B.subtilis DNA terminator,TerI, becomes considerably distorted upon binding RTP. Binding of the first dimer of RTP to the B site of TerI causes the DNA to become slightly unwound and bent by approximately 40 degrees. Binding of a second dimer of RTP to the A site causes the bend angle to increase to approximately 60 degrees . We have used this new data to construct two plausible models that might explain how the ternary terminator complex can block DNA replication in a polar manner. In the first model, polarity of action is a consequence of the two RTP-DNA half-sites having different conformations. These different conformations result from different RTP-DNA contacts at each half-site (due to the intrinsic asymmetry of the terminator DNA), as well as interactions (direct or indirect) between the RTP dimers on the DNA. In the second model, polar fork arrest activity is a consequence of the different affinities of RTP for the A and B sites of the terminator DNA, modulated significantly by direct or indirect interactions between the RTP dimers.

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 degrees 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 (K2,4) of (1.2 +/- 0.1) x 10(6) 1/mol was obtained, a value of (9.4 +/- 0.7) x 10(4) 1/mol was obtained for K4.6 and for all reactions forming oligomers higher than the hexamer an average approximate value of (2.7 +/- 0.4) x 10(5) 1/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 2-14 g/l, thus further justifying the use of the Adams and Fujita approximation for this protein over this concentration range.

Beta-lactoglobulin B: a proposed standard for the study of reversible self-association reactions in the analytical ultracentrifuge?

Bovine beta-lactoglobulin B is proposed for use as a standard in the measurement of reversible self-association reactions in the analytical ultracentrifuge. The protein is well understood on a molecular level, is readily obtainable, and is stable under harsh conditions. Bovine beta-lactoglobulin B undergoes a simple monomer-dimer equilibrium which can be predictably controlled and consistently reproduced. In this investigation bovine beta-lactoglobulin B has been studied via sedimentation equilibrium experiments in the XL-A analytical ultracentrifuge at 5-30 degrees C in buffers of ionic strength 0.1-0.2 M and pH 2.0-3.0. Samples subjected to a number of different treatments and storage methods all yielded similar results. Molar equilibrium constants for the association reaction were determined by nonlinear regression fitting of a monomer-dimer model of association either to concentration versus radius data, using the programs NONLIN and ORIGIN, or to Omega versus concentration data using the program DUGOM. At 20 degrees C and pH 2.6, over the ionic strength range 0. 1-0.2 M, the equilibrium constant for the association reaction ranges between 1 x 10(4) and 5 x 10(4) M-1. The parameters of nonideal self-association behavior were found to be independent of the particular analysis strategy. Fitting to the concentration distribution, the apparent weight-average molecular weight, or the Omega function all returned identical parameters.

Multimer formation as a consequence of separate homodimerization domains: the human c-Jun leucine zipper is a transplantable dimerization module.

Human c-Jun and c-Fos leucine zipper domains were examined for their ability to serve as autonomous dimerization domains as part of a heterologous protein construct. Schistosoma japonicum glutathione S-transferase (GST) was fused to recombinant Jun leucine zipper (rJunLZ) and Fos leucine zipper (rFosLZ) domains. SDS-PAGE 'snapshot' analyses based on disulphide linkage of monomers demonstrated the ability of rJunLZ to function as a dimerization motif in a foreign protein environment. Steric hindrance prevented formation of rJunLZ-GST::rFosLZ-GST heterodimers whereas rJunLZ-GST::rFosLZ and rJunLZ:: rFosLZGST formed readily. Furthermore, rJunLZGST generated homodimers suggesting fusion protein heterodimers interact differently to homodimers. Gel filtration chromatography confirmed that GST is a dimer in solution and that attachment of a leucine zipper domain allows further interactions to take place. Sedimentation equilibrium analyses showed that GST is a stable dimer (K(a) > 10(6) M(-1)) with no higher multimeric forms. rFosLZ-GST weakly associates beyond a dimer (K(a) approximately 4 x 10(4) M(-1)) and rJunLZ-GST associates indefinitely (K(a) approximately 4 x 10(5) M(-1)) [corrected], consistent with an isodesmic model of association. The interaction of these leucine zippers independently of GST association demonstrates their utility in the modification of proteins when multimer formation is desired.

