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.     

Site-specific replacement of amino acid residues within the CD binding loop of rat oncomodulin

Relative to the same site in oncomodulin, the CD ion-binding domain of rat parvalbumin exhibits much greater affinity for Ca2+ and Mg2+. As part of an effort to understand the structural basis for these differences, site-specific variants of oncomodulin have been prepared in which the amino acid residues at positions 52, 54, 57, 59, and 60 have been replaced with the residues present at the corresponding positions in rat parvalbumin. The proteins resulting from the single-site substitutions at residues 52, 54, and 57 are indistinguishable from the wild-type protein on the basis Eu3+ luminescence spectroscopy, and none of the three variants displays increased affinity for Ca2+. By contrast, the substitutions at residues 59 and 60 perturb both the Eu3+ luminescence parameters and the Ca2+ and Mg2+ affinities, and these differences are amplified when both replacements are simultaneously incorporated into the protein. The Eu3+ 7F0----5D0 spectrum of the double variant (D59E/G60E) at pH 5.0, with a maximum at 5796 A and pronounced shoulder at 5791 A, strongly resembles that obtained with pike parvalbumin. Consistent with this increased parvalbumin-like character, KCa is decreased from 0.78 microM (for the wild-type protein) to 0.41 microM, and KMg is decreased from 3.5 to 0.74 mM. Nevertheless, the affinity of the CD ion-binding domain in D59E/G60E for Ca2+ remains almost 2 orders of magnitude lower than the corresponding site in rat parvalbumin, strongly suggesting that residues besides those present in the binding loop are involved in dictating the metal ion-binding properties of the oncomodulin CD site.     

Interactions between residues in the oncomodulin CD domain influence Ca2+ ion-binding affinity

Despite striking sequence homology with rat parvalbumin, oncomodulin exhibits much lower affinity for Ca2+ ion. We are attempting to identify the structural basis for this difference by systematically substituting the parvalbumin residue for the oncomodulin residue at points of nonidentity. In this paper, we examine two mutations in the helical segments flanking the CD ion-binding loop. Replacement of Asp-45 in the C helix by lysine, to produce D45K, reduces the dissociation constant for Ca2+ at the CD site from 0.81 to 0.53 microM. Replacement of Lys-69 in the D helix by glycine, to afford K69G, similarly reduces KCa to 0.59 microM. Both mutations perturb the Eu3+ 7Fo----5Do spectral parameters. We also examine the consequences of simultaneous mutations involving positions 57, 59, 60, and 69. Ca(2+)-binding assays and Eu3+ luminescence measurements indicate that there is a conformational interaction between residues 57 and 69 and that this interaction is modulated by residues 59 and 60. When the mutations at positions 57, 59, 60, and 69 are combined, the resulting variant exhibits a KCa value for the CD site of 0.25 microM, reflecting a 3-fold increase in affinity relative to the wild-type protein. Moreover, the pK alpha governing the interconversion of low and high pH forms of the Eu3+ 7Fo----5Do spectrum is increased to 8.1, very close to the value of 8.25 determined previously for rat parvalbumin. In this paper, we also complete our survey of single mutations in the CD loop by examining L58I. Replacement of Leu-58 by isoleucine reduces the affinity of the CD site for Ca2+, raising KCa to 2.2 microM. Finally, we revise our previous estimate of the KCa value for Y57F downward, from 0.80 to 0.64 microM. The earlier result is believed to have been inflated by heterogeneity in the preparation, a consequence of proteolysis.     

Circularly polarized luminescence from terbium(III) as a probe of metal ion binding in calcium-binding proteins.

