J.-F. Pechère

Evolutionary diversification of structure and function in the family of intracellular calcium-binding proteins

SummaryThe maximum parsimony method was used to reconstruct the genealogical history of the family of intracellular calcium-binding proteins represented by six major present-day lineages, three of which - calcium dependent modulator protein, heart and skeletal muscle troponin Cs, and alkali light chains of myosin - were found to share a closer kinship with one another than with the other lineages. Similarly, parvalbumins and regulatory light chains of myosin were depicted as more closely related, whereas the branch of intestinal calcium-binding protein proved to have the most distant separation. The computer-generated amino acid sequence for the common ancestor of these six lineages described a four domain protein in which each domain of approximately 40 amino acid residues had a mid-region, 12 residue segment that bound calcium and had properties most resembling those of the calcium dependent modulator protein. It could then be deduced that parvalbumins evolved by deletion of domain I, inactivation of calcium-binding properties in domain II, and acquisition of increased affinity for Ca++ and Mg++ in domains III and IV. Regulatory light chains of myosin lost the cation binding property from three domains, retaining it in I, whereas alkali light chains of myosin lost this ability from each of the four domains. In skeletal muscle troponin C all domains retained their calcium-binding activity; however, like parvalbumins, domains III and IV acquired high affinity properties. Cardiac troponin C lost its binding activity from domain I but otherwise resembled the skeletal muscle form. Finally, intestinal calcium-binding protein evolved by deletion of domains III and IV.Positive selection could be implicated in these evolutionary changes in that the rate of fixation of mutations substantially increased in the mid portions of those domains which were loosing calcium-binding activity. Likewise, when the cation binding sites were changing from low to high affinity, an accelerated rate of fixed mutations was observed. Once this new functional parameter was selected these regions showed a remarkable conservatism, as did those binding sites which were maintaining the lower affinity. Moreover even in sequence regions not directly involved in cation binding, the lineage of troponin C became very conservative over the past 300 million years, perhaps because of the necessity for maintaining specific interfaces in order for the molecule to interact with troponin I and T in a functional thin myofilament. A similar phenomenon was observed in domain II of the regulatory light chains of the myosin lineage suggesting a possible binding site with the heavy chain of myosin.

Muscular parvalbumins: Preparative and analytical methods of general applicability☆

Abstract 1. 1. The muscular parvalbumins present in carp (Cyprinus carpio) myogen have been isolated simultaneously by a three-step procedure involving (NH4)2SO4 fractionation, gel filtration on Sephadex G-75 and chromatography on DEAE cellulose. 2. 2. Disc electrophoresis in 12% polyacrylamide gels was also found useful in analytically separating these parvalbumins, either from whole myogen or at different stages of their purification. 3. 3. The low-molecular-weight fraction of carp myogen was submitted to isoelectric focusing in a sucrose gradient, permitting the isoelectric points of the four major parvalbumins to be determined as being 4.47, 4.37, 4.25 and 3.95 at 2°. 4. 4. The three techniques above were found to be applicable to the isolation and characterization of the parvalbumins from hake (Merluccius merluccius), turbot (Psetta maxima) and frog (Rana esculenta). 5. 5. Their general use in the study of muscular parvalbumins is discussed.

The evolution of muscular parvalbumins investigated by the maximum parsimony method

SummaryPhylogenetic trees requiring the lowest sum of nucleotide replacements and gene duplicative events were constructed from the amino acid sequence data on ten gnathostome parvalbumins (PAR) and two related myofibrillar proteins troponin-C (TNC) and myosin alkali-light-chain (ALC). The origin and differentiation of the structural domains within these proteins were also investigated by the maximum parsimony method and by an alignment statistic for identifying evolutionarily related protein sequences. The results suggest, in agreement with the Weeds-McLachlan model, that tandem duplications in a precursor gene caused a primordial one-domain polypeptide (consisting of two helices with a calcium binding region in between) to double and then quadruple in size. Duplications of the gene coding for this four domain (I–II–III–IV) protein in an early metazoan, pre-gnathostome lineage gave rise to the separate loci for TNC, ALC, and PAR. TNC, which alone retained the Ca-binding function in each of its four domains, evolved much more slowly than either the ALC or PAR lineages. In the PAR lineage the I–II–III–IV structure was degraded, presumably by a partial gene deletion, to the II–III–IV structure during descent to the gnathostome ancestor of parvalbumins. Also during this period the mid region in domain II lost its Ca-binding function and, as it did so, evolved at an accelerated rate over other regions, a pattern indicative of positive selection for a change in function. In turn, from the gnathostome ancestor to the present, the mid regions of domains III and IV, which each retained Ca-bindung function, evolved much more slowly than other regions, a pattern indicative of stabilizing selection for preservation of function. Between the gnathostome and teleost-tetrapod ancestor a gene duplication separated the parvalbumins into anα-lineage and aβ-lineage. During this early vertebrate period PAR genes evolved at the extremely fast rate of 89 nucleotide replacements per 100 codons per 108 years (i.e. 89 NR %), but from the teleost-tetrapod ancestor to the present, bothα- andβ-PAR lineages evolved at a much slower rate, about 8 NR %. The use ofβ-parvalbumins as phylogenetic markers was complicated by presumptive evidence that paralogous (i.e. duplication dependent) gene lineages occur within this group. As a final point, in the genealogy of TNC, ALC, and PAR lineages, a non-random pattern of nucleotide replacements was observed between the reconstructed ancestral and descendant mRNA sequences. The pattern was similar to that observed for other protein genealogies and seems to reflect a bias in the genetic code for guanine to adenine and adenine to guanine transitions (especially at the first nucleotide position of the RNA codons) to produce amino acid substitutions which are compatible with the preservation of protein three-dimensional structure.

