Preparation and characterization of the major isotype of parvalbumin from skeletal muscle of the toad (Bufo bufo japonicus)

The major isotype of parvalbumin has been isolated from the skeletal muscle of the toad, Bufo bufo japonicus. Unlike the skeletal muscle of every frog so far examined (Rana esculenta, Rana temporaria, and Rana catesbeiana), which contains two major isotypes of parvalbumins, toad skeletal muscle has been shown to contain only one isotype, but the content of parvalbumin in toad skeletal muscle was similar to the sum of those of the two isotypes in skeletal muscles of frogs. This feature of toad skeletal muscle is advantageous to clarify the physiological role of parvalbumin. The relative molecular mass of toad parvalbumin was estimated to be 12,200 by SDS-polyacrylamide gel electrophoresis. The isoelectric point was determined to be 4.81 by polyacrylamide gel isoelectric focusing. The amino acid composition indicated that toad parvalbumin corresponds to bullfrog (R. catesbeiana) pI 4.97 parvalbumin, showing that toad parvalbumin is genetically an alpha-parvalbumin. It was also revealed by the amino acid composition that toad parvalbumin is distinctly different from any of the parvalbumins from frogs. The ultraviolet spectrum of toad parvalbumin is consistent with its amino acid composition. The ultraviolet difference spectrum of the Ca2+-loaded form vs. the metal-free form indicates that some Phe residues in the toad parvalbumin molecule are affected by a conformational change associated with Ca2+ binding. On electrophoresis in polyacrylamide gel in 14 mM Tris and 90 mM glycine, the metal-free and Mg2+-loaded forms of toad parvalbumin migrated twice as fast as the Ca2+-loaded form.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of calcium binding proteins, parvalbumin and alpha-lactalbumin, with dipalmitoylphosphatidylcholine vesicles

Interactions of Ca2+ binding proteins, pike (Esox lucius) parvalbumins pI 4.2 and 5.0, and bovine and human alpha-lactalbumins, with dipalmitoylphosphatidylcholine vesicles were studied by means of scanning microcalorimetry and intrinsic tyrosine and tryptophan fluorescence methods. The interactions of pike parvalbumins are modulated by Ca2+ and Mg2+ binding to the protein and induce some changes in the physical properties of both the proteins and liposomes. Liposomes increased thermal stability of Ca2+-loaded parvalbumin and decreased thermal stability of both Mg2+-loaded and metal-free protein. The interaction of parvalbumin with liposomes affects the phase transition from gel to liquid-crystalline state in liposomes. Ca2+-loaded alpha-lactalbumin interacts with liposomes in its native state while the metal-free protein binds to the liposomes mainly in its thermally denatured state. The results of the microcalorimetric and spectrofluorometric studies are supported by data obtained by means of gel-chromatography on Sepharose 4B. It may be suggested that these metal-modulated interactions of Ca2+-binding proteins with membranes have some functional significance.     

Interaction of cupric ion with parvalbumin.

Cod parvalbumin, a calcium-binding protein, possesses a specific Zn2+ (or Cu2+) binding site per molecule. This work employed fluorescence energy transfer techniques to measure the distance between the Zn2+ (Cu2+) site and the stronger Ca(2+)-binding site in parvalbumin. Specifically, the distance between Tb3+ bound at the Ca2+ site and Co2+ bound to the Zn2+ (Cu2+) binding site was 10.3 +/- 0.9 A. Lastly, the effects of Cu2+ on the physico-chemical properties of parvalbumin were studied by measuring the accessibility of protein thiol groups to 5,5'-dithio bis(2-nitrobenzoic acid) and by its affinity for the fluorescent probe 4,4'-bis[1-(phenylamino)-8-naphthalene sulfonic acid] dipotassium salt. The thiol group accessibility decreased and the affinity to the fluorescent probe increased upon complexation of Cu2+ to the protein. It appears that the binding of Cu2+ converts parvalbumin to an apo-like state.     

