Kinetics of oxygen binding and subunit assembly for the hemoglobin alpha subunit.

A thorough kinetic characterization of the O2-binding and self-association reactions of alpha-subunits of human hemoglobin A has been performed. All of the rate constants for a five step reaction model linking the monomer-dimer reaction to the O2-binding steps have been determined for the first time. Our analysis of the ligand binding reaction shows that both monomer and dimer have nearly identical intrinsic O2-association and dissociation rate constants and therefore identical affinities for oxygen. During this investigation we discovered a small absorbance difference between the oxy-monomer and oxy-dimer alpha-subunits. This difference spectrum enabled direct measurements of the alpha O2 self-association reaction. We find an association rate constant of, 2.0 10(5) M(-1)s-1, similar to that for other subunit assembly processes in the hemoglobin system. Our results also suggest that the deoxy-subunit assembly kinetics must be similar to that for the oxy-subunit. These kinetic results together with the equilibrium constants obtained for these solution conditions by Ackers and coworkers provides, for the first time, a complete kinetic and thermodynamic description of all the intrinsic ligand binding and association reactions for alpha-subunits.

Cell-mediated immune functions in a patient with MHC class II deficiency.

This report focuses on cell-mediated immune functions in a patient with MHC class II deficiency. The patient described presented with a case of "classical" MHC class II deficiency (T and B cells within the normal range, normal lymphocyte proliferation in response to stimulation with mitogens, gene encoding for MHC class II present, no expression of MHC class II). The absence of MHC class II expression resulted in an incapability of the patient's antigen-presenting cells to function as accessory cells in the presentation of soluble protein antigens, while accessory functions required for the induction of alloantigen-induced lymphocyte proliferation or for the generation of cytotoxic T cells in response to an allostimulus were normal. The patient's T cells responded normally to alloantigenic stimulation and also had the capacity to develop antigen-specific cytotoxic functions. However, the T cells were completely naive with respect to activation by soluble protein antigens, even after presentation by accessory cells derived from the patient's healthy histoidentical brother. In this context it was interesting to note that the patient's CD4-positive cells showed a normal pattern of expression of the 4B4 marker, a marker generally present on memory T cells. These data make it tempting to speculate that in the absence of MHC class II, other cell surface structures may at least partially take over immune functions normally under the control of the MHC class II complex.

The two coiled coils in the isolated rod domain of the intermediate filament protein desmin are staggered. A hydrodynamic analysis of tetramers and dimers.

Desmin protofilaments and the proteolytically derived alpha-helical rod domain have been characterized by high-resolution gel permeation chromatography (GPC) using columns calibrated for the determination of viscosity radii. Additional characterization by chemical cross-linking and the determination of sedimentation values allowed the calculation of the molecular dimensions of the molecular species isolated. In dilute buffers GPC separated desmin rod preparations into two complexes: a dimer species (single coiled coil) with a length of 50 +/- 5 nm and a tetramer species (two coiled coils) with a length of 65 +/- 5 nm. Thus the two coiled coils in the tetramer are staggered by approximately 15 nm. The hydrodynamically derived lengths of the rod dimer and tetramer are supported by electron microscopy after metal shadowing. The hydrodynamic properties of desmin protofilaments follow that of the rod tetramer. The data on the hydrodynamic analysis of the rod tetramer of desmin in solution are in full agreement with the structural information recently deduced from paracrystals of the rod of glial fibrillary acid protein [Stewart, M., Quinlan, R.A. & Moir, R.D. (1989) J. Cell Biol. 109, 225-234]. Our results explain the inhomogeneity of molecules encountered in previous electron microscopical analyses.

Time-resolved solution X-ray scattering of tobacco mosaic virus coat protein: kinetics and structure of intermediates.