Structural and biochemical studies of human galanin: NMR evidence for nascent helical structures in aqueous solution.

The 30-residue human neuropeptide, galanin, was shown to bind to rat insulinoma RINm5F cells and to inhibit glyceraldehyde-stimulated insulin secretion from these cells in a manner quantitatively similar to that of porcine galanin. Neither human nor porcine galanin stimulated Ca2+ mobilization in cultured human small cell lung carcinoma cells. Sedimentation equilibrium analysis of human galanin showed that it was strictly monomeric in aqueous solution, indicating that the peptide interacts with its receptor(s) as a monomer. The monomeric nature of the peptide makes it especially suitable for structural studies using NMR. Nuclear Overhauser enhancement spectroscopy experiments performed on galanin dissolved in aqueous solution (150 mM KCl, pH 4) at both 33 and 3 degrees C indicate that certain regions of the peptide are capable of adopting detectable levels of short-range structure in rapid equilibrium with random coil. At 33 degrees C, the short-range structures include a nascent helix spanning residues 3-11 which incorporates a hydrophobic core from residues 6-11. Residues 14-18 and 22-30 display sequential NH-NH and C beta H-NH connectivities, indicating that these regions of the peptide adopt nonrandom conformations by significantly populating the alpha-region of conformational space. However, no medium-range dipolar connectivities indicative of nascent helix or turn conformations were observed. At 3 degrees C, almost all residues significantly populate the alpha-region of conformational space, and the nascent helix between residues 3 and 11, with its hydrophobic core, is retained.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions of myelin basic protein with palmitoyllysophosphatidylcholine: characterization of the complexes and conformations of the protein.

The stoichiometry of palmitoyllysophosphatidylcholine/myelin basic protein (PLPC/MBP) complexes, the location of the protein in the lysolipid micelles, and the conformational changes occurring in the basic protein and peptides derived from it upon interaction with lysolecithin micelles were investigated by circular dichroic spectropolarimetry, ultracentrifugation, electron paramagnetic resonance (EPR) and 31P, 13C, and 1H nuclear magnetic resonance spectroscopy (NMR), and electron magnetic resonance spectroscopy (NMR), and electron microscopy. Ultracentrifugation measurements indicated that well-defined complexes were formed by the association of one protein molecule with approximately 141 lysolipid molecules. Small-angle X-ray scattering data indicated that the PLPC/MBP complexes form particles with a radius of gyration of 3.8 nm. EPR spectral parameters of the spin labels 5-, and 16-doxylstearate incorporated into lysolecithin/basic protein aggregates, and 13C- and 1H-NMR relaxation times of PLPC indicated that the addition of the protein did not affect the environment and location of the labels and the organization of the lysolipid micelles. The data suggested that MBP lies primarily near the surface of the micelles, with segments penetrating beyond the interfacial region into the hydrophobic interior, but without any part of the protein being protected against rapid exchange of its amide groups with the aqueous environment. The basic protein acquired about 20% alpha-helix when bound to lysolipid micelles. Circular dichroic spectra of sequential peptides derived by cleavage of the protein revealed the formation of alpha-helical regions in the association with lysolecithin. Specific residues in myelin basic protein that participated in binding to the micelles were identified from magnetic resonance data on changes in the chemical shifts and intensities of assigned resonances, and line broadening of peaks by fatty acid spin-labels incorporated into the micelles.

Reinvestigation of the thermodynamics of spectrin self-association.