Characterisation in vivo of the reactive thiol groups of the lactose permease from Escherichia coli and a mutant; exposure, reactivity and the effects of substrate binding

The reactivity and accessibility of the reactive thiol groups of the native lactose permease and a mutant have been studied in a number of circumstances and with a number of reagents, in particular using the specific thiol-disulphide exchange reaction. Seven different reactive states of the thiol in the native protein have been characterised by their different second-order rate constants. Interconversion between these states is dependent on the magnitude of the protonmotive force, pH and substrate binding. In the absence of galactoside, reactivity is controlled by an ionisation with apparent pKa 9.3. This pKa is not affected by the protonmotive force, but it is lowered in the presence of external galactoside. The conformation adopted by the permease when in equilibrium with saturating galactoside appears to be different from that of the intermediate that accumulates during net turnover. In the former state, the reactivity of the thiol group is depressed, whereas in the latter state it is enhanced. The thiol group of the native protein is buried in a hydrophobic environment that has a dielectric constant considerably lower than that of water. The environment is not greatly perturbed by changes in the magnitude of the protonmotive force, but it is affected by the binding of galactoside. In a strain which carries the YUN mutation (Wilson, T.H. and Kusch, M. (1972) Biochim. Biophys. Acta 255, 786-797), two reactive thiols were characterised. The more reactive of the two is more exposed than the thiol group of the native molecule and is in an environment that has a dielectric constant close to that of water. The less reactive thiol appears to be more deeply buried than that of the native protein. Thus the mutation appears to produce a conformation change in the central portion of the polypeptide chain that results in greater exposure of the reactive thiol to the aqueous environment.     

Ca-inhibited binding of melittin with parvalbumin. A new role for parvalbumins?

It was found that pike parvalbumins pI 4.2 and 5.0 bind amphiphilic peptide melittin extracted from bee venom in an extraordinary Ca-dependent manner: in apo-state the protein forms a tight equimolar complex with melittin (Ka = 10(6) M-1 at 18 degrees C); in Ca- (and Mg-) loaded state it does not take place. Heating of the protein up to temperatures above the denaturation temperature of apo-parvalbumin does not change the stoichiometry of the complex but increases its association constant by an order of magnitude (Ka = 1.2.10(7) M-1 at 44 degrees C). Isolated Ca-binding domain of parvalbumin, 38-108, retains the ability for Ca-inhibited binding of equimolar quantities of melittin. The possible function of parvalbumin in vivo is suggested: Ca-inhibited interactions with some intracellular components.     

The effect of calcium and magnesium ions on the interaction of calcium-binding proteins parvalbumin and alpha-lactalbumin with dipalmitoylphosphatidylcholine vesicles

Interactions of the calcium binding proteins, like parvalbumins pI 4.2 and p15.0 and bovine and human alpha-lactalbumins, with dipalmitoylphosphatidylcholine vesicles have been studied by means of scanning microcalorimetry and intrinsic tryptophan, tyrosine and phenylalanine fluorescence. The interactions are modulated by the Ca2+ and Mg2+ binding to the proteins and induce some changes in the physical properties of both the proteins and the liposomes. The liposomes increase the thermal stability of the Mg2+-loaded and metal-free parvalbumin. Ca2+-loaded alpha-lactalbumin interacts with the liposomes in its native state, while the metal-free protein binds to the liposomes mainly in its thermally denatured state. The interactions of both proteins with the liposomes affect the phase transition from gel to liquid-crystalline state in the liposomes. The results of the microcalorimetric and spectrofluorometric studies are corroborated by the data obtained by means of gel-chromatography on Sepharose 4B.     

Structural studies of metal-free metallothionein

We report the first molecular dynamics calculations on the structure of metal-free betaalpha recombinant human metallothionein, with comparison to the two isolated fragments, alpha-rhMT and beta-rhMT, starting from a linear synthesized strand as well as a demetallated conformation. Following a 5000 ps MM3/MD calculation, the cysteine side chains were found to populate the outside surface of the metal-free protein, regardless of the initial conformation. The polypeptide backbone adopted a random coil conformation when starting from the linear strand, however, it retained a significant amount of secondary structure when starting from the demetallated conformation. We propose that the inverted cysteinyl sulfur orientation facilitates the binding of the metal ions to form the proteolytically stable, metallated protein.     

