Biomedical subjects

T M Schuster

Publications and source records attributed to T M Schuster.

At least 19 recordsLinked to original sources

PubMed1999

Biophysical characterization of a designed TMV coat protein mutant, R46G, that elicits a moderate hypersensitivity response in Nicotiana sylvestris.

The hypersensitivity resistance response directed by the N' gene in Nicotiana sylvestris is elicited by the tobacco mosaic virus (TMV) coat protein R46G, but not by the U1 wild-type TMV coat protein. In this study, the structural and hydrodynamic properties of R46G and wild-type coat proteins were compared for variations that may explain N' gene elicitation. Circular dichroism spectroscopy reveals no significant secondary or tertiary structural differences between the elicitor and nonelicitor coat proteins. Analytical ultracentrifugation studies, however, do show different concentration dependencies of the weight average sedimentation coefficients at 4 degrees C. Viral reconstitution kinetics at 20 degrees C were used to determine viral assembly rates and as an initial assay of the rate of 20S formation, the obligate species for viral reconstitution. These kinetic results reveal a decreased lag time for reconstitution performed with R46G that initially lack the 20S aggregate. However, experiments performed with 20S initially present reveal no detectable differences indicating that the mechanism of viral assembly is similar for the two coat protein species. Therefore, an increased rate of 20S formation from R46G subunits may explain the differences in the viral reconstitution lag times. The inferred increase in the rate of 20S formation is verified by direct measurement of the 20S boundary as a function of time at 20 degrees C using velocity sedimentation analysis. These results are consistent with the interpretation that there may be an altered size distribution and/or lifetime of the small coat protein aggregates in elicitors that allows N. sylvestris to recognize the invading virus.

Capsid Proteins↗

PubMed1996

New revolutions in the evolution of analytical ultracentrifugation.

The use of the biophysical technique of analytical ultracentrifugation has recently undergone a resurgence. The commercial availability of the Beckman optima XL-A and XL-I analytical ultracentrifuges along with the continued growth in computing ability and analysis software has led to the expanded use of analytical ultracentrifugation and its capabilities. The genetic revolution and the search for further understanding of macromolecular interactions have again brought analytical ultracentrifugation to the forefront of macromolecular characterization.

Macromolecular Substances↗

PubMed1992

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.

Chemical Phenomena↗

PubMed1990

Preparation and properties of recombinant DNA derived tobacco mosaic virus coat protein.

Recombinant DNA derived tobacco mosaic virus (vulgare strain) coat protein (r-TMVP) was obtained by cloning and expression in Escherichia coli and was purified by column chromatography, self-assembly polymerization, and precipitation. SDS-PAGE, amino terminal sequencing, and immunoblotting with polyclonal antibodies raised against TMVP confirmed the identify and purity of the recombinant protein. Isoelectric focusing in 8 M urea and fast atom bombardment mass spectrometry demonstrated that the r-TMVP is not acetylated at the amino terminus, unlike the wild-type protein isolated from the tobacco plant derived virus. The characterization of r-TMVP with regard to its self-assembly properties revealed reversible endothermic polymerization as studied by analytical ultracentrifugation, circular dichroism, and electron microscopy. However, the details of the assembly process differed from those of the wild-type protein. At neutral pH, low ionic strength, and 20 degrees C, TMVP forms a 20S two-turn helical rod that acts as a nucleus for further assembly with RNA and additional TMVP to form TMV. Under more acidic conditions, this 20S structure also acts as a nucleus for protein self-assembly to form viruslike RNA-free rods. The r-TMVP that is not acetylated carries an extra positive charge at the amino terminus and does not appear to form the 20S nucleus. Instead, it forms a 28S four-layer structure, which resembles in size and structure the dimer of the bilayer disk formed by the wild-type protein at pH 8.0, high ionic strength, and 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗

PubMed1989

On the theory of gel electrophoresis of DNA: extension and evaluation of the Lumpkin-DèJardin-Zimm model.

