Emanuel Margoliash

“Homology” in proteins and nucleic acids: A terminology muddle and a way out of it

“Homology” has the precise meaning in biology of “having a common evolutionary origin,” but it also carries the loose meaning of “possessing similarity or being matched.” Its rampant use in the loose sense is clogging the literature on protein and nucleic acid sequence comparisons with muddy writing and, in some cases, muddy thinking In its precise biological meaning, “homology” is a concept of quality. The word asserts a type of relationship between two or more things. Thus, amino acid or nucleotide sequences are either homologous or they are not. They cannot exhibit a particular “level of homology” or “percent homology.” Instead, two sequences possess a certain level of similarity. Similarity is thus a quantitative property. Homologous proteins or nucleic acid segments can range from highly similar to not recognizably similar (where similarity has disappeared through divergent evolution). If using “homology” loosely did not interfere with our thinking about evolutionary relationships, the way in which we use the term would be a rather unimportant semantic issue. The fact is, however, that loose usage in sequence comparison papers often makes it difficult to know the authors intent and can lead to confusion for the reader (and even for the author). There are three common situations in which hazards arise by using “homology” to mean similarity. The first case is the most obvious offense but perhaps the least troublesome. Here an author identifies sequence similarities (calling them homologies) but claims that the sequences being compared are not evolutionarily related. Some awkward moments occur in such a paper, since the author claims both homology (i.e., similarity) and nonhomology (i.e., lack of a common ancestor). Nonetheless, the author’s ideas are likely to be clear since arguments against common ancestry are presented explicitly. A second case is one in which an author points out similarities (again called homologies) but does not address the issue of evolutionary origins. The reader, seeing the term “homology,” may infer that the author is postulating coancestry when that is not the authors intent. The final case occurs most frequently and is the most subtle and therefore most troublesome. Here, similarities (called homologies) are used to support a hypothesis of evolutionary homology. In this case, the two meanings of homology seem to overlap, and it is almost inevitable that the thinking of author and reader alike will be intrusively distorted as follows. Similarity is relatively straightforward to document. In comparing sequences, a similarity can take the form of a numerical score (O/o amino acid or nucleotide positional identity, in the simplest approach) or of a probability associated with such a score. In comparisons of three-dimensional structures, a typical numerical description is root-mean-square positional deviation between compared atomic positions. A similarity, then, can become a fully documented, simple fact. On the other hand, a common evolutionary origin must usually remain a hypothesis, supported by a set of arguments that might include sequence or three-dimensional similarity. Not all similarity connotes homology but that can be easily overlooked if similarities are called homologies. Thus, in this third case, we can deceive ourselves into thinking we have proved something substantial (evolutionary homology) when, in actuality, we have merely established a simple fact (a similarity, mislabeled as homology). Homology among similar structures is a hypothesis that may be correct or mistaken, but a similarity itself is a fact, however it is interpreted. We believe that the concepts of evolutionary homology and sequence or three-dimensional similarity can be kept distinct only if they are referred to with different words. We therefore offer the following recommendations: *Sequence similarities (or other types of similarity) should simply be called similarities. They should be documented by appropriate statistical analysis. In writing about sequence similarities the following sorts of terms might be used: a level or degree of similarity; an alignment with optimized similarity; the % positional identity in an alignment; the probability associated with an alignment. *Homology should mean “possessing a common evolutionary origin” and in the vast majority of reports should have no other meaning. Evidence for evolutionary homology should be explicitly laid out, making it clear that the proposed relationship is based on the level of observed similarity, the statistical significance of the similarity, and possibly other lines of reasoning. One could argue that the meaning of the term “homology” is itself evolving. But if that evolution is toward vagueness and if it results in making our scientific discourse unclear, surely we should intervene. With a collective decision to mend our ways, proper usage would soon become fashionable and therefore easy. We believe that we and our scientific heirs would benefit significantly.