A number of different experimental techniques have been used to probe the details of structural changes on the binding of Ca(II) to the large number of known calcium-binding proteins. The use of luminescent lanthanide(III) ions, especially terbium(III) and europium(III), as substitutional replacement for calcium(II), has led to a number of useful experiments from which important details concerning the metal ion coordination sites have been obtained. This work is concerned with the measurement of the circularly polarized luminescence (CPL) from the 5D4----7F5 transition of Tb(III) bound to the calcium binding sites of bovine trypsin, bovine brain calmodulin, and frog muscle parvalbumin. It is demonstrated that it is possible to make these polarization measurements from very dilute solutions (less than 20 microM) and monitor structural changes as equivalents of Tb(III) are added. It is shown that the two proteins that belong to the class of "EF-hand" structures (calmodulin and parvalbumin) possess quite similar CPL line shapes, whereas Tb(III) bound to trypsin has a much different band structure. CPL results following competitive and consecutive binding of Ca(II) and Tb(III) bound to calmodulin are also reported and yield information concerning known differences between the sequence of binding of these two species.     

Terbium as a luminescent probe of metal-binding sites in protein kinase C

In the present report, we demonstrate that Tb3+ binds to protein kinase C and serves as a luminescent reporter of certain cationic metal-binding sites. Tb3+ titration of 50 nM protein kinase C results in a 20-fold enhancement of Tb3+ luminescence which is half-maximal at 12 microM Tb3+. A Kd of approximately 145 nM was determined for Tb3+ binding to the enzyme. The excitation spectrum of bound Tb3+ exhibits a peak at 280 nm characteristic of energy transfer from protein tryptophan or tyrosine residues. The luminescence of this complex can be markedly decreased by other metals, including Pb2+ (IC50 = 25 microM), La3+ (IC50 = 50 microM), Hg2+ (IC50 = 300 microM), Ca2+ (IC50 = 6 mM), and Zn2+ (IC50 greater than 10 mM), and chelation of Tb3+ by 2 mM EGTA. Tb3+ binding to protein kinase C is correlated with its inhibition of protein kinase activity (IC50 = 8 microM), r = 0.99) and phorbol ester binding (IC50 = 15 microM, r = 0.98). Tb3+ inhibition of protein kinase C activity cannot be overcome by excess Ca2+, but can be partially overcome with excess phosphatidylserine or by chelation of Tb3+ with EGTA. Tb3+ noncompetitively inhibits phorbol ester binding by decreasing the maximal extent of binding without significantly altering binding affinity. The results suggest that the Tb3(+)-binding site is at or allosterically related to the enzyme's phosphatidylserine-binding site, but is distinct from the phorbol ester-binding domain and the Ca2(+)-binding site that regulates enzyme activity.     

Oncomodulin and parvalbumin. A comparison of their interactions with europium ion

The 7F0----5D0 transition of Eu3+ was used to probe the metal-binding domains of rat oncomodulin and rat parvalbumin. Two distinct differences between the two proteins were observed. The first relates to the pH-dependent behavior of their 7F0----5D0 spectra, a phenomenon noted previously for other paravalbumins. In the case of rat parvalbumin, the spectral features associated with both metal-binding sites titrate concomitantly (pK alpha = 8.2); however, in the case of oncomodulin, the two sites titrate sequentially (pK alpha = 6.3 for the CD site; pK alpha = 8.3 for EF site). The proteins also contrast with regard to their discrimination for Eu3+ over Ca2+. The CD and EF sites in rat parvalbumin both display a large preference for Eu3+: (KCa/KEu)CD = 143 +/- 11 and (KCa/KEu)EF = 191 +/- 30. However, in the case of oncomodulin, although the EF site of oncomodulin greatly prefers the trivalent lanthanide ion (KCa/KEu = 300 +/- 80), the CD site exhibits a relatively minor preference (KCa/KEu = 11 +/- 1).     

An interdomain distance in cardiac troponin C determined by fluorescence spectroscopy