Ca2+-binding parvalbumin in rat testis. Characterization, localization, and expression during development

Parvalbumin, a Ca2+-binding protein, was isolated from rat testis. This is the first demonstration of the protein in endocrine glands. By using a rat parvalbumin cDNA probe, parvalbumin mRNA was demonstrated in the testis, indicating that the protein is synthesized in this tissue and that testis parvalbumin is a product of the same gene as the one encoding for muscle parvalbumin. Parvalbumin was localized by immunohistochemical methods in the Leydig cells and in the acrosome region of maturing spermatids (stages 1-15). The expression of parvalbumin during testis development was followed. High parvalbumin protein and mRNA levels were found at stages of highest Leydig cell activity, i.e. at late fetal stages until birth and again around postnatal day 50. This suggests that parvalbumin may be involved in the production of testosterone in Leydig cells, a process which is highly dependent on calcium.     

Remodeling of the AB site of rat parvalbumin and oncomodulin into a canonical EF-hand.

Parvalbumin (PV) and the homologous protein oncomodulin (OM) contain three EF-hand motifs, but the first site (AB) cannot bind Ca2+. Here we aimed to recreate the putative ancestral proteins [D19-28E]PV and [D19-28E]OM by replacing the 10-residue-long nonfunctional loop in the AB site by a 12-residue canonical loop. To create an optical conformational probe we also expressed the homologs with a F102W replacement. Unexpectedly, in none of the proteins did the mutation reactivate the AB site. The AB-remodeled parvalbumins bind two Ca2+ ions with strong positive cooperativity (nH = 2) and moderate affinity ([Ca2+]0.5 = 2 microM), compared with [Ca2+]0.5 = 37 nM and nH = 1 for the wild-type protein. Increasing Mg2+ concentrations changed nH from 2 to 0.65, but without modification of the [Ca2+]0. 5-value. CD revealed that the Ca2+ and Mg2+ forms of the remodeled parvalbumins lost one-third of their alpha helix content compared with the Ca2+ form of wild-type parvalbumin. However, the microenvironment of single Trp residues in the hydrophobic cores, monitored using intrinsic fluorescence and difference optical density, is the same. The metal-free remodeled parvalbumins possess unfolded conformations. The AB-remodeled oncomodulins also bind two Ca2+ with [Ca2+]0.5 = 43 microM and nH = 1.45. Mg2+ does not affect Ca2+ binding. Again the Ca2+ forms display two-thirds of the alpha-helical content in the wild-type, while their core is still strongly hydrophobic as monitored by Trp and Tyr fluorescence. The metal-free oncomodulins are partially unfolded and seem not to possess a hydrophobic core. Our data indicate that AB-remodeled parvalbumin has the potential to regulate cell functions, whereas it is unlikely that [D19-28E]OM can play a regulatory role in vivo. The predicted evolution of the AB site from a canonical to an abortive EF-hand may have been dictated by the need for stronger interaction with Mg2+ and Ca2+, and a high conformational stability of the metal-free forms.     

Comparison of metal ion-induced conformational changes in parvalbumin and oncomodulin as probed by the intrinsic fluorescence of tryptophan 102