[18] Mitochondrial cytochrome c: Preparation and activity of native and chemically modified cytochromes c

Publisher Summary This chapter discusses the preparation and activity of native and chemically modified cytochromes c. Cytochrome c is extracted from ground and homogenized tissue with a dilute solution of aluminum sulfate at pH 4.5, the trivalent cations effectively displacing the protein even at low ionic strength. Aluminum ions are precipitated as the hydroxide at slightly alkaline pH, and each is replaced in solution by three monovalent ammonium ions. The cytochrome c is purified by (NH 4 ) 2 SO 4 fractionation and cation-exchange chromatography. This method of extraction is suited to vertebrate and invertebrate tissues, but for plant materials, protists, or fungi, special cytolysis procedures are often required before use of a similar scheme. Although both these aspects of cytochrome c function are interesting, it seems that the biologically significant evolutionary variations of cytochrome c structure predominantly affect the mechanics of protein–protein interaction with its physiological oxidants and reductants, rather than the mechanism of electron transfer.

A method for estimating the number of invariant amino acid coding positions in a gene using cytochrome c as a model case

This paper shows, within the limitations of the assumption stated below, that approximately 27–29 of the unmutated codons which determine the amino acids of cytochrome c are invariant because of biological requirements. A mutation is defined here as the change of a single base in the sequence of a trinucleotide codon, which change alters the amino acid coded for. Codons, if any, in which mutations would be vigorously selected against are termed invariant codons. We assume that, subject to one adjustment, those mutations in the cytochrome c gene which survived in the descent of todays species are randomly distributed among the variable codons. The one adjustment arises from the possibility that a very few codon positions may exhibit frequencies of mutation sufficiently great to justify the exclusion of these codons from the overall distribution on the grounds that the frequency of mutation occurring in these few positions is clearly inconsistent with the assumption of randomness. There are 5 out of the total 110 codons in the cytochrome c structural gene which have clearly sustained an abnormally large number of mutations.

A centrosymmetric projection at 4 Å of horse heart oxidized cytochrome c

Abstract Horse heart ferricytochrome c has been crystallized in space group P41 with one molecule per asymmetric unit and cell dimensions: a = b = 58.45 A , c = 42.34 A . A derivative search concentrating on transition metal complexes, organomercurials and labeled carboxymethylation reagents has produced two isomorphous single-site derivatives, PtCl42− and mersalyl. Data from the hk0 zone out to a resolution of 4 A have been collected, heavy metal sites found and phases found and refined. The refinement behavior of mean figure of merit and Kraut R factor has been compared. The hk0 projection has been calculated and interpreted, and the interpretation checked with salt difference studies. The molecule appears to be a sphere of 31 A diameter, with a center of packed hydrophobic side chains, a polypeptide chain framework and an outer covering of packed hydrophilic side groups.

Interaction of cytochrome c with cytochrome c oxidase: An understanding of the high- to low-affinity transition

The steady-state kinetics of high- and low-affinity electron transfer reactions between various cytochromes c and cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) preparations were studied spectrophotometrically and polarographically. The dissociation constants for the binding of the first and second molecules of horse cytochrome c (I = 15 mM) are 5.10(-8) M and 1.10(-5) M, respectively, close to the spectrophotometric Km values and consistent with the controlled binding model for the interaction between cytochrome c and cytochrome oxidase (Speck, S.H., Dye, D. and Margoliash, E. (1984) Proc. Natl. Acad. Sci. USA 81, 346-351) which postulates that the binding of a second molecule of cytochrome c weakens that of the first, resulting in low-affinity kinetics. While the Km of the polarographically assayed high-affinity reaction is comparable to that observed spectrophotometrically, the low-affinity Km is over an order of magnitude smaller and cannot be attributed to the binding of a second molecule of cytochrome c. Increasing the viscosity has no effect on the Vmax of the low-affinity reaction assayed polarographically, but increases the Km. Thus, the transition from high- to low-affinity kinetics is dependent on the frequency of productive collisions, as expected for a hysteresis model ascribing the transition to the trapping of the oxidase in a primed state for turnover. At ionic strengths above 150 mM, the rate of cytochrome c oxidation decreases without any correlation to the calculated net charge of the cytochrome c, indicating rate-limiting rearrangement of the two proteins in proximity to each other.

