A comparison of the properties of the binary and ternary complexes formed by calmodulin and troponin C with two regulatory peptides of phosphorylase kinase.

The regulatory peptides Phk13 (301-327) and a modified form of Phk5 (342-367) from the gamma-subunit of glycogen phosphorylase kinase form binary and ternary complexes with both calmodulin and the related muscle protein troponin C. Neither peptide appears to affect to a major extent a fluorescent probe linked to Cys-27 of wheat germ calmodulin. Phk13, but not Phk5, significantly modifies the properties of a probe joined to Cys-98 of troponin C. A comparison by means of radiationless energy transfer of the average separations of Trp-16 of Phk5 from specific groups in the N- and C-terminal halves of calmodulin and troponin C indicate significant changes upon going from the 1:1 binary complex to the 1:1:1 ternary complex with Phk13. A comparison of the effects of addition of Phk13 to calmodulin, troponin C, and their binary complexes with Phk5 suggests that the conformation of Phk13 is similar in the binary and ternary complexes.

The interaction of calmodulin with regulatory peptides of phosphorylase kinase.

The regulatory peptides Phk13 (301-327) and Phk5 (342-367) have been synthesized and their interaction with calmodulin studied. In the case of Phk13 modified forms were also synthesized in which a tryptophan group was placed at position 4 or 21, as well as a form with tryptophan at position 4 and nitrotyrosine at position 21. From tryptic digestion, circular dichroism, and radiationless energy transfer measurements, it appears that Phk13 forms an elongated complex with calmodulin in which the peptide is in a non-helical conformation, probably bent into a hairpin-shaped structure, the connecting strand of calmodulin is extended and exposed to the action of proteolytic enzymes, and the peptide makes contact with both the N- and C-terminal half-molecules of calmodulin. In contrast, the Phk5 peptide has an alpha-helical conformation in the complex, which is relatively compact in shape.

Nature of the intermediate in the 3-oxo-delta 5-steroid isomerase reaction.

The role of Tyr-14 of 3-oxo-delta 5-steroid isomerase (KSI) was probed by analysis of the spectra of 3-amino-1,3,5(10)-estratrien-17 beta-ol (4) and equilenin (5) bound to the active site of KSI. The ultraviolet spectrum of 4 bound to KSI is identical to that for 4 in neutral solution. This observation indicates that Tyr-14 does not protonate the amine group of 4 at the active site. By analogy, it is argued that the 3-oxo group of steroid substrates for KSI is not protonated during the reaction. In contrast, the fluorescence excitation spectra of 5 bound to KSI show characteristics of an ionized phenol, even at pH values as low as 3.8. It is concluded that the pKa of equilenin is perturbed from its value in solution of 9 to less than or equal to 3.5 at the active site of KSI. Similarly, the pKa of the intermediate dienol in the KSI reaction should be lowered to less than or equal to 4.5 when it is bound to KSI. Thus, the function of Tyr-14 as an electrophilic catalyst is likely the stabilization of the anion of the dienol by hydrogen bonding rather than by proton transfer.

The interaction of auramine O with calmodulin: location of the binding site on the connecting strand.

The cationic dye auramine O forms a fluorescent complex with Ca(2+)-liganded calmodulin. One moderately strong binding site is present, as well as one or more weaker sites. The binding site for auramine O is different from those for toluidinyl-naphthalene sulfonate. The dependence of binding upon electrolyte concentration suggests a substantial electrostatic component of the free energy of binding. The splitting of the bond between residues 77 and 78 by trypsin digestion abolishes auramine O binding; the N- and C-terminal half-molecules have virtually no binding capacity. This suggests that the primary binding site is located near the midpoint of the connecting strand and includes elements of both half-molecules. Thrombin digestion, which splits calmodulin between residues 106 and 107, also substantially reduces auramine O binding; this may be interpreted in terms of the stabilization of the structure of the connecting strand by interaction with residues within binding domain IV. The binding affinity at pH 5.0, where the helical organization of the connecting strand may be intact, is greater than at neutral pH.

A mass spectrometry method for mapping the interface topography of interacting proteins, illustrated by the melittin-calmodulin system.

The shielding of lysine groups from acetylation by acetic anhydride has been used to identify the regions of calmodulin in contact with melittin in the 1:1 complex. The estimation of the degree of acetylation was done by examining cyanogen bromide and cyanogen bromide/trypsin digests by mass spectrometry. Evidence was obtained that lysines-21, -75, and -148 are protected to some extent, with the implication that both the N- and C-terminal lobes and the connecting strand are involved in the interaction.

The interaction of troponin C with phosphofructokinase. Comparison with calmodulin.

