Purification and characterization of a novel calcium-binding protein from the extrapallial fluid of the mollusc, Mytilus edulis.

In the bivalve mollusc Mytilus edulis shell thickening occurs from the extrapallial (EP) fluid wherein secreted shell matrix macromolecules are thought to self-assemble into a framework that regulates the growth of CaCO(3) crystals, which eventually constitute approximately 95% of the mature shell. Herein is the initial report on the purification and characterization of a novel EP fluid glycoprotein, which is likely a building block of the shell-soluble organic matrix. This primary EP fluid protein comprises 56% of the total protein in the fluid and is shown to be a dimer of 28,340 Da monomers estimated to be 14.3% by weight carbohydrate. The protein is acidic (pI = 4.43) and rich in histidine content (11.14%) as well as in Asx and Glx residues (25.15% total). The N terminus exhibits an unusual repeat sequence of histidine and aspartate residues that occur in pairs: NPVDDHHDDHHDAPIVEHHD approximately. Ultracentrifugation and polyacrylamide gel electrophoresis demonstrate that the protein binds calcium and in so doing assembles into a series of higher order protomers, which appear to have extended structures. Circular dichroism shows that the protein-calcium binding/protomer formation is coupled to a significant rearrangement in the protein's secondary structure in which there is a major reduction in beta-sheet with an associated increase in alpha-helical content of the protein. A model for shell organic matrix self-assembly is proposed.

Analytical ultracentrifugation.

Analytical ultracentrifugation is one of the most powerful, though as yet underexploited, techniques available to molecular biology and biochemistry. This overview describes applications for analytical ultracentrifugation along with important considerations relating to experimental design.

Analysis of transport experiments using pseudo-absorbance data.

The measurement of the concentration distribution of a macromolecule across a solution column by absorption optics usually requires optical transmission profiles of both the sample solution and the buffer, measured under identical conditions, to calculate the absorbance as the logarithm of the ratio of reference to sample intensity. For transport experiments, however, where the changes in the local macromolecule concentration with time are measured, a reference buffer intensity is not necessarily required. We demonstrate that the logarithm of the light transmitted through the sample solution, referred to as pseudo-absorbance, can suffice to determine macromolecular transport parameters of interest, with little loss of precision. Local changes in illumination of the sample column or in the detection efficiency of the transmitted light, as well as temporal fluctuations of the light source intensity can be well-described by consideration of time-invariant and radial-invariant signal components in the pseudo-absorbance data, using the systematic noise decomposition techniques developed recently (Schuck, P., and Demeler, B. (1999) Biophys. J. 76, 2288-2296). The practical use of the method is demonstrated with double-sector and single-sector sedimentation velocity experiments, and with analytical electrophoresis experiments. It is shown that pseudo-absorbance analysis can increase the capacity of a sedimentation velocity experiment in ultracentrifugation, and, in general, can considerably simplify the requirements of optical design.

Biophysical characterization of interactions between the core binding factor alpha and beta subunits and DNA.

Core binding factors (CBFs) play key roles in several developmental pathways and in human disease. CBFs consist of a DNA binding CBFalpha subunit and a non-DNA binding CBFbeta subunit that increases the affinity of CBFalpha for DNA. We performed sedimentation equilibrium analyses to unequivocally establish the stoichiometry of the CBFalpha:beta:DNA complex. Dissociation constants for all four equilibria involving the CBFalpha Runt domain, CBFbeta, and DNA were defined. Conformational changes associated with interactions between CBFalpha, CBFbeta, and DNA were monitored by nuclear magnetic resonance and circular dichroism spectroscopy. The data suggest that CBFbeta 'locks in' a high affinity DNA binding conformation of the CBFalpha Runt domain.

Modern applications of analytical ultracentrifugation.

Analytical ultracentrifugation is a classical method of biochemistry and molecular biology. Because it is a primary technique, sedimentation can provide first-principle hydrodynamic and first-principle thermodynamic information for nearly any molecule, in a wide range of solvents and over a wide range of solute concentrations. For many questions, it is the technique of choice. This review stresses what information is available from analytical ultracentrifugation and how that information is being extracted and used in contemporary applications.