The kinetics of assembly and disassembly of tobacco mosaic virus coat protein (TMVP) following temperature jumps have been studied by small-angle X-ray scattering and turbidimetry. The structures of the principal aggregates of TMVP oligomers (A protein), intermediate size (helix I) and large size helical rods (helix II), have been characterized by their average radii of gyration of thickness, cross section, and shape obtained from the corresponding regimes of the small-angle scattering pattern. This structural information was obtained within seconds after the temperature-induced initiation of either polymerization or depolymerization and allowed us to detect transient intermediates. This methodology made it possible to observe and characterize the structure of a principal intermediate. Taken together with other kinetic information, these data suggest that polymerization of TMVP under virus self-assembly conditions may proceed via a single-layered helical nucleus that contains about 20 subunits. Previous studies have shown that overshoot polymerization of TMVP can occur and results in metastable long helical viruslike rods which subsequently depolymerize and then form short helical rods, depending on the conditions of the final equilibrium state. The longer rods (helix II) are overshoot polymers which form within seconds and contain 17 1/3 subunits per turn (helix IIB), in contrast to the subunit packing arrangement of 16 1/3 subunits per turn found in the shorter helical rods (helix IA). The latter packing arrangement is the one found in TMV. An overall polymerization scheme is proposed for the formation of these two helical forms of TMVP.

The separation and characterization of two forms of Torpedo electric organ calelectrin.

Two methods for extracting calelectrin, a Ca2+-regulated membrane-binding protein from the electric organ of Torpedo marmorata, have been compared and the more promising one was modified to increase the yield to 7-8 mg.kg-1 wet weight of tissue, that is 4-5 times greater than the original method. The calelectrin so obtain could be resoloved into a minor component (designated L-calelectrin) eluted from an anion-exchange column at relatively low ionic strength (100 mM NaCl) and a major component (H-calelectrin) eluted at higher ionic strength (300 mM NaCl). The two forms were also separated by chromatography on a hydrophobic resin. Electrophoresis on cellulose acetate indicated that L-calelectrin had a lower mean isoelectric point that the H-form and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate showed that under reducing conditions (presence of 5% beta-mercaptoethanol) both forms migrated as single species, the L-form having a lower apparent relative molecular mass (Mr 32,000) that the H-form (34,000). Under non-reducing conditions, there was no change in the migration of L-calelectrin but the H-form was resolved into two components of Mr 34,000 and 32,000. The addition of 2 mM Ca2+ had no effect on the migration of either form. Both forms were equally recognized by an anti-calelectrin antiserum and were microheterogeneous with respect to their isoelectric points (pH 4.3-5.5) in two-dimensional gel electrophoresis. Physical measurements were carried out on the major H-form. The Stokes radius was estimated to be 3nm, corresponding to an apparent Mr of 44,000. It was unaffected by changes in ionic strength, pH or Ca2+ concentration. Analytical ultracentrifugation gave a sedimentation constant of 2.9 S and an apparent Mr of 36,000. Measurements of circular dichroism indicated that 78% of the molecule was in the alpha-helix configuration and 22% in random coil. Ca2+ had no significant effect on the conformation.

Universal calibration of gel permeation chromatography and determination of molecular shape in solution.

Gel permeation chromatography (GPC) has become a routine technique for both biology and polymer chemistry. By comparison our theoretical perception of the separation principle of GPC is still immature and conflicting and so is the assessment of the analytical informational content of this method. In order to discriminate between the various parameters that might influence GPC and thus to decide among the numerous propositions of calibration, several odd biopolymers (tropomyosin, spectrin, DNA, tobacco mosaic virus, alpha-actinin, ovomucoid) were selected. They were characterized by analytical ultracentrifugation as well as quasielastic light scattering, and they were compared to globular proteins including icosahedral viruses (tomato bushy stunt virus, turnip yellow mosaic virus, Q beta, MS2) on several different HPLC column matrices. The results demonstrate that the universal calibration principle of GPC is the viscosity radius, i.e., the molecular volume times a shape function which is defined by the intrinsic viscosity. Alternate propositions such as molecular weight, second virial coefficient, diffusion coefficient (Stokes radius), radius of gyration, mean linear projected length, contour length, and related measures seem to be excluded on the basis of the evidence presented. These results help to focus the physical picture which seems to govern GPC. Finally it is demonstrated that GPC is a versatile and unique tool with which to characterize molecular shape and dynamics in solution.

Determination of reaction volumes and polymer distribution characteristics of tobacco mosaic virus coat protein.