Using fundamental concepts of hydrodynamics in porous media, we have rederived the Lumpkin-DèJardin-Zimm (LDZ) model for the gel electrophoresis of reptating, infinitely long, worm-like chains, such as DNA. The force balance provides a constraint for evaluating the correlation among the segment-to-field angles of a given molecular conformation. We have used an approximate analytical expression to account for this correlation in order to apply the present derivation to finite chain lengths. The resulting extended LDZ model predicts a nonlinear variation of electrophoretic mobility (mu) with reciprocal chain length (1/Lc) at low electric field strengths, similar to the one observed. The present derivation is valid only at low electric field strengths, and the predictions of the extended LDZ model fit data for a dimensionless electric field strength, E1*, of less than 1.23. An empirically useful criterion for determining the onset of reptation is also described. The present treatment shows how size-exclusion effects can be included in future theories. Models based on reptation alone are shown to predict a discontinuity in the molecular chain length dependence of mobility at a critical molecular size. Such discontinuities are not observed experimentally.

Chemical Phenomena↗

PubMed1988

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.

Capsid Proteins↗

PubMed1988

Structure and function of disk aggregates of the coat protein of tobacco mosaic virus.

Experiments have been carried out on the coat protein of tobacco mosaic virus (TMVP) to test for the occurrence of the previously postulated RNA-induced direct switching, during in vitro assembly of tobacco mosaic virus (TMV), of the subunit packing from the cylindrical bilayer disk to the virus helical arrangement. No evidence was found for such RNA-induced switching and no evidence for the direct participation of the bilayer disk in either the nucleation or elongation phases of the in vitro virus assembly. Instead, virus assembly proceeds by an initiation step involving the binding of the RNA to the previously characterized two-plus turn helical aggregate that is formed from small oligomers of subunits. However, a bilayer disk, which has been characterized in high ionic strength crystals, has been observed in low ionic strength virus assembly solutions only as a transient species upon depolymerization of dimers of bilayer disks formed in solution at high ionic strength, and not as an equilibrium species of TMVP.

Capsid Proteins↗

PubMed1988

Quaternary interactions in hemoglobin beta subunit tetramers. Kinetics of ligand binding and self-assembly.

We have investigated the rates of monomer in equilibrium with tetramer self-association of oxygenated beta SH subunits of human hemoglobin A as well as the influence of self-association on the binding kinetics for O2 and CO. A 4 beta in equilibrium with 2 beta 2 in equilibrium with beta 4 assembly pathway can be used to describe the association equilibria and kinetics. We have determined all four elementary rate constants for this assembly pathway at 15 degrees C in 0.1 M Tris-HCl, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4. These data imply that a significant amount (approximately 17%) of beta 2 can be present. Laser photolysis kinetic studies of O2 binding indicate that the O2 association rate constant is unaffected by the degree of self-association. In contrast, photolysis of beta CO solutions shows an overall rate of CO binding that increases at higher protein concentrations. These data are consistent with a concentration-dependent equilibrium between two protein species with CO association rates differing by a factor of 2.5, but they do not appear to be compatible with a direct assignment of different CO binding rates to the different assembly states. Rather, we believe the data imply that CO binding to beta oligomers is heterogeneous, with both a fast binding and a slow binding form being present in single association states. The fast binding form predominates (approximately equal to 87%) in beta 4, while the beta monomer has very little or none of the fast binding form. We propose that the slow binding component within beta 4 may be those subunits with rotationally disordered hemes (La Mar, G. N., Yamamoto, Y., Jue, T., Smith, K. M., and Pandey, R. K. (1985) Biochemistry 24, 3826-3831). The implications of these findings for the use of isolated subunits as models for the subunits within "R state" hemoglobin tetramers are discussed.

Algorithms↗

PubMed1987

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.

Capsid↗

PubMed1987

Isolation and characterization of nucleoprotein assembly intermediates of tobacco mosaic virus.

During assembly of tobacco mosaic virus from pure RNA and 20S capsid protein aggregates under conditions where protein is limiting, partially assembled intermediates of specific sizes accumulate; these were isolated on sucrose density gradients. The earliest intermediate found in substantial quantity sedimented at 56 S and was shown, by measurement of its buoyant density and of the length of the RNA segment protected by the capsid protein from nuclease digestion, to consist of RNA that is 13% encapsidated (corresponding to a rod length of about 39 nm); the next intermediate sediments at 78 S and is 18% encapsidated (corresponding to a rod length of about 54 nm). Studies of the distribution of intermediates at various input ratios of protein/RNA indicated that their accumulation results from decreases in the rate constants for protein binding that are local to specific points in the course of encapsidation. After extensive nuclease digestion, the RNA still associated with the first intermediate was shown to include a portion that is unencapsidated. This segment of the RNA may be a region of stable secondary that confers the nuclease resistance despite the lack of protection by capsid protein. Such RNA secondary structure, if it exists, would also cause the accumulation of intermediates by imposing an energy barrier to subsequent rod elongation.