The thermodynamics of the self-association reactions of human spectrin have been reinvestigated by means of sedimentation equilibrium over the temperature range 18-40 degrees C. The experimental data were analysed in terms of a cooperative isodesmic model of association. The van't Hoff plot showed that the standard change in enthalpy for the heterodimer-tetramer step was temperature-dependent, leading to an estimate of -8.5 kJ mol-1 K-1 for the change in molar heat capacity, delta Cp. Curvature in the van't Hoff plots, not detected in previous studies, was revealed through the increased precision of the data and the wider temperature range examined. On the assumption that delta Cp reflects hydrophobic interactions in the tetramer that cannot be formed in the heterodimer, it can be estimated that approximately 50 CH2 groups per heterodimer participate in hydrophobic interactions in the tetramer that cannot be formed in the heterodimer.

Conformational stability of the endothelin/sarafotoxin family of peptides.

There are significant differences between the structures reported for members of the endothelin/sarafotoxin family of peptides, but also for the same peptides studied by different groups, raising the possibility that some of the differences are attributable to variation in solution conditions rather than intrinsic structural heterogeneity. We have shown, using circular dichroism spectroscopy and equilibrium sedimentation, that the secondary structures of these peptides are little affected by wide variations in pH, or by self-association. Although acetonitrile has a pronounced effect on the extent of peptide self-association it does not appear to alter the backbone structure of sarafotoxin SRTb, and has only minor effects on endothelin-1 and endothelin-3. The observed conformational variation thus appears largely to reflect sequence-dependent differences.

The solution structure of sarafotoxin-c. Implications for ligand recognition by endothelin receptors.

The solution structure of sarafotoxin-c has been determined using NMR spectroscopy. A total of 112 interproton distance constraints derived from two-dimensional NMR spectra were used to calculate a family of structures using a combination of distance geometry and dynamical simulated annealing calculations. The structures reveal a well defined alpha-helix extending from Glu9 to Cys15 and an N-terminal region (Cys1-Asp8) that is tightly constrained by disulfide bonds to Cys residues in the central helix. In contrast, the C-terminal region (His16-Trp21) does not adopt a defined conformation in the final family of structures. This is consistent with the paucity of NMR-derived structural constraints obtained for this region and leads to the suggestion that the C-terminal region oscillates rapidly between a number of substantially different conformers. It is proposed that differences between the central helix of the endothelin and sarafotoxin isopeptides might be important in binding of these ligands by the G protein-coupled endothelin receptors.

The concentration dependence of the activity coefficient of the human spectrin heterodimer. A quantitative test of the Adams-Fujita approximation.

The heterodimer and tetramer states of human spectrin, in equilibrium at 30 degrees C, have been resolved by means of sedimentation velocity experiments at low temperature. This has allowed quantification of the concentrations of each oligomer at equilibrium, as a function of the total concentration of spectrin. In separate sedimentation equilibrium experiments, the thermodynamic activity of the heterodimer has been estimated as a function of the total spectrin concentration through the use of the Omega function. Combination of the concentration and thermodynamic activity of the spectrin heterodimer allowed estimation of the activity coefficient of the heterodimer as a function of total spectrin concentration. Over the accessible concentration range of 0-16 g/L, the logarithm of the activity coefficient of the heterodimer was linear in the total concentration, and the apparent equilibrium constant for tetramer formation was only weakly dependent on concentration, implying that a single virial coefficient is sufficient to describe the nonideality of this system over this concentration range.

A proton nuclear magnetic resonance study of the mobile regions of human erythroid spectrin.

