Conformational changes between the active-site and regulatory light chain of myosin as determined by luminescence resonance energy transfer: The effect of nucleotides and actin

Myosin is thought to generate movement of actin filaments via a conformational change between its light-chain domain and its catalytic domain that is driven by the binding of nucleotides and actin. To monitor this change, we have measured distances between a gizzard regulatory light chain (Cys 108) and the active site (near or at Trp 130) of skeletal myosin subfragment 1 (S1) by using luminescence resonance energy transfer and a photoaffinity ATP-lanthanide analog. The technique allows relatively long distances to be measured, and the label enables site-specific attachment at the active-site with only modest affect on myosin’s enzymology. The distance between these sites is 66.8 ± 2.3 Å when the nucleotide is ADP and is unchanged on binding to actin. The distance decreases slightly with ADP-BeF3, (−1.6 ± 0.3 Å) and more significantly with ADP-AlF4 (−4.6 ± 0.2 Å). During steady-state hydrolysis of ATP, the distance is temperature-dependent, becoming shorter as temperature increases and the complex with ADP⋅Pi is favored over that with ATP. We conclude that the distance between the active site and the light chain varies as Acto-S1-ADP ≈ S1-ADP > S1-ADP-BeF3 > S1-ADP-AlF4 ≈ S1-ADP-Pi and that S1-ATP > S1-ADP-Pi. The changes in distance are consistent with a substantial rotation of the light-chain binding domain of skeletal S1 between the prepowerstroke state, simulated by S1-ADP-AlF4, and the post-powerstroke state, simulated by acto-S1-ADP.

Determination of the distance between two spin labels attached to a macromolecule.

An EPR "spectroscopic ruler" was developed using a series of alpha-helical polypeptides, each modified with two nitroxide spin labels. The EPR line broadening due to electron-electron dipolar interactions in the frozen state was determined using the Fourier deconvolution method. These dipolar spectra were then used to estimate the distances between the two nitroxides separated by 8-25 A. Results agreed well with a simple alpha-helical model. The standard deviation from the model system was 0.9 A in the range of 8-25 A. This technique is applicable to complex systems such as membrane receptors and channels, which are difficult to access with high-resolution NMR or x-ray crystallography, and is expected to be particularly useful for systems for which optical methods are hampered by the presence of light-interfering membranes or chromophores.

Statistical analysis of shape through triangulation of landmarks: A study of sexual dimorphism in hominids

Two objects with homologous landmarks are said to be of the same shape if the configuration of landmarks of one object can be exactly matched with that of the other by translation, rotation/reflection, and scaling. In an earlier paper, the authors proposed statistical analysis of shape by considering logarithmic differences of all possible Euclidean distances between landmarks. Tests of significance for differences in the shape of objects and methods of discrimination between populations were developed with such data. In the present paper, the corresponding statistical methodology is developed by triangulation of the landmarks and by considering the angles as natural measurements of shape. This method is applied to the study of sexual dimorphism in hominids.

A Polymer Model for the Structural Organization of Chromatin Loops and Minibands in Interphase Chromosomes

A quantitative model of interphase chromosome higher-order structure is presented based on the isochore model of the genome and results obtained in the field of copolymer research. G1 chromosomes are approximated in the model as multiblock copolymers of the 30-nm chromatin fiber, which alternately contain two types of 0.5- to 1-Mbp blocks (R and G minibands) differing in GC content and DNA-bound proteins. A G1 chromosome forms a single-chain string of loop clusters (micelles), with each loop ∼1–2 Mbp in size. The number of ∼20 loops per micelle was estimated from the dependence of geometrical versus genomic distances between two points on a G1 chromosome. The greater degree of chromatin extension in R versus G minibands and a difference in the replication time for these minibands (early S phase for R versus late S phase for G) are explained in this model as a result of the location of R minibands at micelle cores and G minibands at loop apices. The estimated number of micelles per nucleus is close to the observed number of replication clusters at the onset of S phase. A relationship between chromosomal and nuclear sizes for several types of higher eukaryotic cells (insects, plants, and mammals) is well described through the micelle structure of interphase chromosomes. For yeast cells, this relationship is described by a linear coil configuration of chromosomes.

Measuring genome evolution

The determination of complete genome sequences provides us with an opportunity to describe and analyze evolution at the comprehensive level of genomes. Here we compare nine genomes with respect to their protein coding genes at two levels: (i) we compare genomes as “bags of genes” and measure the fraction of orthologs shared between genomes and (ii) we quantify correlations between genes with respect to their relative positions in genomes. Distances between the genomes are related to their divergence times, measured as the number of amino acid substitutions per site in a set of 34 orthologous genes that are shared among all the genomes compared. We establish a hierarchy of rates at which genomes have changed during evolution. Protein sequence identity is the most conserved, followed by the complement of genes within the genome. Next is the degree of conservation of the order of genes, whereas gene regulation appears to evolve at the highest rate. Finally, we show that some genomes are more highly organized than others: they show a higher degree of the clustering of genes that have orthologs in other genomes.

A unified statistical framework for sequence comparison and structure comparison

We present an approach for assessing the significance of sequence and structure comparisons by using nearly identical statistical formalisms for both sequence and structure. Doing so involves an all-vs.-all comparison of protein domains [taken here from the Structural Classification of Proteins (scop) database] and then fitting a simple distribution function to the observed scores. By using this distribution, we can attach a statistical significance to each comparison score in the form of a P value, the probability that a better score would occur by chance. As expected, we find that the scores for sequence matching follow an extreme-value distribution. The agreement, moreover, between the P values that we derive from this distribution and those reported by standard programs (e.g., blast and fasta validates our approach. Structure comparison scores also follow an extreme-value distribution when the statistics are expressed in terms of a structural alignment score (essentially the sum of reciprocated distances between aligned atoms minus gap penalties). We find that the traditional metric of structural similarity, the rms deviation in atom positions after fitting aligned atoms, follows a different distribution of scores and does not perform as well as the structural alignment score. Comparison of the sequence and structure statistics for pairs of proteins known to be related distantly shows that structural comparison is able to detect approximately twice as many distant relationships as sequence comparison at the same error rate. The comparison also indicates that there are very few pairs with significant similarity in terms of sequence but not structure whereas many pairs have significant similarity in terms of structure but not sequence.