Capsid↗

PubMed1986

Disk aggregates of tobacco mosaic virus protein in solution: electron microscopy observations.

Previous studies of the coat protein of tobacco mosaic virus (TMVP) have shown that TMVP presumably exists as linear stacks of two-ring cylindrical disks in the 0.7 M ionic strength buffer used for crystallizing the disks for X-ray diffraction studies [Raghavendra, K., Adams, M.L., & Schuster, T.M. (1985) Biochemistry 24, 3298-3304]. The spectroscopic and sedimentation studies of solutions of TMVP under these crystallizing conditions have demonstrated a long-term metastability of these disk aggregates when they are placed in 0.1 M ionic strength buffers, as are used for reconstituting tobacco mosaic virus from TMVP and viral RNA. The present work describes an electron microscopic study of TMVP disk aggregates under the same solution conditions employed in the previous spectroscopic and sedimentation studies. The results show that in the pH 8.0 0.7 M ionic strength crystallization buffer TMVP exists as stacks of disks which range in size from about 6 to 24 layers, corresponding to 3-12 2-layer disk aggregates having 17 subunits per layer. These TMVP aggregates persist in a metastable form in 0.1 M ionic strength virus reconstitution buffer with no apparent changes in structure of the stacked disks. The results are consistent with the conclusions of the solution physical-chemical studies which suggest that the disk structure may not be related to the 20S TMVP aggregate that is the nucleation species in virus

Crystallization↗

PubMed1985

Sedimentation equilibrium measurements of the intermediate-size tobacco mosaic virus protein polymers.

Short-column sedimentation equilibrium methods have been applied for the first time to tobacco mosaic virus (TMV) protein (0.1 M ionic strength orthophosphate) at pH 6.5 and at pH 7.0 to estimate molecular weights. Previous sedimentation velocity experiments at pH 6.5, 20 degrees C have led to the conclusion that the major boundary with an S0(20),w value of 24.4 +/- 0.1 S consists of a distribution of polymers which are mainly three-turn, 48-51-subunit helical rod aggregates. The directly measured z-average molecular weights together with sedimentation velocity data are entirely consistent with this assignment of a three-turn aggregate. Molecular weights have also been determined under two conditions where a large mass fraction of the protein sediments with an S0(20),w value of 20.3 +/- 0.2 S. At pH 6.5, 6-8 degrees C, the aggregates in this boundary are metastable and correspond to 50-60% of the preparation. At pH 7.0, 20 degrees C at equilibrium, 65-75% of the protein sediments at 20.3 S. The 20.3S boundary is very similar under both conditions and is interpreted as being composed of a distribution of protein aggregates centered about 39 +/- 2 subunits. This result is important in the interpretation of previous kinetic measurements of TMV self-assembly. The current view is that the 34-subunit structure of TMV protein, in the form of a cylindrical disk which is made up of two 17-subunit layers and has been characterized in single-crystal X-ray diffraction studies, plays a central role in the initial binding steps with RNA. The present results are not consistent with the view that there is a significant concentration of the TMV protein disk structure in solution under the usual conditions of TMV self-assembly.

Hydrogen-Ion Concentration↗

PubMed1985

Tobacco mosaic virus protein aggregates in solution: structural comparison of 20S aggregates with those near conditions for disk crystallization.