Long-Range Electron Transfer at Fixed and Known Distance within Protein Complexes

Abstract We have used zinc-substituted hemoproteins to study long-range electron transfer between redox centers at fixed and known distances. The photo-excited zinc triplet state in one subunit of the α1-β2 electron transfer complex of [Zn,Fe] hybrid hemoglobin transfers an electron to its partner aquoferriheme subunit at a rate, kt=100 s−1. The temperature dependence of this electron transfer from 77K to 313K is indicative of non-adiabatic electron tunnelling in which the accompanying nuclear rearrangements proceed by nuclear tunnelling. For the complex between zinc-substituted yeast cytochrome c peroxidase (CCP) and native yeast cytochrome c , electron transfer occurs at a rate, kt=138 s−1, compared to kt = 17 s−1 in the complex between the yeast enzyme and horse cyt c . The difference demonstrates the species specificity involved in physiological electron transfer. Oxidation of ferroporphyrin by the zinc porphyrin radical is more rapid, and, for the yeast cytochrome, occurs with a rate, kh ∼ 104 s−1.

The isolation of bovine-heart cytochrome c oxidase subunits Dependence on phospholipid and cholate content

The polypeptide chains of bovine-heart cytochrome c oxidase were preparatively isolated by a simple large-scale procedure based on gel permeation chromatography in the presence of sodium dodecyl sulphate. The resolution of the subunits as a function of the cholate and phospholipid content of the preparation was investigated. Cholate, and to a lesser extent, phospholipids interfere with the separation of the subunits; however, they do not prevent dissociation of the enzyme by SDS. Bovine-heart cytochrome c oxidase consists of six major subunits (estimated molecular weights in thousands: 40, 25, 20, 14, 12 and 10). In addition, the enzyme preparation contains at least five minor constituents, present in less than stoichiometric amounts. The first two of the three large subunits, all of which are hydrophobic, have amino-terminal N-formylmethionine. Subunit III, however, has a free methionine N-terminus.

Active Carboxylic Acid-Terminated Alkanethiol Self-Assembled Monolayers on Gold Bead Electrodes for Immobilization of Cytochromes c

It is extremely difficult to immobilize cytochrome c (cyt c) on carboxylic acid-terminated alkanethiol self-assembled monolayers (HOOC-SAM) on gold bead electrodes prepared in a hydrogen flame. We found that simple pretreatment of a HOOC-SAM/gold bead electrode by potential cycling in buffer solution in the range ±300 mV prior to immobilization of the protein facilitated stable cyt c binding to HOOC-SAMs. The stability of cyt c on the HOOC-SAMs is independent of the topology of the gold surface.

Expression of recombinant cytochromes c from various species in Saccharomyces cerevisiae: post-translational modifications

A complete protocol for the expression of recombinant cytochrome c genes from yeast, Drosophila melanogaster, and rat in a yeast strain, GM-3C-2, which does not express its own cytochromes c is described. The construction of the expression vectors, transformation and large-scale growth of the yeast, and preparation and purification of the recombinant cytochromes c are described. It was found that, contrary to the way yeast modifies its own cytochromes c, the recombinant proteins were partially acetylated at their N-terminus, except for the drosophila protein, which remained entirely unblocked. Furthermore, the yeast and rat proteins were close to fully trimethylated at lysine 72, while the drosophila protein could be separated chromatographically into forms containing tri-, di-, mono-, and unmethylated lysine 72 showing corresponding resonances in the NMR spectrum. These observations emphasize that, in employing expression procedures to obtain native or mutant forms of cytochrome c, it is essential to identify the variety and extent of post-translational modifications and to separate the preparation into pure monomolecular species. Otherwise, it may become impossible to distinguish between the influence of a site-directed mutation and unexamined post-translational modifications.

Antibodies to Rabbit Cytochrome c Arising in Rabbits

Antibodies reactive with rabbit cytochrome c have been observed in rabbits immunized with several heterologous cytochromes. Such antibodies have also been observed in rabbits immunized with rabbit cytochrome c conjugated to bovine gamma globulin. The serum of a rabbit immunized with human cyto chrome c reacted with the cytochrome c of the same rabbit.