Biomedical subjects
Publications and source records attributed to R F Doolittle.
A hidden Markov model (HMM) approach was used to identify potential candidates in sequence databases for fibronectin type III domains in plants, a kingdom heretofore bereft of these structures. Fortuitously, one of the proteins uncovered had already had a crystal structure published, allowing direct structural confirmation of the existence of this domain in plants.
Unexpected relationships among the various aminoacyl-tRNA synthetases continue to be uncovered. The question arises - is this mainly the result of promiscuous exchange, or is the confusion really a reflection of the differential loss of past duplications? Phylogenetic analysis may yet provide the answer.
Fibrinogen is a 340 kDa glycoprotein found in the blood plasma of all vertebrates. It is transformed into a fibrin clot by the action of thrombin. Recent X-ray structures of core fragments of both fibrinogen and fibrin have revealed many details about this polymerization event. These include structures of a 30 kDa recombinant gammaC domain, an 86 kDa fragment D from human fibrinogen and a cross-linked double-D fragment from fibrin.
Recently reported X-ray structures for large core fragments derived from human fibrinogen and fibrin make it possible to correlate structural and functional anomalies of known genetic variants. Here we examine a variety of amino acid replacements previously reported for hereditary dysfibrinogenemias, most of which are associated with impaired fibrin polymerization. For many of these we have modeled in the mutant amino acid and considered the structural consequences. We have also examined the cases of a small deletion and a large insertion, as well as the impact of substitutions in the GPRPam ligand that was co-crystallized with fragment double-D.
A recent study of the divergence times of the major groups of organisms as gauged by amino acid sequence comparison has been expanded and the data have been reanalyzed with a distance measure that corrects for both constraints on amino acid interchange and variation in substitution rate at different sites. Beyond that, the availability of complete genome sequences for several eubacteria and an archaebacterium has had a great impact on the interpretation of certain aspects of the data. Thus, the majority of the archaebacterial sequences are not consistent with currently accepted views of the Tree of Life which cluster the archaebacteria with eukaryotes. Instead, they are either outliers or mixed in with eubacterial orthologs. The simplest resolution of the problem is to postulate that many of these sequences were carried into eukaryotes by early eubacterial endosymbionts about 2 billion years ago, only very shortly after or even coincident with the divergence of eukaryotes and archaebacteria. The strong resemblances of these same enzymes among the major eubacterial groups suggest that the cyanobacteria and Gram-positive and Gram-negative eubacteria also diverged at about this same time, whereas the much greater differences between archaebacterial and eubacterial sequences indicate these two groups may have diverged between 3 and 4 billion years ago.
In blood coagulation, units of the protein fibrinogen pack together to form a fibrin clot, but a crystal structure for fibrinogen is needed to understand how this is achieved. The structure of a core fragment (fragment D) from human fibrinogen has now been determined to 2.9 A resolution. The 86K three-chained structure consists of a coiled-coil region and two homologous globular entitles oriented at approximately 130 degrees to each other. Additionally, the covalently bound dimer of fragment D, known as 'double-D', was isolated from human fibrin, crystallized in the presence of a Gly-Pro-Arg-Pro-amide peptide ligand, which simulates the donor polymerization site, and its structure solved by molecular replacement with the model of fragment D.
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The thrombin-catalysed conversion of fibrinogen into a fibrin gel is common to all extant vertebrates. Because fibrin formation is both temporary and risky, an effective scheme for fibrinolysis evolved concomitantly. In this regard, the fibrinogen molecule is well adapted both for network polymerization and for subsequent dismantling. The question is, has it always been so? It has long been known that the three non-identical chains that compose vertebrate fibrinogen are descended from a common ancestor, and the original molecule must have been either a homotrimer or a dimer thereof. Three-dimensional studies on core fragments of fibrinogen are revealing new insights about both fibrin formation and its destruction. These studies are also showing exactly what structural changes have accompanied changes in function for the various domains. Chief among these is the reversal of direction for the alpha chain after replacement of its C-terminal domain.
Amino acid substitution tables are essential for the proper alignment of protein sequences, and alignment scores based on them can be transformed into distance measures by various means. In the simplest case, the negative log of the score is used. This Poisson relationship assumes that all sites are equally likely to change, however. A more accurate relationship would correct for different rates of change at each residue position. Recently, Grishin (J. Mol. Evol. 41:675-679, 1995) published a set of simple equations that correct for various circumstances, including different rates of change at different sites. We have used these equations in conjunction with similarity scores that take into account constraints on amino acid interchange. Simulation studies show a linear relationship between these calculated distances and the numbers of allowed mutations based on the observed variation of rate at all sites in various proteins.