Rotational and translational motion of troponin C.

Time resolved fluorescence anisotropy and sedimentation velocity has been used to study the rotational and translational hydrodynamic behavior of two mutants of chicken skeletal troponin C bearing a single tryptophan residue at position 78 or 154 in the metal-free-, metal-bound-, and troponin I peptide (residues 96-116 of troponin I)-ligated states. The fluorescence anisotropy data of both mutants were adequately described by two rotational correlation times, and these are compared with the theoretically expected values based on the rotational diffusion of an idealized dumbbell. These data imply that the motion of the N- and C-terminal domains of troponin C are independent. They also suggest that in the metal-free, calcium-saturated and calcium-saturated troponin I peptide-bound states, troponin C is elongated, having an axial ratio of 4-5. Calcium or magnesium binding to the high affinity sites alone reduces the axial ratio to approximately 3. However, with calcium bound to sites III and IV and in the presence of a 1:1 molar ratio of the troponin I peptide, troponin C is approximately spherical. The metal ion and troponin I peptide-induced length changes in troponin C may play a role in the mechanism by which the regulatory function of troponin C is effected.

Visualizing ion relaxation in the transport of short DNA fragments.

Ion relaxation plays an important role in a wide range of phenomena involving the transport of charged biomolecules. Ion relaxation is responsible for reducing sedimentation and diffusion constants, reducing electrophoretic mobilities, increasing intrinsic viscosities, and, for biomolecules that lack a permanent electric dipole moment, provides a mechanism for orienting them in an external electric field. Recently, a numerical boundary element method was developed to solve the coupled Navier-Stokes, Poisson, and ion transport equations for a polyion modeled as a rigid body of arbitrary size, shape, and charge distribution. This method has subsequently been used to compute the electrophoretic mobilities and intrinsic viscosities of a number of model proteins and DNA fragments. The primary purpose of the present work is to examine the effect of ion relaxation on the ion density and fluid velocity fields around short DNA fragments (20 and 40 bp). Contour density as well as vector field diagrams of the various scalar and vector fields are presented and discussed at monovalent salt concentrations of 0.03 and 0.11 M. In addition, the net charge current fluxes in the vicinity of the DNA fragments at low and high salt concentrations are briefly examined and discussed.

Solution assembly of the pseudo-high affinity and intermediate affinity interleukin-2 receptor complexes.

The high affinity interleukin-2 receptor is composed of three cell surface subunits, IL-2Ralpha, IL-2Rbeta, and IL-2Rgamma. Functional forms of the IL-2 receptor exist, however, that enlist only two of the three subunits. On activated T-cells, the alpha- and beta-subunits combine as a preformed heterodimer (the pseudo-high affinity receptor) that serves to capture IL-2. On a subpopulation of natural killer cells, the beta- and gamma-subunits interact in a ligand-dependent manner to form the intermediate affinity receptor site. Previously, we have demonstrated the feasibility of employing coiled-coil molecular recognition for the solution assembly of a heteromeric IL-2 receptor complex. In that study, although the receptor was functional, the coiled-coil complex was a trimer rather than the desired heterodimer. We have now redesigned the hydrophobic heptad sequences of the coiled-coils to generate soluble forms of both the pseudo-high affinity and the intermediate affinity heterodimeric IL-2 receptors. The properties of these complexes were examined and their relevance to the physiological IL-2 receptor mechanism is discussed.

Analysis of protein and DNA-mediated contributions to cooperative assembly of protein-DNA complexes.