The distance between Ca2+-binding site III in the C-terminal domain and Cys35 in the N-terminal domain in cardiac muscle troponin C (cTnC) was determined with a single-tryptophan mutant using bound Tb3+ as the energy donor and iodoacetamidotetramethylrhodamine linked to the cysteine residue as energy acceptor. The luminescence of bound Tb3+ was generated through sensitization by the tryptophan located in the 12-residue binding loop of site III upon irradiation at 295 nm, and this sensitized luminescence was the donor signal transferred to the acceptor. In the absence of bound cation at site II, the mean interdomain distance was found to be 48-49 A regardless of whether the cTnC was unbound or bound to cardiac troponin I, or reconstituted into cardiac troponin. These results suggest that cTnC retains its overall length in the presence of bound target proteins. The distribution of the distances was wide (half-width >9 A) and suggests considerable interdomain flexibility in isolated cTnC, but the distributions became narrower for cTnC in the complexes with the other subunits. In the presence of bound cation at the regulatory site II, the interdomain distance was shortened by 6 A for cTnC, but without an effect on the half-width. The decrease in the mean distance was much smaller or negligible when cTnC was complexed with cTnI or cTnI and cTnT under the same conditions. Although free cTnC has considerable interdomain flexibility, this dynamics is slightly reduced in troponin. These results indicate that the transition from the relaxed state to an activated state in cardiac muscle is not accompanied by a gross alteration of the cTnC conformation in cardiac troponin.     

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.     

The binding of terbium ions to gelsolin reveals two classes of metal ion binding sites.

Spectroscopically active terbium ions have been used to probe the Ca2+ ion-binding sites on human plasma gelsolin. The luminescence of Tb3+ ions bound to gelsolin is markedly enhanced when excited indirectly at 295 nm due to F?rster type dipole-dipole energy transfer from neighboring tryptophan residues. Titration of this luminescence with increasing concentrations of Tb3+ ions was saturable although the shape of this titration curve was complex indicating the involvement of multiple classes of sites. Luminescence lifetime measurements (obtained by indirect excitation at 295 nm) demonstrate the presence of two classes of sites characterized by a major lifetime of 1.0-1.1 ms and a minor lifetime of 0.7-0.8 ms. However, while the amplitude of the minor lifetime showed a hyperbolic dependence on the Tb3+ ion concentration, the amplitude of the major lifetime showed a strongly sigmoidal dependence. Different classes of Tb3+ ion binding sites can also be distinguished by the different Ca2+ ion concentrations needed to displace Tb3+ ions from these sites on gelsolin. It is proposed that the occupancy of one class of Tb3+ ion binding sites on gelsolin causes a conformational change in gelsolin which then allows a second class of cryptic Tb3+ ion binding sites to be expressed. The implications of these results in terms of the binding of Ca2+ ions to gelsolin and the regulation of the activities of gelsolin by calcium are discussed.     

Lanthanide-binding properties of rat oncomodulin

Oncomodulin, the parvalbumin-like calcium-binding protein frequently expressed in tumor tissue, was isolated from Morris hepatoma 5123tc and studied using the luminescent lanthanide ions, Eu3+ and Tb3+. Titrations of the apoprotein - whether monitored by indirect excitation of bound Tb3+, by direct laser excitation of bound Eu3+, or by quenching of the intrinsic tyrosine fluorescence - all indicated the presence of two high-affinity binding sites for lanthanide ions, as in parvalbumin. Moreover, the appearance of the Eu3+ 7F0----5D0 excitation spectrum of Eu2-oncomodulin was found to be highly pH-dependent, as previously observed with parvalbumin. At pH 5.0, it consists of a single peak centered at 5796 A, having a linewidth of approximately 6 A. At higher pH values, this spectrum is replaced by a broader, more symmetric peak at 5782 A. Oncomodulin, however, was found to differ from parvalbumin in at least one important respect: In contrast to the muscle-associated protein, the affinities of the CD site in oncomodulation for Tb3+ and Ca2+ were found to be rather similar, with KCa/KTb approximately equal to 11 +/- 2.     

Terbium(III) luminescence study of the spatial relationship of tryptophan residues to the two metal ion binding sites of Escherichia coli glutamine synthetase.