Previous X-ray studies (2.8-A resolution) on the crystals of tobacco mosaic virus protein (TMVP) grown from solutions containing high salt have characterized the structure of the protein aggregate as a bilayered cylindrical disk formed by 34 identical subunits [Bloomer, A.C., Champness, J.N., Bricogne, G., Staden, R., & Klug, A. (1978) Nature (London) 276, 362-368]. Under low-salt conditions, 20S aggregates are in equilibrium with 4S species and involved in the efficient nucleation of TMV assembly in vitro [Butler, P.J.G. (1984) J. Gen. Virol. 65, 253-279]. We have investigated by sedimentation velocity and near-UV circular dichroism (CD) measurements the structure of 20S aggregates in low salt (I = 0.1 potassium phosphate at pH 7.0 and 20 degrees C) and the aggregates in high salt [0.2 M (NH4)2SO4 in I = 0.1 tris(hydroxymethyl)aminomethane hydrochloride at pH 8.0 and 20 degrees C, close to the conditions under which TMVP crystallizes as disk aggregates]. At high salt, we observe structures (presumably stacks of disks) having s20,w values around 40, 45, and 50 S, but not the 20S species present in low-salt buffers. The near-UV CD spectrum of 20S aggregates has been obtained for the first time, using computer techniques, from the spectra of the 4S-20S equilibrium mixture and the 4S species. This spectrum of 20S aggregates differs dramatically from that of the stacks of disks examined at both high and low salt (into which the stacks can be returned by dialysis), indicating that the difference is not a solvent effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗

PubMed1984

Diamagnetism of human apo-, oxy-, and (carbonmonoxy)hemoglobin.

In recent years, a controversy has arisen over the magnetic properties of oxyhemoglobin (HbO2) and (carbonmonoxy)hemoglobin (HbCO). At present, it is unclear which, if any, conditions give a completely diamagnetic state for the heme-ligand complex which can be used as a diamagnetic reference state. In order to establish a diamagnetic reference independent of assignments of electronic configurations, we have measured the magnetic susceptibilities of apohemoglobin solutions and powdered iron-free protoporphyrin IX. We have also reexamined the magnetism of HbO2 and HbCO solutions at 20 degrees C and at several ionic strengths. We find no difference in magnetism between HbO2 and HbCO and no changes in their magnetism with solution conditions. Furthermore, relative to the new (apohemoglobin + porphyrin) diamagnetic reference, our data are consistent with complete diamagnetism for both HbO2 and HbCO under all conditions we have studied. Our data imply that any low-lying triplet state must lie at least 900 cm-1 above the diamagnetic ground states. These results disagree strongly with reports of substantial room temperature paramagnetism for HbO2 and a smaller paramagnetism for HbCO which disappears at high ionic strength [see Cerdonio, M., Morante, S., Vitale, S., Giacometti, G., & Brunori, M. (1982) in Hemoglobin and Oxygen Binding (Ho, C., Ed.) pp 63-68, Elsevier/North-Holland, Amsterdam, and references cited therein].

Apoproteins↗

PubMed1982

Sequence specificity of trinucleoside diphosphate binding to polymerized tobacco mosaic virus protein.

The binding of trinucleoside diphosphates to long helical rods of tobacco mosaic virus (TMV) protein is shown to depend on base sequence, 5' AAG 3' binding being the strongest of the 25 trinucleoside diphosphate sequences measured. As TMV has a stoichiometry of three nucleotides per protein subunit, the sequence of TMV RNA suggested to be the nucleation site for self-assembly of the virus has three possible binding frames. From our binding constant data the most likely frame is predicted and shown to have two contiguous AAG sequences in a hairpin loop region.

Base Sequence↗

PubMed1981

Association-dependent absorption spectra of oxyhemoglobin A and its subunits.

A number of factors which lower the oxygen affinity of hemoglobins are also known to produce shifts of the absorption band maxima of the oxyheme. We have studied the variation of the absorption spectra of oxygenated alpha SH and beta SH subunits of human Hb A as a function of their state of association (i.e. alpha, alpha 2, beta, beta 4, alpha beta, or alpha 2 beta 2), and have attempted to correlate the spectral changes with changes to O2 affinity. These studies were carried out under solution conditions (0.1 M Tris, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4, 10 degrees C) where detailed thermodynamic data for subunit association and oxygen binding are available (Ackers, G. K. (1980) Biophys. J. 32, 331-346). Concentration-difference spectra reveal that the visible and Soret absorption band maxima of beta O2 are slightly red shifted relative to beta 4O8. A unique feature of this spectral change is that the red shift is accompanied by an increase in the ratio of the peak absorbances of the visible alpha and beta spectral bands. By measuring the spectral change as a function of concentration, an association constant of 6.4 +/- 1.9 X 10(15) M-3 was determined for the 4(beta O2) in equilibrium beta 4O8 equilibrium. In contrast, no spectral differences were found between alpha O2 and alpha 2O4 or between oxy alpha beta dimers and oxyHb. Mixing experiments show that the spectrum of oxyHb differs from the average of either alpha O2 + beta O2 or alpha O2 + beta 4O8, but is closer to the former. A comparison between these spectral data and the reported O2 affinities of these species shows that affinity and oxyheme spectra are not correlated.