The participation of parvalbumins in the activation—relaxation cycle of vertebrate fast skeletal-muscle

Although the physiological role of muscular parvalbumins [l-3] remains an open question, several studies [4,5] have restricted the range of possibilities and suggested that these proteins constitute a non-interacting regulatory system of the sarcoplasmic Ca*-level which operates essentially in fast contracting muscles so as to facilitate their cyclic relaxation. In addition, a scheme has been proposed [S] , in which the affinity of parvalbumins for Ca*’ (thus their possible competition with troponin C for this ion) could be modulated by changes in hydrogen ion-concentration associated with the contraction process. This allows undisturbed activation of the myofibrils at higher pH, while ensuring their complete relaxation at lower pH. Experiments reported recently in the literature [6,7] have substantiated the validity of several steps of the above scheme. Thus, they have shown that calcium-free parvalbumins indeed have the potential to block contraction or ATPase activity of Ca’+-activated myofibrils. Sarcoplasmic reticulum, in turn, is able to remove efficiently all the Ca*-ions which can be bound by parvalbumins. The first experiments reported in the present communication were designed independently in order

Conformational studies on muscular parvalbumins. II. Nuclear magnetic resonance analysis.

Summary NMR spectroscopy (proton resonance at 100 and 270 MHz) has been used to observe conformational features in muscular parvalbumins from hake (Merluccius merluccius) and carp (Cyprinus carpio), in the native state, in the denatured state (6M guanidinium chloride or heating) and after almost complete removal of calcium ions. From these observations it appears that the removal of the strongly bound calcium ions leads to a structure very similar to that obtained by chemical or thermal denaturation. On the other hand, the NMR spectrum of the native carp parvalbumins can be interpreted in the light of the recent X-Ray data obtained elsewhere for this protein. Some aspects of the primary structure of these parvalbumins, such as the presence of an N-terminal acetyl residue, have also been investigated with the NMR technique.

The amino acid sequence of the major parvalbumin from hake muscle.

Abstract A sequence of 107 amino acids has been determined in the single chain of the major parvalbumin (pI 4.36) from hake ( Merluccius merluccius ) muscle as a result of studies on peptides obtained after tryptic, chymotryptic, peptic and cyanogen bromide cleavages of the protein.

Conformational studies on muscular parvalbumins: I. Optical rotatory dispersion and circular dichroism analysis(*)

Summary The optical rotatory dispersion and the circular dichroism of solutions of the major parvalbumins from hake (Merluccius merluccius) and thornback ray (Raja clavata) have been studies in the visible and ultraviolet regions. From the results of such measurements, the helical content of these two proteins was estimated to lie around 30–40 p. cent. This and other features of their secondary structure are discussed in the light of independently known structural properties of parvalbumins.

Binding of calcium by parvalbumin fragments.

Parvalbumin fragments from carp pI 4.47 parvalbumin corresponding to its residues 1--75 and 76--108 bind Ca2+ with affinities corresponding to Kd 0.9 . 10(-4) M and Kd 3 . 10(-3) M, respectively.

Troponins C from reptile and fish muscles and their relation to muscular parvalbumins

Parvalbumins are sarcoplasmic acidic proteins, with a MW around 12 000, which are abundant in the white muscles of fishes and amphibians. The amino acid sequence of eight parvalbumins has confirmed their homology and has allowed the building of a phylogenetic tree, incorporating two major lines of genetic descent, o~ and/~ (see ref. [1 ] for a review). Similarities between parvalbumins and TN-C*, the calcium-sensitizing factor of the myofibrillar troponin-trepomyosin regulatory system [2], have been noted on several occasions [3,4], leading to the suggestion that these two types of protein might be structurally and functionally related, and that they

Investigation of some physico-chemical properties of muscular parvalbumins by means of the luminescence of their phenylalanyl residues

The influence of pH, temperature and Ca2+-release on the phenylalanyl and tyrosyl fluorescene of muscular parvalbumins from white muscles of hake and carp has been investigated. Within the pH range from 7 to 8, Ca2+-saturated parvalbumins show a conformational change registered by fluorescence, that is associated with the release of some of the bound Ca2+. Removal of Ca2+ by means of EGTA (ethyleneglycolbis-(aminoethylether)tetra-acetic acid) considerably narrows the region of protein nativity, increases the accessibility of their chromophores to quencher ions (Cs+ and CNS-) and decreases their stability against heat denaturation. The usefulness of measurements of the phenylalanine fluorescence and of the tyrosine-phenylalanine energy transfer in the investigation of these and other proteins is discussed.