The luminescence of Tb(III) was used to explore the topography of the metal ion sites of Escherichia coli glutamine synthetase and the relationship between these sites and tryptophan residues of the enzyme. By irradiation of tryptophan residues at 295 nm and measurement of the resulting Tb(III) luminescence at 544 nm, a biphasic curve was obtained upon titrating apoenzyme with Tb(III) indicating sequential binding of Tb(III) ions to the two binding sites of glutamine synthetase. The luminescence intensity was greater in the second region of the titration curve which is mostly due to energy transfer from Trp-158 to the second Tb(III) binding site of the enzyme. By use of the F?rster equation for energy transfer from donor Trp to acceptor Tb(III), distances from Trp-57 to Tb(III) at the n1 and n2 sites were calculated, by using a mutant enzyme in which Trp-158 was replaced by Ser, to be 16.4 and 15.7 A, respectively; distances from Trp-158 to Tb(III) at the n1 and n2 sites were calculated, by using a mutant enzyme in which Trp-57 was replaced by Leu, to be 16.8 and 9.5 A, respectively. All the distances are in reasonably good agreement with the crystal structure distances from Salmonella typhimurium glutamine synthetase except the distance from Trp-158 to the second Tb(III) binding site. The discrepancies may result from a slightly different conformation of glutamine synthetase in solution and in the crystal and/or a slightly different conformation for trivalent Ln(III) binding compared to divalent Mn(II) binding.     

A comparative spectroscopic study of tryptophan probes engineered into high- and low-affinity domains of recombinant chicken troponin C.

The spectral properties of three tryptophan-substituted mutants of recombinant chicken troponin C are compared. Site-specific mutagenesis was used to introduce a tryptophan residue into the high-affinity (Ca2+/Mg2+) domain of troponin C at residue position 105, thereby creating the mutant phenylalanine-105 to tryptophan (F105W). The spectral properties of F105W and a double mutant, F29W/F105W, were compared with the mutant phenylalanine-29 to tryptophan (F29W). Since wild-type chicken troponin C does not naturally contain either tyrosine or tryptophan residues, the tryptophan substitutions behaved as site-specific reporters of metal ion binding and conformational change. The residues that occupy positions 29 and 105 are at homologous locations in low-affinity and high-affinity domains, preceding the first liganding residues of binding loops I and III, respectively. Mutant proteins were examined by a combination of absorbance and fluorescence methods. Calcium induced significant changes in the near-UV absorbance spectra, fluorescence emission spectra, and far-UV circular dichroism of all three mutant proteins. Magnesium induced significant changes in the spectral properties of only F105W and F29W/F105W proteins. Tryptophan substitutions allowed Ca(2+)-specific and Ca(2+)/Mg(2+) sites to be titrated independently of one another. Results indicate that there is no interaction between the two binding domains under conditions where troponin C is isolated from the troponin complex. Magnesium-induced changes in the environment of the tryptophan reporter at position 105 were significantly different from those induced by calcium. This suggests that calcium and magnesium differ in their influence on the conformation of the high-affinity, Ca(2+)/Mg(2+) sites.     

A new intrinsic fluorescent probe for proteins Biosynthetic incorporation of 5-hydroxytryptophan into oncomodulin

The tryptophan analog, 5-hydroxytryptophan (5HW), has a significant absorbance between 310–320 nm, which allows it to act as an exclusive fluorescence probe in protein mixtures containing a large number of tryptophan residues. Here for the first time a method is reported for the biosynthetic incorporation of 5HW into an expressed protein, the Y57W mutant of the Ca²⁺ binding protein, oncomodulin. Fluorescence anisotropy and time-resolved fluorescence decay measurements of the interaction between anti-oncomodulin antibodies and the 5HW-incorporated oncomodulin conveniently provide evidence of complex formation and epitope identification that could not be obtained with the natural amino acid. This report demonstrates the significant potential for the use or 5HW as an intrinsic probe in the study of structure and dynamics of protein—protein interactions.     

A luminescent affinity tag for proteins based on the terbium(III)-binding peptide

Genetically encoded tags attached to proteins of interest are widely exploited for proteome analysis. Here, we present Tb(3+)-binding peptides (TBPs) which can be used for both luminescent measurements and affinity purification of proteins. TBPs consist of acidic amino acid residues and tryptophan residues which serve as Tb(3+)-binding sites and sensitizers for Tb(3+) luminescence, respectively. The Tb(3+) complexes of TBPs fused to a target protein exhibited luminescence characteristic of Tb(3+) by excitation of the tryptophan residue, and fusion proteins fused to one of the TPBs were successfully isolated from Escherichia coli cell lysate by affinity chromatography with a Tb(3+)-immobilized solid support.     