The calcium-induced conformational changes of the 108-amino acid residue proteins, cod III parvalbumin and oncomodulin, were compared using tryptophan as a sensitive spectroscopic probe. As native oncomodulin is devoid of tryptophan, site-specific mutagenesis was performed to create a mutant protein in which tryptophan was placed in the identical position (residue 102) as the single tryptophan residue in cod III parvalbumin. The results showed that in the region probed by tryptophan-102, cod III parvalbumin experienced significantly greater changes in conformation upon decalcification compared to the oncomodulin mutant, F102W. Addition of 1 eq of Ca2+ produced greater than 90% of the total fluorescence response in F102W, while in cod III parvalbumin, only 74% of the total was observed. Cod III parvalbumin displayed a negligible response upon Mg2+ addition. In contrast, F102W did respond to Mg2+, but the response was considerably less when compared to Ca2+ addition. Time-resolved fluorescence showed that the tryptophan in both proteins existed in at least two conformational states in the presence of Ca2+ and at least three conformational states in its absence. Comparison with quantum yield measurements indicated that the local electronic environment of the tryptophan was significantly different in the two proteins. Collectively, these results demonstrate that both cod III parvalbumin and oncomodulin undergo Ca2(+)-specific conformational changes. However, oncomodulin is distinct from cod III parvalbumin in terms of the electronic environment of the hydrophobic core, the magnitude of the Ca2(+)-induced conformational changes, and the number of calcium ions required to modulate the major conformational changes.     

Developmental and Functional Studies of Parvalbumin and Calbindin D28K in Hypothalamic Neurons Grown in Serum-Free Medium

The Ca2+-binding proteins parvalbumin (Mr = 12K) and calbindin D28K [previously designated vitamin D-dependent Ca2+-binding protein (Mr = 28K)] are neuronal markers, but their functional roles in mammalian brain are unknown. The expression of these two proteins was studied by immunocytochemical methods in serum-free cultures of hypothalamic cells from 16-day-old fetal mice. Parvalbumin is first detected in all immature neurons, but during differentiation, the number of parvalbumin-immunoreactive neurons greatly declines to a level reminiscent of that observed in vivo, where only a subpopulation of neurons stains for parvalbumin. In contrast, calbindin D28K was expressed throughout the period investigated only in a distinct subpopulation of neurons. Depolarization of fully differentiated hypothalamic neurons in culture resulted in a dramatic decrease of parvalbumin immunoreactivity but not of calbindin D28K immunoreactivity. The parvalbumin staining was restored on repolarization. Because the anti-parvalbumin serum seems to recognize only the metal-bound form of parvalbumin, the loss of immunoreactivity may signal a release of Ca2+ from intracellular parvalbumin during depolarization of the cells. We suggest that parvalbumin might be involved in Ca2+-dependent processes associated with neurotransmitter release.     

The conformations of the manganese transport regulator of Bacillus subtilis in its metal-free state

The manganese transport regulator (MntR) from Bacillus subtilis binds cognate DNA sequences in response to elevated manganese concentrations. MntR functions as a homodimer that binds two manganese ions per subunit. Metal binding takes place at the interface of the two domains that comprise each MntR subunit: an N-terminal DNA-binding domain and a C-terminal dimerization domain. In order to elucidate the link between metal binding and activation, a crystallographic study of MntR in its metal-free state has been undertaken. Here we describe the structures of the native protein and a selenomethionine-containing variant, solved to 2.8 A. The two structures contain five crystallographically unique subunits of MntR, providing diverse views of the metal-free protein. In apo-MntR, as in the manganese complex, the dimer is formed by dyad-related C-terminal domains that provide a conserved structural core. Similarly, each DNA-binding domain largely retains the folded conformation found in metal bound forms of MntR. However, compared to metal-activated MntR, the DNA-binding domains move substantially with respect to the dimer interface in apo-MntR. Overlays of multiple apo-MntR structures indicate that there is a greater range of positioning allowed between N and C-terminal domains in the metal-free state and that the DNA-binding domains of the dimer are farther apart than in the activated complex. To further investigate the conformation of the DNA-binding domain of apo-MntR, a site-directed spin labeling experiment was performed on a mutant of MntR containing cysteine at residue 6. Consistent with the crystallographic results, EPR spectra of the spin-labeled mutant indicate that tertiary structure is conserved in the presence or absence of bound metals, though slightly greater flexibility is present in inactive forms of MntR.     

