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.

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+.

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.

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.

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.

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.

A novel trypsin inhibitor from the hemolymph of the horseshoe crab Limulus polyphemus.

Trypsin inhibitory activity from the hemolymph of Limulus polyphemus was found to co-purify with coagulogen (the clottable protein in blood coagulation) after acidification, ammonium sulfate precipitation, and gel filtration. Limulus trypsin inhibitor (LTI) was separated from coagulogen by ion-exchange chromatography on carboxymethyl-Sephadex. LTI is an inhibitor of trypsin (Ki = 3.3 nM) on both high and low molecular weight substrates. It also inhibits chymotrypsin but has little or no effect on thrombin, thermolysin, pepsin, or papain, nor does LTI inhibit the proteolytic cascade produced in endotoxin-stimulated Limulus amoebocyte lysate coagulation. Electrophoresis under nonreducing conditions on denaturing polyacrylamide gel yields a doublet migrating with an estimated Mr of 20,000. Under reducing conditions, a single broad band migrates with an estimated Mr of 15,000. The native structure is a monomer of moderate asymmetry with a molecular weight of 16,300 and a so20,w = 1.5(5), as determined by analytical ultracentrifugation. The amino acid composition of LTI yields a calculated molecular weight of 15,680 and a calculated partial specific volume of 0.71(7) ml/g. LTI does not contain methionine, tryptophan, or detectable levels of reducing carbohydrate. The NH2-terminal sequence (V-S-P-P-F-I-K-Q-T-K-F-S-T-X-F-L-G-X-S-S) consists primarily of hydrophobic amino acid residues. Comparison of the amino acid composition and amino-terminal sequence of LTI with those of other known protease inhibitors reveals no significant similarity to other trypsin inhibitors. The novel physical characteristics suggest that LTI represents a new type of protease inhibitor.

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.

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.

The molecular weight and oligomerization of rabbit thrombomodulin as assessed by sedimentation equilibrium.

Lubrol-solubilized rabbit thrombomodulin has been examined by equilibrium sedimentation in buffers that include sufficient D2O to make the detergent neutrally buoyant. Data were acquired at rotor speeds from 12,000 to 28,000 rpm from two thrombomodulin preparations, at protein concentrations from 0.01 to 0.07%, and in buffer containing 0.01 to 0.23% Lubrol. Examination of the data from different rotor speeds shows that the thrombomodulin exists as a heterogeneous mixture containing monomer (Mr 65,000), trimer, and higher oligomers. The oligomers do not equilibrate over the time scale of the experiment. The weight fraction as monomer varies from preparation to preparation, and appears to be independent of detergent concentration. Thus, experimenters should be cautious when interpreting binding or kinetic results obtained under similar buffer conditions.

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.

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.

Rapid precision interferometry for the analytical ultracentrifuge. I. A laser controller based on a phase-lock-loop circuit.

This is the first of a series of manuscripts presenting methods to enable rapid reduction of data from the Rayleigh interference optical system of the Beckman Model E analytical ultracentrifuge. Here we present a pulsed laser controller for the ultracentrifuge. This laser controller uses a phase-lock-loop to provide properly timed light pulses over the speed range of 3000 to 60,000 rpm; it effectively resolves one rotor revolution into 4096 discrete angular positions. The circuit has been designed so that the laser light bursts occur at selectable angular positions of the rotor that are independent of rotor speed even under conditions of maximum acceleration or deceleration. We have used this controller in our laboratory over a 7-year period for both photographic and real-time collection at interferometric data from the ultracentrifuge.

Rapid precision interferometry for the analytical ultracentrifuge. III. Determination of period of rotation, frequency of rotation, and elapsed time.

The approaches presented in this series of papers make possible rapid gathering, reduction, and analysis of data from the Rayleigh interference optical system of an analytical ultracentrifuge. Instrumentation described in this paper provides some of the timing and measurement circuits necessary for a microprocessor or minicomputer to determine the rotor frequency, rotor period, and elapsed time of an experiment. It includes simple but effective circuits to generate precise rotor timing pulses that are useful for synchronization of pulsed light sources. Circuits to control photographic operations in the ultracentrifuge are described briefly. All of these circuits are interfaced to a simple microcomputer address/data bus. An adapter between this bus and a Q-bus (for a DEC LSI-11/2 or LSI 11/23 microcomputer) is also described. The circuits presented have been used in this laboratory over a 3-year period. They have proven reliable and form an integral part of the real-time data acquisition systems that have been constructed.

Rapid precision interferometry for the analytical ultracentrifuge. II. A laser controller based on a rate-multiplying circuit.

A laser controller that uses a fixed frequency clock and a digital rate-multiplying circuit to synchronize the triggering of a pulsed laser to the spinning of an analytical ultracentrifuge rotor has been designed. The circuit is simple, inexpensive, and virtually free of any adjustments. It tracks rotors undergoing full acceleration or deceleration. At constant rotor speed it provides triggering that is accurate and reproducible to better than 0.5 microseconds. The settings of this controller are independent of rotor speed over the full range of the ultracentrifuge.

Ca2+ dependence of the interactions between protein C, thrombin, and the elastase fragment of thrombomodulin. Analysis by ultracentrifugation.

The two-way and three-way interactions among active-site-blocked bovine thrombin, bovine protein C, and the elastase fragment of rabbit thrombomodulin (elTM) were examined by analytical ultracentrifugation at 23.3 degrees C in 100 mM NaCl, 50 mM Tris (pH 7.65), and 1 mM benzamidine, in the presence of 0 to 5 mM calcium chloride. Thrombin and elTM form a tight (Kd less than 10(-8) M) 1:1 complex in the absence of Ca2+ that weakens with the addition of Ca2+ (Kd approximately 4 microM in 5 mM Ca2+). Without Ca2+, thrombin and protein C form a 1:1 complex (Kd approximately 1 microM) and what appears to be a 1:2 thrombin-protein C complex. The Kd for the 1:1 complex weakens over 100-fold in 5 mM CaCl2. Protein C and elTM form a Ca(2+)-independent 1:1 complex (Kd approximately 80 microM). Nearly identical binding to thrombin and elTM is observed when active-site-blocked activated bovine protein C is substituted for protein C. Thrombin inhibited by diisopropyl fluorophosphate and thrombin inhibited by a tripeptide chloromethyl ketone exhibited identical behavior in binding experiments, suggesting that the accessibility of protein C to the substrate recognition cleft of these two forms of thrombin is nearly equal. Human protein C binds with lower affinity than bovine protein C. Ternary mixtures also were examined. Protein C, elTM, and thrombin form a 1:1:1 complex which dissociates with increasing [Ca2+]. In the absence of Ca2+, protein C binds to the elTM-thrombin complex with an apparent Kd approximately 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)