Localization of a fibrinogen calcium binding site between gamma-subunit positions 311 and 336 by terbium fluorescence

Calcium is required for effective fibrin polymerization. The high affinity Ca2+ binding capacity of fibrinogen was directly localized to the gamma-chain by autoradiography of nitrocellulose membrane blots of fibrinogen subunits incubated with 45Ca2+. Terbium (Tb3+) competitively inhibited 45Ca2+ binding to fibrinogen during equilibrium dialysis, accelerated fibrin polymerization, and limited fibrinogen fragment D digestion by plasmin. The intrinsic fluorescence of Ca2+-depleted fibrinogen was maximally enhanced by Ca2+ and Tb3+, but not by Mg2+, at about 3 mol of cation/mol of fibrinogen. Protein-bound Tb3+ fluorescence at 545 nm was maximally enhanced by resonance energy transfer from tryptophan (excitation at 290 nm) at about 2 mol of Tb3+mol of fibrinogen and about 1 mol of Tb3+/mol of plasmic fragment D94 (Mr 94,000). Fibrinogen fragments D78 (Mr 78,000) and E did not show effective enhancement of Tb3+ fluorescence, suggesting that the Ca2+ site is located within gamma 303 to gamma 411, the peptide which is absent in fragment D78 but present in D94. When CNBr fragments of the carboxyamidated gamma-subunit were assayed for enhancement of Tb3+ fluorescence, peptide CBi (gamma 311-336) bound 1 mol of Tb3+/mol of CBi. Thus, the Ca2+ site is located within this peptide. The sequence between gamma 315 and gamma 329 is homologous to the calmodulin and parvalbumin Ca2+ binding sites.     

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.     

Site-specific substitution of glutamate for aspartate at position 59 of rat oncomodulin

Replacement of the aspartate residue at position 59 of rat oncomodulin by glutamate by oligonucleotide-directed mutagenesis has afforded a protein which more closely resembles rat parvalbumin, at least judged by its interaction with the luminescent lanthanide ion Eu3+. The single-peak 7F0----5D0 spectrum observed at pH 5.0 with the fully bound wild-type protein is replaced by one which clearly shows two features at 5791 and 5796 A, arising from Eu3+ ions bound at the CD and EF sites, respectively. Furthermore, the pH dependence of the spectrum is substantially altered; the pKa observed for the CD domain, in which aspartate 59 residues, is shifted upward from pH 6.0 for the wild-type recombinant protein to pH 6.8 in the D59E mutant. Moreover, the maximum in the high-pH spectrum is shifted from 5781 to 5784 A. All three changes are indicative of a CD binding domain having increased parvalbumin-like character. Interestingly, however, the D59E substitution has only a modest effect on the Ca2+- and Mg2+-binding properties of the CD domain. For the wild-type protein, KCa = 7.8 x 10(-7) M and KMg = 3 x 10(-3) M. These affinities are more than an order of magnitude weaker than those seen for various parvalbumins and substantiate previous claims for calcium specificity made for the oncomodulin CD domain. Replacement of aspartate 59 by glutamate resulted in minor increases in affinity of the CD domain for Ca2+ (KCa = 5.5 x 10(-7) M) and Mg2+ (KMg = 1 x 10(-3) M). These findings strongly suggest that residues in oncomodulin besides aspartate 59 are important determinants of the observed calcium specificity of the CD calcium-binding domain. The consequences of the substitution at residue 59 appear to be confined to the CD domain. For the EF site in wild-type recombinant oncomodulin, KCa = 4.2 x 10(-8) M and KMg = 1.6 x 10(-4) M. The corresponding values for the D59E site-specific variant are identical within experimental error (KCa = 4.2 x 10(-8) M and KMg = 1.8 x 10(-4) M).     