Carboxyhemoglobin↗

PubMed1981

Resonance Raman and absorption spectroscopic detection of distal histidine--fluoride interactions in human methemoglobin fluoride and sperm whale metmyoglobin fluoride: measurements of distal histidine ionization constants.

The pH dependence of the resonance Raman and absorption spectra of human methemoglobin fluoride (HbIIIF) and sperm whale metmyoglobin fluoride (MbIIIF) has been examined. Both the Raman and absorption spectra of HbIIIF and MbIIIF indicate the existence at alkaline pH of an equilibrium between the hydroxide and fluoride complexes. The absorption maxima of HbIIIF and MbIIIF solutions shift to longer wavelengths as the pH is decreased from neutrality. The Raman data show a corresponding shift of the 461- and 468-cm-1 Fe-F vibrational stretching peaks at pH 7.0 [Asher, S. A., & Schuster, T. M. (1979) Biochemistry 18, 5377] to 399 and 407 cm-1 at acid pH in MbIIIF and HbIIIF, respectively. These shifts are interpreted to result from protonation of the distal histidine and the formation of a hydrogen bond to the fluoride ligand. Measurements of the pH dependence of the absorption and resonance Raman spectra give distal histidine ionization constants (apparent) corresponding to pK = 5.1 (+/- 0.1) for HbIIIF and pK = 5.5 (+/- 0.1) for MbIIIF. An examination of the distal histidine pK values and the frequency of the hydrogen-bonded Fe--F stretching vibration at pH 5.0 of HbIIIF with and without inositol hexaphosphate indicates little difference in the distal histidine--heme distance between the so-called R and T quaternary forms of HbIIIF. These results indicate that the changes in the electronic spectrum of HbIIIF that occur upon switching from the R to the T form do not result from alterations in (1) the iron--fluoride bond distance, (2) the iron out-of-heme plane distance, or (3) the distal histidine--fluoride distance.

Animals↗

PubMed1981

Differences in iron-fluoride bonding between the isolated subunits of human methemoglobin fluoride and sperm whale metmyoglobin fluoride as measured by resonance Raman spectroscopy.

The heme geometries of the isolated alpha and beta subunits of human methemoglobin fluoride (HbIIIF) and sperm whale metmyoglobin fluoride (MbIIIF) have been examined by exciting their Raman spectra within their ca. 6000-A charge-transfer absorption bands. The Fe-F stretching vibration at 471 cm-1 in the beta subunits shifts to 466 cm-1 in the alpha subunits and to 461 cm-1 in MbIIIF. The Fe-F bond is estimated to elongate by 0.02 A in the alpha subunits and 0.03 AZ in MbIIIF compared with that in the beta subunits. This bond elongation is interpreted to result from an increased iron displacement toward the proximal histidine side of the heme in the series MbIIIF greater than alpha greater than beta. A comparison of the isolated subunit spectra with that of tetrameric HbIIIF indicates little change occurs in isolated subunit heme geometry upon association into tetrameric HbIIIF. A correlation is found between the gamma max of the 600-A charge-transfer absorption band and the Fe-F bond length. Elongation of the Fe-F bond is associated with a shift of the absorption spectral maximum to a longer wavelength. However, the absorption spectral shift induced by the inositsol hexaphosphate induced R leads to T conversion does not result from a change in the Fe-F stretching frequency (+/- 0.5 cm-1). In contrast, frequency shifts are observed for heme macrocyclic vibrational modes. The data are interpreted to indicate that the effect of the R leads to T conversion in HbIIIF is to perturb heme macrocycle conformation without altering the heme out-of-plane iron distance or the Fe-F bond length.

Animals↗