The effect of added NaCl (0-150 mM) and temperature (6-65 degrees C) on the conformation of erythrocyte spectrin was investigated using 400 MHz 1H NMR. The relatively narrow resonances (20-40 Hz linewidth) in the spectra arising from protons in regions of the molecule undergoing rapid motions were selectively detected using either the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence without water presaturation or a simple pi/2 pulse sequence with water presaturation. The T2 relaxation of these protons was not influenced by changes in solution conditions (0-150 mM NaCl, 6-37 degrees C) indicating that their motions were independent of the overall shape of the molecule. Significant increases in the areas of the aliphatic peaks for spectrin samples at fixed salt concentrations occurred as the temperature was raised from 6 to 37 degrees C. The increases were independent of the state of polymerization of spectrin and were greater in the absence of added salt above 25 degrees C. The changes reflect increasing numbers of mobile residues, probably due to partial unfolding of spectrin's repeated structural unit. At temperatures above 37 degrees C, sharp increases in the areas of the spectral envelopes reflect cooperative unfolding of spectrin. Comparison with results previously obtained in this laboratory using CD and ORD indicate that at least part of the lost structure is alpha-helical.

A 1H NMR spectroscopic comparison of gamma S- and gamma B-crystallins.

Two-dimensional 1H NMR spectroscopic studies are presented on bovine gamma S- and gamma B-crystallin. In gamma S-crystallin, the four N-terminal residues have great flexibility compared with the rest of the molecule and assume a random coil conformation. NMR spectroscopy and electrospray mass spectrometry show that the N-terminal residue is acetylated. Thus, gamma S-crystallin is similar to the acidic beta-crystallins in having a flexible N-terminal extension and an N-terminus that is blocked with an acetyl group but no C-terminal extension. In addition to the short N-terminal extension in gamma S-crystallin, other unassigned resonances are also observed in the NMR spectra. In gamma B-crystallin, however, cross-peaks in the NH to alpha-CH region of the spectrum are essentially restricted to the last three residues of the C-terminal domain. The NMR data imply that gamma S-crystallin has a more flexible structure than gamma B-crystallin. Sedimentation equilibrium studies on gamma S-crystallin are consistent with this proposal. Resonances from the N-terminal extension of gamma S-crystallin are not affected by the presence of alpha-crystallin implying that this region is not involved in interactions between the two molecules. It is concluded that gamma S-crystallin shares structural properties which are intermediate between the beta- and gamma-crystallins.

Quantitative assessment of the association of the alpha-I fragment of spectrin with oligomers of intact spectrin.

The alpha-I fragment of human spectrin that carries the binding site on the alpha-chain of spectrin for the beta-chain has been purified from limited trypsin digests of spectrin by means of FPLC. The self-association of spectrin and the binding of the alpha-I fragment to spectrin heterodimers and to tetramers have been quantified through the use of gel electrophoresis, staining with Coomassie Blue, and quantification of the bound dye following elution with pyridine. The parameters of self-association were found to be consistent with those estimated from sedimentation equilibrium analysis. The data were consistent with a model in which both self-association and the binding of the alpha-I fragment are considered to occur through an intermediate in which the alpha-beta interface is initially dissociated. The alpha-beta interface in the heterodimer was found to be less stable than that of higher oligomers by approx. 3 kJ/mol.

Enhancement of self-association of human spectrin by polyethylene glycol.

1. In the presence of polyethylene glycol, the self-association of human spectrin is enhanced in a manner that depends approx. exponentially on the mass concentration of polyethylene glycol. 2. For a given mass concentration, the enhancement is independent of the molecular weight of the polyethylene glycol. 3. These data are consistent with the operation of excluded volume effects, and support the contention that the association of spectrin is likely to be increased in the presence of the high concentration of hemoglobin within the erythrocyte in vivo.

The self-association of ovine erythrocyte spectrin.

1. Spectrin extracted from ovine erythrocyte membranes at low temperature shows association behaviour similar to that reported for human and bovine erythrocytes. 2. The spectrin tetramer is the predominant oligomer, the dimer is well represented, and smaller amounts of hexamer and higher oligomers are present. 3. The estimates of parameters describing the self-association of purified ovine spectrin studied by sedimentation equilibrium analysis were found to be indistinguishable from those obtained for human spectrin under the same conditions, within the precision of the measurements. 4. The data suggest that the cooperative isodesmic model may be general for spectrin, and not a peculiarity of the human.