The cooperative assembly of protein-DNA complexes is a widespread phenomenon that is of particular significance to transcriptional regulation. Assembly of these complexes is controlled by the chemistry of the macromolecular interactions. In this sense, transcriptional regulation is a chemical issue. The purpose of this review is to present an analytical approach designed to understand this regulation from a chemical perspective. By investigating the solution interactions between all combinations of molecules, protein-protein, protein-ligand, and protein-DNA, and the interplay between them, it is possible to determine the relative free energies of the different configurations of the regulatory complex. This governs their distribution and thereby controls the biological activity. To illustrate the approach, we will address the molecular basis for cooperativity in the bacteriophage lambda, lysogenic-lytic switch mechanism, a system that has long served as a paradigm for gene regulation. The driving force for cooperativity in the assembly of gene regulatory complexes is generally thought to be provided by direct protein-protein interactions. However, other interactions mediated by both proteins and DNA are also involved and may be critical to the regulatory mechanism. We will review advances over the past several years in the application of biophysical chemical methods to investigate protein-protein and protein-DNA interactions. Many of these applications were first employed for the lambda system. In addition to describing the physical basis for the methods, we will focus on the unique information that can be gained and how to combine the information obtained from several techniques to develop a comprehensive view of the critical regulatory interactions.

An apparatus for membrane-confined analytical electrophoresis.

A membrane-confined analytical electrophoresis apparatus for measuring the solution charge of macromolecules has been described previously (T. M. Laue et al., Anal. Biochem. 1989, 182, 377-382). Presented here is a design for this apparatus, which permits the on-line acquisition and display of absorbance data from up to 512 positions along an analysis chamber. Concentration distributions of macromolecules in solution can be monitored in the chamber to provide steady-state electrophoresis, electrophoretic mobility and diffusion measurements. Buffer chambers press semipermeable membranes against the open ends of a fused-silica cuvette to form the analysis chamber. This configuration permits both the flow of buffer and the establishment of an electric field across the cuvette, while retaining macromolecules in the field of view. Though a gel may be included in the analysis chamber, none is required for gradient stabilization. The volume of sample required for analysis is 8 microL, most of which is recoverable. Experimental conditions can be varied during study by simply changing the circulating buffer and/or the electric field. The analysis and buffer chambers are held in an aluminum housing that sits in an aluminum water jacket. The water jacket provides temperature control, shielding from external electrical noise and also serves as an optical mask. Plans for the cell assembly, optical system and the computer interface for data acquisition are provided. The assembly and operation of the apparatus and the analysis of data are described.

Overexpression, purification, and biophysical characterization of the heterodimerization domain of the core-binding factor beta subunit.

Core-binding factors (CBF) are heteromeric transcription factors essential for several developmental processes, including hematopoiesis. CBFs contain a DNA-binding CBF alpha subunit and a non-DNA binding CBF beta subunit that increases the affinity of CBF alpha for DNA. We have developed a procedure for overexpressing and purifying full-length CBF beta as well as a truncated form containing the N-terminal 141 amino acids using a novel glutaredoxin fusion expression system. Substantial quantities of the CBF beta proteins can be produced in this manner allowing for their biophysical characterization. We show that the full-length and truncated forms of CBF beta bind to a CBF alpha DNA complex with very similar affinities. Sedimentation equilibrium measurements show these proteins to be monomeric. Circular dichroism spectroscopy demonstrates that CBF beta is a mixed alpha/beta protein and NMR spectroscopy shows that the truncated and full-length proteins are structurally similar and suitable for structure determination by NMR spectroscopy.

Linkage between operator binding and dimer to octamer self-assembly of bacteriophage lambda cI repressor.

Cooperative binding of the bacteriophage lambda cI repressor dimer to specific sites of the phage operators OR and OL controls the developmental state of the phage. Cooperativity has long been thought to be mediated by self-assembly of repressor dimers to form tetramers which can bind simultaneously to adjacent operators. More recently, we demonstrated that when free repressor dimers self-associate in solution, tetramer is an intermediate in a concerted assembly reaction leading to octamer as the predominant higher order species [Senear, D. F., et al. (1993) Biochemistry 32, 6179-6189]. Even as a minority component in the assembly reaction, tetramer can account for pairwise cooperativity. In a similar manner, were it able to bind all three operators simultaneously, octamer could account for three-way cooperativity. In fact, based solely on repressor self-assembly, the naive prediction is that the repressor-OR interactions should be substantially more cooperative than they are. Evidently, there are unfavorable contributions to cooperativity from processes other than repressor self-assembly. Here, we focus on coupling between repressor self-association and operator binding as one possible unfavorable contribution to cooperativity. Sedimentation equilibrium analysis was used to compare the dimer-octamer association reactions of a repressor dimer-OR1 complex and free repressor dimer. Fluorescence anisotropy was used to investigate OR1 binding to free dimers and dimers assembled as higher order species. The results of these experiments indicate a significant and salt-dependent unfavorable contribution generated by such coupling. Since the oligonucleotides used in these experiments are the size of single operator sites, this coupling is mediated by the protein, not by the DNA. This mechanism does not account for an additional, salt-independent, unfavorable contribution which we presume is transmitted via the DNA. Thus, unfavorable contributions generated by structural transitions in both macromolecules serve to moderate the effect of self-association alone. We speculate that this is a general feature of cooperative protein-DNA interactions.