Phosphofructokinase (PFK) is a calmodulin (CaM)-binding protein [Mayr & Heilmeyer (1983) FEBS Lett. 195, 51-57]. We found that troponin C (TnC), which is homologous to CaM, also binds PFK and affects PFK's catalytic activity, aggregation states and conformational changes as CaM does in most cases. PFK titration of N-acetylaminoethyl-5-naphthylamido-1-sulphonate ('AEDANS')-TnC showed that its apparent dissociation constant is comparable with that of PFK-CaM. Fluorescent labels were also used to probe contact regions on TnC and CaM. It is likely that the C-terminal end of the connecting strand of the TnC molecule is close to PFK in the binary complex. Hydrophobic regions of TnC and CaM also possibly play roles in the binding and polymerization of PFK. TnC and CaM deactivate PFK through accelerating PFK conformational change as well as through accelerating PFK tetramer dissociation, as implied in the results of activity, light-scattering, fluorescence and c.d. experiments. The intact molecule of CaM appears to be required to deactivate PFK, because neither half of the CaM molecule has an effect on PFK activity.

Ligand interactions in the ArsA protein, the catalytic component of an anion-translocating adenosinetriphosphatase.

The ars operon of the conjugative R-factor R773 produces resistance to arsenicals in cells of Escherichia coli. The operon encodes an oxyanion pump which is composed of a membrane subunit, the 45.5-kDa ArsB protein, and a catalytic subunit, the 63-kDa ArsA protein. Purified ArsA protein is an arsenite(antimonite)-stimulated ATPase. From its amino acid sequence, as deduced from the nucleotide sequence, the ArsA protein has four tryptophanyl residues which could serve as intrinsic fluorescent probes for the study of substrate-induced conformational changes. Both static and dynamic measurements of tryptophan fluorescence were performed with the ArsA protein. Results from static anisotropy measurements indicated differences in molecular motion with addition of ATP, SbO2-, or Mg2+. These results were supported by time decay measurements of fluorescence anisotropy. The results of time decay measurements indicated a shorter correlation time, reflecting localized motion in the vicinity of the probe, and a longer correlation time, which could have arisen from rotation of the major portion of the molecule. The longer correlation time changed with addition of the various effectors, especially MgCl2, suggesting that binding of Mg2+ decreases probe mobility.

Substrate-induced dimerization of the ArsA protein, the catalytic component of an anion-translocating ATPase.

The ArsA protein, the catalytic component of the plasmid-encoded resistance system for removal of the toxic oxyanions arsenite, antimonite, and arsenate from bacterial cells, catalyzes oxyanion-stimulated ATP hydrolysis. Three lines of evidence suggest that the ArsA protein functions as a homodimer. First, the ArsA protein was modified with 5'-p-fluorosulfonyl-benzoyladenosine (FSBA). Antimonite potentiated FSBA inhibition, while ATP or ADP afforded partial protection. ATP and antimonite together provided complete protection, indicating interaction of the anion- and nucleotide-binding sites. The estimated Ki values for FSBA were 0.4 mM in the absence of antimonite and 0.1 mM in the presence of antimonite, suggesting that the binding of antimonite increased the affinity of ArsA protein for FSBA. Incorporation of [14C]FSBA was examined. Extrapolation of the amount of FSBA required to inactivate the protein indicated that 1 mol of FSBA was sufficient to inhibit the activity of 1 mol of ArsA protein in the absence of substrates, while only 0.5 mol was required in the presence of the anionic substrate antimonite. Second, chemical cross-linking of the 63-kDa ArsA protein with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline resulted in formation of a species approximately twice the size of the monomer in the presence of antimonite but not ATP. Third, determination of the average mass of the ArsA protein in solution by light scattering demonstrated that the average species was 66 kDa in the absence of substrates. In the presence of antimonite the weight average molecular mass increased to a mass in excess of 100 kDa. These results are consistent with the ArsA protein existing in an equilibrium between monomer and dimer, with the equilibrium favoring dimerization upon binding of the anionic substrate. Moreover, total loss of ATPase activity in the half-modified enzyme suggests that the catalytic sites on each monomer must interact.

Intensity and anisotropy decays of the tyrosine calmodulin proteolytic fragments, as studied by GHz frequency-domain fluorescence.

Frequency-domain fluorescence measurements to 2 GHz were able to recover and account for essentially all of the intrinsic tyrosine anisotropy of calmodulin and its proteolytic fragments containing one or two tyrosine residues. Low-temperature measurements have detected a very rapid initial anisotropy decay in the 2-tyrosine species which may be attributed to radiationless energy transfer between the two tyrosines. The observed values of the rotational correlation times indicate that both tyrosines of calmodulin possess considerable mobility, which decreases in the presence of Ca2+ and at low temperatures.