Conformation of complexes of thiamin pyrophosphate with divalent cations as studied by nuclear magnetic resonance spectroscopy.

The binding of Ni-2+ and Mn-2+ to thiamin phosphate and thiamin pyrophosphate (thiamin-PP) has been compared with the binding of these ions to oxythiamin phosphate and oxythiamin pyrophosphate, analogues of thiamin in which the C-4 amino group has been replaced by an -OH group. The replacement of the NH2 group results in reduced basicity of N-1 of the pyrimidine ring of oxythiamine derivatives. The effects of pD, ligand concentration, and temperature on the binding of metal ions to N-1 have been studied by observing the metal ion-induced shifting and broadening of the C-6-H signal of these compounds. The results indicate the following: (a) the metal ion is held near N-1, resulting in a "folded" conformation, because of a favorable bonding interaction between N-1 and the metal ion rather than for general conformational reasons alone; and (b) the amount of "folded" conformation present in the different pyrophosphate complexes at neutral pH follows the order: Ni-2+-thiamin-PP greater than Mn-2+-thiamin-PP greater than Mn-2+-oxythiamin-PP and Ni-2+-oxythiamin-PP It is concluded that the strength of the metal ion-pyrimidine interaction in the "folded" conformation depends strongly both on the coordination affinity of the metal ion and on the basicity of N-1. Since the interaction of the phosphate-bound metal ion with the pyrimidine ring in the Mg-2+-thiamin-PP complex is probably weaker than the corresponding interaction in the Mn-2+-thiamin-PP complex, these results predict that the Mg-2+-thiamin-PP complex in solution, at neutral pH, exists predominantly in an "unfolded" conformation.     

Electrospray ionization mass spectrometry: analysis of the Ca2+-binding properties of human recombinant alpha-parvalbumin and nine mutant proteins

A set of 10 different recombinant human parvalbumins was used to establish a method for the investigation of the Ca2+-binding properties of proteins by electrospray ionization mass spectrometry (ESI-MS). Human PVWT was found to bind 2 mol Ca2+ ions/mol of protein, whereas its mutants (PVE101V, PVD90A, PVE62V, PVD51A, PVD90A,E101V, PVE62V,E101V, PVD51A,D90A, PVD51A,E62V, PVD51A,E62V, D90A,E101V) containing inactivating substitutions in the Ca2+-binding loops bind 0 or 1 Ca2+ ion per protein molecule, depending on the degree of inactivation. These findings fully agree with previously reported results obtained by flow dialysis experiments. The RP-HPLC desalted metal-free proteins were analyzed in 10 mM ammonium acetate at pH 7.0. The experimental conditions were optimized with the recombinant parvalbumin test system before analyzing the Ca2+-binding properties of rat and murine parvalbumins in muscle tissue extracts. ESI-MS revealed that (i) rat and murine alpha-parvalbumins each bind specifically two Ca2+ ions per protein molecule and (ii) both extracted parvalbumins were found to be posttranslationally modified; each protein is acetylated at the N-terminus. Finally, during our investigations of the murine parvalbumin a sequencing error was detected at the C-terminus where the amino acid at position 109 is Ser and not Thr as mentioned in the SwissProt data base (Accession No. P32848). This work demonstrates the great potential of the ESI-MS technique as a sensitive, specific, and rapid method for direct identification and determination of the stoichiometry of Ca2+-binding proteins and other metalloproteins.     

Metal-dependent protein folding: metallation of metallothionein

Metallothionein (MT), a low molecular mass, cysteine-rich protein, is a model system for metal ion-directed folding due to its diverse metal binding properties. In this minireview, the current status of theoretical and experimental studies that have focused primarily on the initial metallation steps involving the metal-free, or apo, MT and divalent metals, Zn²⁺ and Cd²⁺ is described. Apo-MT has recently been reported to be present in the cell in quantities equal to that of the metallated protein, which might indicate a potential role for the protein in the absence of metals. Molecular mechanics-molecular dynamics (MM3/MD) calculations carried out on the demetallation of cadmium-coordinated MT isoform 1a indicate structural stability of the metal-free protein with significant retention of the backbone conformation imposed by the metal-thiolate clusters present in the metallated holo-protein. Significantly, the cysteinyl sulfurs were found inverted to the outside of a quite compact sphere. In contrast, MM3/MD calculations of apo-MT starting from a linear strand did not possess any structural stability and can be described as a random coil conformation. Evidence for the sequence of metallation is discussed, together with current experimental data to support either a cooperative or sequential binding mechanisms.     