Soluble monomeric P-selectin containing only the lectin and epidermal growth factor domains binds to P-selectin glycoprotein ligand-1 on leukocytes.

Under shear stress, leukocytes use P-selectin glycoprotein ligand-1 (PSGL-1) to tether to and roll on P-selectin expressed on activated platelets or endothelial cells. P-selectin has an NH2-terminal lectin domain, an epidermal growth factor (EGF)-like motif, nine consensus repeats (CRs), a transmembrane domain, and a cytoplasmic tail. To determine whether the CRs are required for P-selectin to bind PSGL-1, we expressed a soluble protein (Lec-EGF) that contained only the lectin and EGF domains, plus a short C-terminal epitope tag. Electron microscopy and hydrodynamic analysis confirmed that Lec-EGF was monomeric, as previously shown for soluble P-selectin (sPS) that contained the lectin and EGF domains plus all nine CRs. Fluid-phase Lec-EGF or sPS inhibited binding of oligomeric125I-labeled membrane-derived P-selectin (mPS) to PSGL-1 on neutrophils and binding of 125I-PSGL-1 to immobilized mPS. The IC50 for inhibiting binding of mPS to neutrophils was fivefold greater for Lec-EGF than for sPS, whereas the IC50 for inhibiting binding of mPS to purified PSGL-1 was indistinguishable for Lec-EGF and sPS. Under static or shear conditions, neutrophils used PSGL-1 to tether to or roll on Lec-EGF that was captured by an immobilized monoclonal antibody to the C-terminal epitope. These data show that P-selectin requires only the lectin and EGF domains to bind to PSGL-1.

Comparison of backbone dynamics of reduced and oxidized Escherichia coli glutaredoxin-1 using 15N NMR relaxation measurements.

NMR-based structure determination of Escherichia coli glutaredoxin-1 in its reduced and oxidized forms revealed only subtle structural differences between the two forms. In an effort to characterize the role dynamics may play in the functioning of the protein, the backbone dynamics of both the reduced and oxidized forms of E. coli glutaredoxin-1 have been characterized using inverse-detection two-dimensional 15N-1H NMR spectroscopy. Longitudinal (T1) and transverse (T2) 15N relaxation time constants and steady-state [1H]-15N NOEs were measured for a majority of the protonated backbone nitrogen atoms. These data were analyzed by using a model-free formalism to determine the generalized order parameter (S2), the effective correlation time for internal motions (tau(e)), 15N exchange broadening contributions (R(ex)), and the overall molecular rotational correlation time (tau(m)). Sedimentation equilibrium measurements showed the reduced protein to be monomeric whereas the oxidized form could be fit to a monomer-dimer equilibrium. In order to try and assess the effect of dimerization on the dynamical parameters, the measurements on the oxidized protein have been carried out at two concentrations with very different monomer/dimer ratios. There is increased motion on both nano-picosecond and micro-millisecond time scales in the reduced form relative to the oxidized form, consistent with a more rigid oxidized protein. The increase in motion in the reduced protein correlates with its decreased thermodynamic stability. The role of the observed differences in the dynamic behavior in the two forms, particularly in the active site, in glutaredoxin-1's role as a protein disulfide reductant is discussed.

Advances in sedimentation velocity analysis.