Distribution of separations between groups in an engineered calmodulin.

An engineered calmodulin (VU-9-CaM) has been prepared in which a tryptophan group is present at position 99 and a tyrosine at position 138. The tyrosine was converted to nitrotyrosine. Timedomain dynamic fluorescence measurements were made of energy transfer from the tryptophan donor to the nitrotyrosine acceptor. These were analyzed to yield the parameters characterizing the distribution of separations between the two groups, which are located on Ca(2+)-binding domains III and IV. Their mean separation is in reasonable agreement with the crystallographic value.

Perturbation of molecular species distribution in steady states supported by a flow of energy. Models analogous to Ca2(+)-dependent ATPase and phosphorylase b.

When an allosteric macromolecular system is capable of existing in two conformations, both of which may be converted into energy-storing forms by the binding of a substrate or by the absorption of radiant energy, then a kinetic process may occur, such as an enzymic conversion of the substrate into products, which liberates energy and selectively depletes one or more of the forms of the macromolecule. Upon a continuous supply of energy, a steady state, or pseudoequilibrium, is reached during which the selective depletion of molecular species results effectively in a directional flow of energy through the system. This perturbs the distribution of the various molecular species. This effect may simulate both positive and negative binding cooperativity, and mimic the presence of multiple binding sites with different affinities even in monomeric, monovalent systems. Specific model systems are presented analogous to the transport of Ca2+ by sarcoplasmic reticulum and the allosteric behavior of phosphorylase b.

The interaction of cyclosporin and calmodulin.

The interaction of cyclosporin A and dansyl cyclosporin A with bovine and wheat germ calmodulin has been monitored by measurements of induced changes in dansyl and bound toluidinyl naphthalene sulfonate fluorescence. The interaction is Ca2(+)-dependent and 1:1. Measurements of the efficiency of radiationless energy transfer from bound dansyl cyclosporin A to an acceptor group located on Cys-27 of wheat germ calmodulin suggest that the primary binding site is not located on the N-terminal lobe (residues 1-65). However, studies with proteolytic fragments of calmodulin indicate that elements of the N-terminal half-molecule (residues 1-77) may be involved in the stabilization of the binding site. The binding of cyclosporin alters the physical properties of calmodulin and, in particular, reduces the localized rotational mobility of a fluorescent probe.

The interaction of calmodulin with the C-terminal M5 peptide of myosin light chain kinase.

The interaction with calmodulin of the 17-residue C-terminal fragment M5 of myosin light chain kinase has been studied by several physical techniques. Circular dichroism measurements suggest that M5 exists within the complex primarily as an alpha-helix. Fluorescence intensity measurements of the single tryptophan of M5 (Trp-4) indicate that it is in a relatively nonpolar environment and is shielded from solvent. Dynamic measurements of fluorescence anisotropy decay indicate that Trp-4 changes from a freely rotating fluorophore to one which is largely immobilized upon complex formation. Static fluorescence measurements show that 2,6-TNS is displaced from its binding site on calmodulin by M5. The binding of M5 also partially inhibits the proteolytic scission by trypsin of the bond between residues 77 and 78.

The secondary structure of turkey gizzard myosin light chain kinase and the nature of its interaction with calmodulin.

The enzymatic activities of native myosin light chain kinases are subject to modification by interaction with Ca2(+)-calmodulin (CaM). The interaction between myosin light chain kinase isolated from turkey gizzard (tgMLCK) and calmodulin isolated from bovine testes (CaMbt) and wheat germ (CaMwg) has been examined by means of the intrinsic tryptophan fluorescence of tgMLCK and the fluorescence of extrinsic fluorescent labels located at Cys-27 and Tyr-139 of CaMwg and Tyr-99 of CaMbt. Static and dynamic fluorescence measurements provide evidence for the involvement of the former two sites in the zone of contact with lesser involvement of the site marked by the probe at Tyr-99. Complex formation protected the primary cleavage site in CaMbt (Lys-77) from proteolysis by trypsin. These results are consistent with involvement of the N- and C-terminal lobes of CaM in stabilization of the complex with tgMLCK, but cannot rule out participation of the connecting strand in the interaction. CD measurements extending to 175 nm, obtained using synchroton radiation, indicate the following secondary structure content for tgMLCK: 17 +/- 2% alpha-helix, 22 +/- 3% antiparallel beta-sheet, 3 +/- 1% parallel beta-sheet, 24 +/- 2% beta-turns, and 34 +/- 2% random coil. Similar measurements of the CD spectra of CaMbt and of the 1:1::CaMbt:tgMLCK complex presently indicate that neither protein undergoes major secondary structure rearrangement during their interaction, although subtle changes in the CD spectrum of tgMLCK appear to be correlated with the interaction with CaM.