Adjacent pore-lining residues within sodium channels identified by paired cysteine mutagenesis.

The pores of voltage-gated ion channels are lined by protein loops that determine selectivity and conductance. The relative orientations of these "P" loops remain uncertain, as do the distances between them. Using site-directed mutagenesis, we introduced pairs of cysteines into the P loops of micro1 rat skeletal muscle sodium channels and sought functional evidence of proximity between the substituted residues. Only cysteinyl residues that are in close proximity can form disulfide bonds or metal-chelating sites. The mutant Y401C (domain I) spontaneously formed a disulfide bond when paired with E758C in the P loop of domain II; the same residue, when coupled with G1530C in domain IV, created a high-affinity binding site for Cd2+ ions. The results provide the first specific constraints for intramolecular dimensions of the sodium channel pore.

Inferring phylogenies from DNA sequences of unequal base compositions.

A new method for computing evolutionary distances between DNA sequences is proposed. Contrasting with classical methods, the underlying model does not assume that sequence base compositions (A, C, G, and T contents) are at equilibrium, thus allowing unequal base compositions among compared sequences. This makes the method more efficient than the usual ones in recovering phylogenetic trees from sequence data when base composition is heterogeneous within the data set, as we show by using both simulated and empirical data. When applied to small-subunit ribosomal RNA sequences from several prokaryotic or eukaryotic organisms, this method provides evidence for an early divergence of the microsporidian Vairimorpha necatrix in the eukaryotic lineage.

Unusual horizontal transfer of a long interspersed nuclear element between distant vertebrate classes

We have shown previously by Southern blot analysis that Bov-B long interspersed nuclear elements (LINEs) are present in different Viperidae snake species. To address the question as to whether Bov-B LINEs really have been transmitted horizontally between vertebrate classes, the analysis has been extended to a larger number of vertebrate, invertebrate, and plant species. In this paper, the evolutionary origin of Bov-B LINEs is shown unequivocally to be in Squamata. The previously proposed horizontal transfer of Bov-B LINEs in vertebrates has been confirmed by their discontinuous phylogenetic distribution in Squamata (Serpentes and two lizard infra-orders) as well as in Ruminantia, by the high level of nucleotide identity, and by their phylogenetic relationships. The horizontal transfer of Bov-B LINEs from Squamata to the ancestor of Ruminantia is evident from the genetic distances and discontinuous phylogenetic distribution. The ancestor of Colubroidea snakes is a possible donor of Bov-B LINEs to Ruminantia. The timing of horizontal transfer has been estimated from the distribution of Bov-B LINEs in Ruminantia and the fossil data of Ruminantia to be 40–50 My ago. The phylogenetic relationships of Bov-B LINEs from the various Squamata species agrees with that of the species phylogeny, suggesting that Bov-B LINEs have been maintained stably by vertical transmission since the origin of Squamata in the Mesozoic era.

Estimation of evolutionary distances under stationary and nonstationary models of nucleotide substitution

Estimation of evolutionary distances has always been a major issue in the study of molecular evolution because evolutionary distances are required for estimating the rate of evolution in a gene, the divergence dates between genes or organisms, and the relationships among genes or organisms. Other closely related issues are the estimation of the pattern of nucleotide substitution, the estimation of the degree of rate variation among sites in a DNA sequence, and statistical testing of the molecular clock hypothesis. Mathematical treatments of these problems are considerably simplified by the assumption of a stationary process in which the nucleotide compositions of the sequences under study have remained approximately constant over time, and there now exist fairly extensive studies of stationary models of nucleotide substitution, although some problems remain to be solved. Nonstationary models are much more complex, but significant progress has been recently made by the development of the paralinear and LogDet distances. This paper reviews recent studies on the above issues and reports results on correcting the estimation bias of evolutionary distances, the estimation of the pattern of nucleotide substitution, and the estimation of rate variation among the sites in a sequence.

Microsatellite variability and genetic distances.

We analyze the within- and between-population dynamics of the distribution of the number of repeats at multiple microsatellite DNA loci subject to stepwise mutation. Analytical expressions for moments up to the fourth order within a locus and the variance of between-locus variance at mutation-drift equilibrium have been obtained. These statistics may be used to test the appropriateness of the one-step mutation model and to detect between-locus variation in the mutation rate. Published data are compatible with the one-step mutation model, although they do not reject the two-step model. Using both multinomial sampling and diffusion approximations for the analysis of the genetic distance introduced by Goldstein et al. [Goldstein, D. B., Linares, A. R., Cavalli-Sforza, L. L. & Feldman, M. W. (1995) Proc. Natl. Acad. Sci. USA 92, 6723-6727], we show that this distance follows a chi 2 distribution with degrees of freedom equal to the number of loci when there is no variation in mutation rates among the loci. In the presence of such variation, the variance of the distance is obtained. We conclude that the number of microsatellite loci required for the construction of phylogenetic trees with reliable branch lengths may be several hundred. Also, mutations that change repeat scores by several units, even though extremely rare, may dramatically influence estimates of population parameters.