Luminescence studies of Tb3+ bound to the high affinity sites of the Ca2+-ATPase of sarcoplasmic reticulum

Direct excitation of lanthanide luminescence with a pulsed dye laser has been used to probe the molecular environment of the high affinity sites of the sarcoplasmic reticulum Ca2+-ATPase. The direct excitation spectrum of Tb3+ bound to these sites has been determined and a luminescence lifetime of approximately 1 ms measured. Measurements of the difference in lifetime of the Tb X ATPase complex in H2O and D2O indicate that there are approximately 2 H2O molecules in the first coordination sphere of Tb3+ bound at the high affinity sites of the ATPase. The results are compared with the properties of Tb3+ binding to high affinity sites of other Ca2+ binding proteins. The binding constant of Tb3+ to the ATPase is in the range of 0.3-5.0 X 10(8) M-1 as inferred from the KI for inhibition of ATP hydrolysis, in agreement with a previous report (Highsmith, S. R., and Head, M. R. (1983) J. Biol. Chem. 258, 6858-6862). The values of the Ca2+ binding constant (approximately 2 X 10(6) M-1) and the cooperative nature (n = 1.9) of Ca2+ protection of Tb3+ inhibition indicate that Tb3+ and Ca2+ compete for the high affinity sites of the ATPase. The results demonstrate that directly-excited Tb3+ luminescence provides unique information on the environment of the Ca2+ binding-transport sites of the SR ATPase.     

Absorption and fluorescence spectroscopic studies of the Ca2(+)-dependent lipid binding protein p36: the annexin repeat as the Ca2+ binding site

The existence of a single tryptophan residue in the protein p36, a member of a recently characterized family of Ca2+ binding proteins called annexins, is exploited to provide unique spectroscopic information on the annexin repeat motif and its role in Ca2+ binding. The differences in ultraviolet absorption and fluorescence excitation upon Ca2+ binding are interpreted solely in terms of this tryptophan, which, in view of the pronounced blue-shifts and the presence of vibronic structure, seems to reside in a highly nonpolar environment. The fluorescence emission from the protein is correspondingly blue-shifted, and it is found to transfer energy in resonance with Tb3+ absorption lines in the near-ultraviolet. This effect allows us to locate the Tb3+ and, by implication, the Ca2+ binding site to within ca. 8 A of the tryptophan residue.     

Structure of oncomodulin refined at 1.85 A resolution. An example of extensive molecular aggregation via Ca2+

The crystal structure of oncomodulin, a 12,000 Mr protein isolated from rat tumours, has been determined by molecular replacement using the carp parvalbumin structure as a starting model. Refinement was performed by cycles of molecular fitting and restrained least-squares, using area-detector intensity data to 1.85 A resolution. For the 5770 reflections in the range 6.0 to 1.85 A, which were used in the refinement, the crystallographic R-factor is 0.166. The refined model includes residues 2 to 108, three Ca2+ and 87 water molecules per oncomodulin molecule. The oncomodulin backbone is closely related to that of parvalbumin; however, some differences are found after a least-squares fit of the two backbones, with root-mean-square (r.m.s.) deviations of 1 to 2 A in residues 2 to 6, 59 to 61 of the CD loop, 87, 90 and 108. The overall r.m.s. deviation of the backbone residues 5 to 108 is 0.62 A. Each of the two Ca2+ atoms that are bound to the CD and EF loops is co-ordinated to seven oxygen atoms, including one water molecule. The third Ca2+ is also seven-co-ordinated, to five oxygen atoms belonging to three different oncomodulin molecules and to two water molecules which form hydrogen bonds to a fourth oncomodulin; thus, this intermolecular Ca2+ and its equivalents interlink the molecules into zigzag layers normal to the b axis with a spacing of b/2 or 32.14 A. No such extensive molecular aggregation has been reported for any of the related Ca-binding regulatory proteins of the troponin-C family studied thus far. The Ca-O distances in all three polyhedra are in the range 2.07 A to 2.64 A, indicating tightly bound Ca polyhedra.