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Surveillance of SARS-CoV-2 through reported positive RT-PCR tests is biased due to non-random testing. Prevalence estimation in population-based samples corrects for this bias. Within this context, the pooled testing design offers many advantages, but several challenges remain with regards to the analysis of such data. We developed a Bayesian model aimed at estimating the prevalence of infection from repeated pooled testing data while (i) correcting for test sensitivity; (ii) propagating the uncertainty in test sensitivity; and (iii) including correlation over time and space. We validated the model in simulated scenarios, showing that the model is reliable when the sample size is at least 500, the pool size below 20, and the true prevalence below 5%. We applied the model to 1.49 million pooled tests collected in Switzerland in 2021-2022 in schools, care centres, and workplaces. We identified similar dynamics in all three settings, with prevalence peaking at 4-5% during winter 2022. We also identified differences across regions. Prevalence estimates in schools were correlated with reported cases, hospitalizations, and deaths (coefficient 0.84 to 0.90). We conclude that in many practical situations, the pooled test design is a reliable and affordable alternative for the surveillance of SARS-CoV-2 and other viruses.

Carbon pricing is a key component of current climate policy agendas. There are a variety of societal and health impacts from carbon pricing interventions (e.g. from improved air quality). A better understanding of potential health impacts and how they depend on context and policy design is crucial to improve the political feasibility and fairness of carbon pricing. Recent reviews have synthesized evidence on the effectiveness, equity and perceptions of carbon pricing and on the health co-benefits of mitigation. This review provides a narrative structured synthesis of the health impacts of carbon pricing. We identified 58 relevant publications of which all were modelling studies. We classify review findings into policy-relevant categories, synthesizing information on how carbon pricing affects health outcomes when implemented in different contexts, in isolation or as part of policy mixes. Findings suggest that internalization of health co-benefits in optimal price level estimates could lead to substantial mitigation in some regions. There are also opportunities to design carbon pricing to improve health outcomes, including through progressive or targeted use of revenues to improve food security, subsidize healthier diets or promote active transportation. Revenue use, price differentiation, market size and permit allocation of emissions trading schemes (ETS), and interaction with other public health or mitigation policies all influence health outcomes. Overall, the health impacts of carbon pricing are highly context-specific and further evidence is needed, particularly on health inequalities and ex-post analysis. However, existing evidence suggests that it is possible to design health-beneficial carbon pricing policies, thus enhancing policy acceptability and feasibility., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Previous molecular phylogenetic analyses have resolved the Australian bloodwood eucalypt genus Corymbia (~100 species) as either monophyletic or paraphyletic with respect to Angophora (9-10 species). Here we assess relationships of Corymbia and Angophora using a large dataset of chloroplast DNA sequences (121,016 base pairs; from 90 accessions representing 55 Corymbia and 8 Angophora species, plus 33 accessions of related genera), skimmed from high throughput sequencing of genomic DNA, and compare results with new analyses of nuclear ITS sequences (119 accessions) from previous studies. Maximum likelihood and maximum parsimony analyses of cpDNA resolve well supported trees with most nodes having >95% bootstrap support. These trees strongly reject monophyly of Corymbia, its two subgenera (Corymbia and Blakella), most taxonomic sections (Abbreviatae, Maculatae, Naviculares, Septentrionales), and several species. ITS trees weakly indicate paraphyly of Corymbia (bootstrap support <50% for maximum likelihood, and 71% for parsimony), but are highly incongruent with the cpDNA analyses, in that they support monophyly of both subgenera and some taxonomic sections of Corymbia. The striking incongruence between cpDNA trees and both morphological taxonomy and ITS trees is attributed largely to chloroplast introgression between taxa, because of geographic sharing of chloroplast clades across taxonomic groups. Such introgression has been widely inferred in studies of the related genus Eucalyptus. This is the first report of its likely prevalence in Corymbia and Angophora, but this is consistent with previous morphological inferences of hybridisation between species. Our findings (based on continent-wide sampling) highlight a need for more focussed studies to assess the extent of hybridisation and introgression in the evolutionary history of these genera, and that critical testing of the classification of Corymbia and Angophora requires additional sequence data from nuclear genomes.

We assembled the plastome of the temperate, Southern Hemisphere liana Muehlenbeckia australis from high throughput sequencing data (paired-end Illumina reads) generated from total genomic DNA sequencing libraries. M . australis ' chloroplast genome sequence (GenBank: MG604297) is 163,484 bp in length and composed of long single copy (LSC; 88,166 bp) and short single copy (SSC; 13,486 bp) regions flanked by inverted repeats (IR; 30,916 bp each) typical for angiosperms. The plastome includes 131 genes comprising 83 protein-coding genes, 37 transfer RNA genes, eight ribosomal RNA genes, two possible pseudogenes, psbL and rpl23 with internal stop codons, and truncated repeats of ndhF and rps19 at IR boundaries., Competing Interests: The authors report no conflicts of interest., (© 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

The buckwheat family Polygonaceae is a diverse group of plants and is a good model for investigating biogeography, breeding systems, coevolution with symbionts such as ants and fungi, functional trait evolution, hybridization, invasiveness, morphological plasticity, pollen morphology and wood anatomy. The main goal of this study was to obtain age estimates for Polygonaceae by calibrating a Bayesian phylogenetic analysis, using a relaxed molecular clock with fossil data. Based on the age estimates, we also develop hypotheses about the historical biogeography of the Southern Hemisphere group Muehlenbeckia. We are interested in addressing whether vicariance or dispersal could account for the diversification of Muehlenbeckia, which has a "Gondwanan" distribution. Eighty-one species of Polygonaceae were analysed with MrBayes to infer species relationships. One nuclear (nrITS) and three chloroplast markers (the trnL-trnF spacer region, matK and ndhF genes) were used. The molecular data were also analysed with Beast to estimate divergence times. Seven calibration points including fossil pollen and a leaf fossil of Muehlenbeckia were used to infer node ages. Results of the Beast analyses indicate an age of 110.9 (exponential/lognormal priors)/118.7 (uniform priors) million years (Myr) with an uncertainty interval of (90.7-125.0) Myr for the stem age of Polygonaceae. This age is older than previously thought (Maastrichtian, approximately 65.5-70.6 Myr). The estimated divergence time for Muehlenbeckia is 41.0/41.6 (39.6-47.8) Myr and its crown clade is 20.5/22.3 (14.2-33.5) Myr old. Because the breakup of Gondwana occurred from 95-30 Myr ago, diversification of Muehlenbeckia is best explained by oceanic long-distance and maybe stepping-stone dispersal rather than vicariance. This study is the first to give age estimates for clades of Polygonaceae and functions as a jumping-off point for future studies on the historical biogeography of the family.

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.

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.

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.

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)

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

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.

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.

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.

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.

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)

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.

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.

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.

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.

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.

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].

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.

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.

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.

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.

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.

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.