The intron positions of ten different protein families were examined to determine (the statistical likelihood of) whether spliceosomal introns are the result of random insertion events into previously intronless genes, on the one hand, or the result of random loss from common ancestral introns, on the other. The number of expected matches for the alternative scenarios was calculated for a binomial distribution by considering currently observed introns relative to all possible locations for insertion or loss. Introns occurring at approximately the same location (hereafter called a "match") were tallied for each of the paired proteins. Matches were identified by their positions in the multiple alignment and were defined as any two introns occurring within a window of 11 possible nucleotide positions, thereby allowing for possible alignment errors and "intron sliding." Matches were tallied from the raw data and compared with the expected number of matches for the two different scenarios. The results suggest that the distribution of introns in genes encoding proteins is due to random insertion and not random loss.
Russell Doolittle is a biocomputing pioneer who was among the first to use computers in the 1960s to compare sequences. He contributed to the discovery of unforeseen sequence similarities, such as the relationship between v-src and v-mos. Doolittle's reflection demonstrates the enormous advances made in bioinformatics, soft- and hardware, and the concurrent explosion in data storage and acceleration of communication. Today's advanced technology was not available at the beginning of Doolittle's career, when no databases existed, and researchers knew by heart most of the sequences which had been published.
Amino acid sequence data from 57 different enzymes were used to determine the divergence times of the major biological groupings. Deuterostomes and protostomes split about 670 million years ago and plants, animals, and fungi last shared a common ancestor about a billion years ago. With regard to these protein sequences, plants are slightly more similar to animals than are the fungi. In contrast, phylogenetic analysis of the same sequences indicates that fungi and animals shared a common ancestor more recently than either did with plants, the greater difference resulting from the fungal lineage changing faster than the animal and plant lines over the last 965 million years. The major protist lineages have been changing at a somewhat faster rate than other eukaryotes and split off about 1230 million years ago. If the rate of change has been approximately constant, then prokaryotes and eukaryotes last shared a common ancestor about 2 billion years ago, archaebacterial sequences being measurably more similar to eukaryotic ones than are eubacterial ones.
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The principal component of blood clots is a protein meshwork called fibrin. The precursor protein, fibrinogen, occurs in a soluble form in the blood plasma where it is activated by thrombin when and if the need arises. More than a century after first being purified, fibrinogen has yet to have its detailed 3-dimensional structure revealed. The situation is changing rapidly, however, and crystallographic studies in progress in several laboratories on a variety of fragments and complexes may soon reveal not only its structure but also the subtleties of how this large glycoprotein is transformed into a fibrin clot.
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The common ancestry of eukaryotes, archaebacteria and eubacteria is well demonstrated by amino acid sequence comparisons of numerous proteins that are common to all three groups. On the other hand, there are a few proteins, like ubiquitin, that are common to eukaryotes and archaebacteria and which have yet to be observed in eubacteria. Some proteins appear to be wholly restricted to eukaryotes; this is especially true of cytoskeletal proteins. Recently, actin has been found by crystallography to be homologous with an ATP-binding domain found in a heat shock protein and several other proteins common to all three urkingdoms. This observation is puzzling on several counts. Most cytoskeletal proteins like actin and tubulin are very slow changing and must have been so for a very long time. How is it, then, that no sequence resemblance can be discerned with their alledged prokaryotic antecedents? The question is addressed by considering two bacterial fts proteins which appear to be related to actin, on the one hand, and tubulin, on the other. One answer may be that the rate of change of these proteins changed dramatically at a key point in their history. Another possibility is that eukaryotes are much older than some of their other proteins indicate.
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Lampreys have two genes for the alpha chains of fibrinogen, the second of which encodes a minor form with a carboxyl-terminal domain homologous to the carboxyl-terminal domains of beta and gamma chains. Initially, we referred to the alternative chain as alpha-II; we now use the designation alpha' in order to facilitate reference to crosslinked dimers. Antisera raised to synthetic peptides based on the cDNA sequence confirmed that the alpha' chain was present in fibrinogen prepared directly from plasma. The same antibodies were used to determine the size and properties of the carboxyl-terminal domain after its release by mild tryptic digestion, a fragment of apparent molecular weight 35,000-40,000 being produced. Unlike fragment D generated in the same digestions, the alpha' fragment did not bind to Gly-Pro-Arg or Gly-Val-Arg peptide affinity columns. During clotting under conditions where factor XIII is active, the alpha' chains became crosslinked very much more rapidly than ordinary alpha chains, the principal product being an apparent dimer, but smaller amounts of higher multimers being detectable. The crosslinking was inhibited by various amines, as well as by peptides that prevent polymerization.