Calmodulin from neurospora crassa. General properties and conformational changes

Calmodulin from Neurospora crassa has been purified to electrophoretic homogeneity. Equilibrium gel filtration experiments suggest that its Ca-binding properties are indistinguishable from those of vertebrate calmodulins. The isoelectric point of 4.04 and electrophoretic behavior under nondenaturing conditions indicate that N. crassa calmodulin is slightly less acidic than its vertebrate counterpart. The amino acid composition is typical of plant calmodulins with the exception that trimethyllysine is absent and that the content of Ser is unusually high. The tryptic peptide map of N. crassa calmodulin reveals an important number of point mutations as compared to vertebrate calmodulin. Differences in primary structure may explain why N. crassa calmodulin is less potent in the activation of myosin light chain kinase than calmodulins from higher organisms. The far UV circular dichroic spectra of the Ca-, Mg-, and metal-free forms of N. crassa calmodulin are similar to those of vertebrate calmodulin; in contrast, the near UV circular dichroic spectra are very different, apparently due to the differences in Tyr content. The single Tyr residue of N. crassa calmodulin, presumably located in position 138, undergoes an inversion of optical chirality upon addition of Ca2+, but not of Mg2+, to the metal-free protein.     

The maintenance of the energized membrane state and its relation to active transport in Escherichia coli.

1. An ATPase mutant of Escherichia coli and two partial revertants of that mutant were examined for the ability to generate a high energy membrane state with D-lactate or ATP, as measured by the quenching of the fluorescent dye quinacrine. 2. All three strains showed reductions in the aerobically-driven quenching of fluorescence compared to the wild type, but the reduction could be reversed by the addition of eitherN,N'-dicyclohexylcarbodiimide or the crude soluble ATPase of the wild type. 3. The mutant exhibited a decreased ability to accumulate sugars and amino acids and showed an increased permeability to protons. 4. One partial revertant showed a slight increase in active transport and a slight decrease in proton permeability. 5. The other partial revertant showed a large increase in transport ability and a large decrease in proton permeability. 6. A model is proposed in which the conformation of the Mg-2+-ATPase is important in the utilization of energy derived from the electron transport chain and this function is independent of the catalytic activity of the Mg-2+-ATPase.     

Conformational differences in myosin, IV.[1-3] Radioactive labeling of specific thiol groups as influenced by ligand binding.

Changes in the mono- and divalentcation-stimulated ATPase activities of myosin progressively labeled with N-ethyl-[2,3-14C2]-maleimide were used to classify the readily reacting thiol groups into 3 types. The results show that one thiol-1 and one thiol-2 group are associated with each of the 2 active sites of myosin. Concentrations of KCl higher than 0.4M and/or temperatures above 10 degrees C lead to exposure of a variable number of thiol groups of a third class not affecting the enzymic properties. Although modification of thiol groups itself results in changes in structure and function of the protein, the patterns of incorporation of N-ethyl-[14C2]-malemide under various conditions of temperature, ionic strength and ligands bound to the protein revealed 9 different conformations of intact myosin. These were distinguished on the basis of the relative reactivity of the 3 different classes of thiol groups. The sequence of blockage of thiol groups reveals that cooperativity between the 2 active sites is induced by binding of a magnesium nucleotide complex to the protein. In the conformation of the long-lived myosin-product intermediate occuring during hydrolysis of Mg-ATP at 25 degrees C, 4 thiol groups of the third class react as well as or even more readily than those of the first and second classes.