A method that allows the quantitative determination of reaction volumes from sedimentation velocity experiments in an analytical ultracentrifuge is presented. Combined with a second method for detecting pressure-induced depolymerization, general characteristics of polymer distributions may be probed. We show that it is possible to determine if a sample is in an equilibrium or metastable state of subunit association. Our approach to probe macromolecular aggregation systems by small pressure perturbations is not restricted to the use of centrifuges. This method has been applied to characterize certain aspects of the polymerization of tobacco mosaic virus coat protein (TMVP). There are at least two helical polymer conformations in RNA-free coat protein rods. The smaller, helix I, polymers are limited to sizes below about 70 subunits (four to five helical turns) and undergo some kind of cooperative conformational change before further subunits may be added indefinitely. In contrast to helix I, the larger helix II polymers occur as broader and skewed size distributions. Under moderately strong polymerization conditions, the equilibrium state can contain both types of helical rods. The reaction volume for the addition of trimers is -220 ml/mol for both types of helical polymers. These results are compared with the results of previous thermodynamic analyses of TMVP polymerization.

Are the terminal domains in intermediate filaments organized as octameric complexes? Reevaluation of a recent suggestion.

Recently L. M. Milam and H. Erickson ((1985) J. Ultrastruc. Res. 90, 251-260) reported the isolation of a particle thought to be an octomeric complex of the terminal domains of desmin. This complex was isolated after trypsin treatment of intact filaments. As the existence of such a complex would place important restrictions on the possible packing modes of subunits within the filament we have repeated their procedure and additionally characterized the particle in question by detailed protein chemical data. We find that the particle is not derived from the terminal domains but instead comprises a portion of the carboxy-terminal half of the alpha-helical rod domain. We further show that the terminal domains are very rapidly digested into small peptides during the trypsin treatment of the filaments. No inferences on the structure of intermediate filament are therefore possible from the data in the original report.

Studies on the mechanism of assembly of tobacco mosaic virus.

Sedimentation and proton binding studies on the endothermic self-association of tobacco mosaic virus (TMV) protein indicate that the so-called "20S" sedimenting protein is an interaction system involving at least the 34-subunit two-turn yield cylindrical disk aggregate and the 49-subunit three-turn helical rod. The pH dependence of this overall equilibrium suggests that disk formation is proton-linked through the binding of protons to the two-turn helix which is not present as significant concentrations near pH 7. There is a temperature-induced intramolecular conformation change in the protein leading to a difference spectrum which is complete in 5 x 10(-6) s at pH 7 and 20 degrees C and is dominated at 300 nm by tryptophan residues. Kinetics measurements of protein polymerization, from 10(-6) to 10(3) s, reveal three relaxation processes at pH 7.0, 20 degrees C, 0.10 M ionic strength K (H) PO4. The fastest relaxation time is a few milliseconds and represents reactions within the 4S protein distribution. The second fastest relaxation is 50-100 x 10(-3) s and represents elementary polymerization steps involved in the formation of the approximately 20 S protein. Analysis of the slowest relaxation, approximately 5 x 10(4) s, suggests that this very slow formation of approximately 20 S protein may be dominated by some first order process in the overall dissociation of approximately 20S protein. Sedimentation measurements of the rate of TMV reconstitution, under the same conditions, show by direct measurements of 4S and approximately 20S incorporation at various 4S to approximately 20S weight ratios that the relative rate of approximately 20S incorporation decreases almost linearly, from 0 to 50% 4S. There appears to be one or more regions of TMV-RNA, approximately 1-1.5 kilobases long, which incorporates approximately 20S protein exclusively. Solutions of approximately 95-100% approximately 20S protein have been prepared for the first time and used for reconstitution with RNA. Such protein solutions yield full size TMV, but at a slower rate than if 4S protein is added. Thus the elongation reaction in TMV assembly, following nucleation with approximately 20S protein, is not exclusively dependent upon the presence of either 4S or approximately 20S protein aggregates. The initial, maximum, rate of reconstitution increases about threefold when the protein composition is changed from 5% to 30% 4S protein, at constant total protein concentration at pH 7.0, 20 degrees C in 0.10 M ionic strength K (H)PO4. The probable binding frame at the internal assembly nucleation site of TMV-RNA has been determined by measuring the association constants for the binding of various trinucleoside diphosphates to helical TMV protein rods. The -CAG-AAG-AAG-sequence at the nucleation site is capable of providing at least 10-14 kcal/mol of sites of binding free energy for the nucleation event in TMV self-assembly.