Search PubMed⌕ Search

Biomedical subjects

T M Schuster

Publications and source records attributed to T M Schuster.

35 records · Page 2Linked to original sources
PubMed1981

Mechanism of tobacco mosaic virus assembly: Incorporation of 4S and 20S protein at pH 7.0 and 20 degrees C.

The mechanism of assembly of tobacco mosaic virus (TMV) has been investigated at pH 7.0 and 20 degrees C by analytical ultracentrifugation. Under these conditions the overall rates of interconversion of 4S and 20S TMV coat protein are sufficiently slow to make possible measurements of the concentrations of remaining 4S and 20S TMV coat protein after addition of homologous RNA to solutions containing, initially, various mass ratios of 20S protein to 4S protein. It has been possible to measure, by schlieren boundary analysis, the relative rates of incorporation of 4S and 20S TMV protein into the growing nucleoprotein rod over the range of initial 20S:4S protein mass ratios from 93:7 to 18:82. The results show that the amount of incorporation of 20S TMV protein depends on the initial 20S:4S mass ratio between approximately 100% and 60% 20S protein but that reconstitution can proceed with approximately 100% 20S TMV protein to form full virus-size rods. However, when the initial protein solutions have less than 60% 20S protein, approximately 80% of the reconstituted nucleoprotein is preferentially formed from 4S coat protein. The remaining approximately 20% appears to require preformed 20S coat protein. These results suggest that a larger region of RNA than previously estimated is involved in the rate-limiting nucleation step in assembly and may explain previously conflicting results concerning the elongation phase of assembly when starting with partially assembled rods.

Journal Article↗
PubMed1980

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.

Base Sequence↗
PubMed1979

Resonance Raman examination of axial ligand bonding and spin-state equilibria in metmyoglobin hydroxide and other heme derivatives.

Resonance Raman spectra and excitation profiles have been obtained within the 5700-6300-A absorption band of purified sperm whale metmyoglobin hydroxide (MbIIIOH) solutions. A large enhancement occurs for a Raman peak at 490 cm-1 which is shown by isotopic substitution of 18O for 16O to be almost purely an Fe-O stretch. The Fe-O vibration in MbIIIOH occurs 5 cm-1 to lower energy than the corresponding vibration at 495 cm-1 in human methemoglobin hydroxide (HbIIIOH) [Asher, S., Vickery, L., Schuster, T., & Sauer, K. (1977) Biochemistry 16, 5849], reflecting differences in ligand bonding between Mb(III) and Hb(III). A larger frequency difference (10 cm-1) exists between MbIIIF and HbIIIF for the Fe-F stretch. We do not observe separate Fe-O or Fe-F stretches from the alpha and beta chains of either HbIIIOH or HbIIIF. Excitation profile measurements for MbIIOH indicate that the 5700-6300-A absorption band is composed of two separate absorption bands which result from a high- and a low-spin form of MbIIIOH. The spin-state-sensitive Raman band at 1608 cm-1 reflects the high-spin species and has an excitation profile maximum at about 6000 A while the low-spin Raman band occurs at 1644 cm-1 and shows an excitation profile maximum at 5800 A. The Fe-O stretch at 490 cm-1 has an excitation profile maximum at about 6000 A. The differences in frequency and Raman cross section between the Fe-X vibrations in MbIIIX and HbIIIX (X = OH-, F-) can be related to increases in the out-of-plane iron distance for the high-spin species of MbIIIX. The shift in the 1644-cm-1 MbIIIOH low-spin state Raman band indicative of the heme core size to 1636 cm-1 in HbIIIOH indicates a larger heme core size in HbIIIOH. Raman frequency shifts are used to estimate differences in bond strain energies between MbIIIX and HbIIIX (X = OH-, F-). Previous resonance Raman excitation profile data can be interpreted in terms of separate contributions from different spin-state species.

Animals↗
PubMed1979

Kinetics and mechanism of tobacco mosaic virus assembly: direct measurement of relative rates of incorporation of 4S and 20S protein.

The mechanism of assembly of tobacco mosaic virus has been investigated under conditions in which the rates of incorporation of the 4S and 20S proteins can each be directly measured by analytical centfrifugation. Under these conditions, pH 6.5, 6.5 degrees C, 0.10 M ionic strength potassium orthophosphate, the protein can be made to exist as a metastable 20S aggregate that is necessary for efficient reconstitution. The overall assembly process consists of an initiation (nucleation) reaction that requires two to three 20S disk aggregates per RNA molecule and is followed by an elongation (growth) reaction. In the elongation phase of assembly the 4S protein is incorporated 50 to 70 times faster than the 20S disk, calculated on the basis of a steady-state kinetic analysis. Therefore, under these conditions, in which the rate of assembly is about 0.06 of that at pH 7, 20 degrees C, 0.10 M ionic strength orthophosphate, the 4S protein preferentially participates in the elongation phase. At this slow reconstitution rate intermediate assembly states (about 70-168 S) can be observed. The kinetics of both protein incorporation and nucleoprotein formation suggest that the elongation process is composed of at least two different, possibly sequential, rate-limiting reactions.

Kinetics↗