On February 20, 1996, a workshop titled "Advances in Sedimentation Velocity Analysis" was held at the Biophysical Society meeting in Baltimore, Maryland, in honor of Professor David Yphantis's 65th birthday. Although he is known more for his work with sedimentation equilibrium, David's work on instrumentation and data analysis is the foundation for many of the recent advances in both equilibrium and velocity sedimentation. Over the years he has trained numerous graduate students, most of whom have gone on to emphasize the use of analytical ultracentrifugation to answer biochemical questions involving macromolecular assembly. His laboratory was one of very few that continued to use and develop analytical ultracentrifugation during its nadir in the 1970s and early 1980s. The rebirth and resurgence of analytical ultracentrifugation owe a great deal to his persistence and enthusiasm. These efforts have borne fruit. In the last five years, through his work at the National Analytical Ultracentrifugation Facility, he has helped train nearly 100 individuals in the delicate art of nonlinear least-squares analysis of equilibrium sedimentation data. Furthermore, the number of researchers using the ultracentrifuge and the number of papers published has skyrocketed in the last few years. This workshop, then, was a way to thank David for his years of devotion to analytical ultracentrifugation.

Characterization of a helix-loop-helix (EF hand) motif of silver hake parvalbumin isoform B.

Parvalbumins are a class of calcium-binding proteins characterized by the presence of several helix-loop-helix (EF-hand) motifs. It is suspected that these proteins evolved via intragene duplication from a single EF-hand. Silver hake parvalbumin (SHPV) consists of three EF-type helix-loop-helix regions, two of which have the ability to bind calcium. The three helix-loop-helix motifs are designated AB, CD, and EF, respectively. In this study, native silver hake parvalbumin isoform B (SHPV-B) has been sequenced by mass spectrometry. The sequence indicates that this parvalbumin is a beta-lineage parvalbumin. SHPV-B was cleaved into two major fragments, consisting of the ABCD and EF regions of the native protein. The 33-amino acid EF fragment (residues 76-108), containing one of the calcium ion binding sites in native SHPV-B, has been isolated and studied for its structural characteristics, ability to bind divalent and trivalent cations, and for its propensity to undergo metal ion-induced self-association. The presence of Ca2+ does not induce significant secondary structure in the EF fragment. However, NMR and CD results indicate significant secondary structure promotion in the EF fragment in the presence of the higher charge-density trivalent cations. Sedimentation equilibrium analysis results show that the EF fragment exists in a monomer-dimer equilibrium when complexed with La3+.

Insights from a new analytical electrophoresis apparatus.

Charge is a fundamental property of macromolecules that is inextricably linked to their structure, solubility, stability, and interactions. Progress has been made on the theoretical and structural aspects of these relationships. However, for several reasons, charge is difficult to measure in solution. Consequently, there is a lack of experimental data that, independent of other macro-ion properties, determines the effective charge. To overcome this problem, novel instrumentation and methods are being developed in our laboratory. Described here is an analytical electrophoresis apparatus that permits both the measurement of electrophoretic mobilities and the determination of steady-state electrophoresis concentration distributions. The latter provides a different-perspective on the processes that influence macro-ion behavior in an electric field. In addition, the apparatus permits the determination of diffusion coefficients either from boundary spreading during transport or from the decay of a concentration gradient. All of these determinations can be made with a single, 8-microL sample in a variety of solvents, thus providing unique insights into the charge properties of a macro-ion. Presented here is a progress report about this emerging technology, including the description of a prototype apparatus and examples of its use with a DNA oligonucleotide.

Turbidimetric studies of Limulus coagulin gel formation.

The turbidity during trypsin-induced coagulin gel formation was studied over a range of wavelengths. The range of wavelengths used (686-326 nm) also made it possible to investigate the dependence of turbidity on wavelength (the wavelength exponent). Using the results from that work, and structural information on coagulin and the coagulin gel from other studies, a model gel-forming system was designed that consists of species for which the turbidity can be calculated relatively simply. These species include small particles (small in all dimensions relative to the wavelength of incident light); long rods and long random coils (particles that are large in just one dimension relative to the wavelength of incident light); and reflective regions (aggregated material that is large in more than one dimension relative to the wavelength of incident light). The turbidimetric characteristics of the real coagulin gel-forming system are compared with those of the model system.