Interactions of troponin I and its inhibitory fragment (residues 104-115) with troponin C and calmodulin.

Fluorescent probes have been used to study the interaction of troponin I and its inhibitory peptide TnIp with troponin C, calmodulin, and the proteolytic fragments of calmodulin. The probes used included the noncovalently bound ligand TNS and the covalently attached labels dansyl and AEDANS. The fluorescence intensity of TNS bound to troponin C, calmodulin, or the calmodulin fragments was greatly enhanced by the presence of TnIp. This effect was used to estimate the corresponding binding constants. It was found that TnIp is bound by the C-terminal half-molecule of calmodulin, TR2C, with an affinity comparable to that of intact calmodulin or troponin C, while the binding affinity of the N-terminal half-molecule, TR1C, was an order of magnitude less, suggesting that the TnIp-containing portion of troponin I combines with the C-terminal half of calmodulin or troponin C. The fluorescence properties of an AEDANS group linked to Cys-98 of troponin C were modified by interaction with troponin I or TnIp. The fluorescence properties of the same group linked to Cys-27 of wheat germ calmodulin were affected by TnI, but not TnIp. TnI had a small effect upon the fluorescence of a dansyl group linked to Met-25 of troponin C. TnIp also inhibited the tryptic hydrolysis of the midpoint of the central connecting strand of calmodulin and troponin C.(ABSTRACT TRUNCATED AT 250 WORDS)

Orientation, accessibility, and mobility of equilenin bound to the active site of steroid isomerase.

The fluorescent aromatic steroid equilenin, which contains a beta-naphthol moiety, is bound by 3-oxo-delta 5-steroid isomerase. The excitation and emission fluorescence spectra of equilenin when bound to the enzyme, as well as the fluorescence decay time, are indicative of ground-state ionization. In view of the high efficiency of tyrosine quenching, which approaches 100%, the beta-naphthol moiety of equilenin must be in proximity to all three tyrosines of steroid isomerase to account for the observed efficiency of radiationless energy transfer. From the observed response to an external quencher, it appears that enzyme-bound equilenin is largely shielded from solvent. Fluorescence anisotropy measurements indicate a high degree of immobilization of the bound ligand. These models are consistent with proposed models of the enzyme-substrate complex.

The effect of a distribution of separations upon intramolecular distances in biopolymers, as determined by radiationless energy transfer.

In a fluorescent donor group and a nonfluorescent acceptor group are incorporated into a biopolymer, so that radiationless energy transfer occurs between the two groups, the apparent separation of the groups, as determined by energy transfer, will be influenced by the existence of a distribution of separations. This might arise from the presence of significant localized flexibility at the sites of attachment of the two groups, or from internal flexibility involving the biopolymer itself. If a Gaussian form is assumed for the distribution of separations of the donor and acceptor groups, the efficiency of transfer is dependent upon the width of the distribution, as well as the average distance between the groups. Significant differences may thereby arise between the true average separation and the separation computed from transfer efficiencies by the usual procedures. The deviations are different for transfer efficiencies computed from quantum yields and from decay times. They become more important with increasing width of the distribution of separations and increasing efficiency of transfer. In general, if a distribution of separations is present, the average separation is most reliably computed by procedures which take into account the effects of this distribution.

The interaction of delta-hemolysin with calmodulin.

Delta-Hemolysin forms a 1:1 complex with Ca2+ -liganded calmodulin. Probably because of the pronounced tendency of delta-hemolysin to self-associate, the apparent binding affinity is much less than that for melittin. Complex formation is reflected by an increase in quantum yield of Trp-15 of delta-hemolysin and by increased shielding from acrylamide quenching. There is, however, no indication of a change in peptide molecular ellipticity. The binding of 2-toluidinyl-naphthalene-6-sulfonate is reduced by complex formation, suggesting the involvement of a hydrophobic region. Complex formation also blocks the proteolysis by trypsin of the bond between residues 77 and 78. The time decays of fluorescence intensity and anisotropy for tryptophan are multiexponential for both free and complexed delta-hemolysin; the average decay time for intensity is substantially increased for the complex. The localized mobility of tryptophan is greatly reduced in the complex. Complex formation appears to involve both the C-terminal lobe and the connecting strand of calmodulin.

Anisotropy decay of fluorescence as an experimental approach to protein dynamics.

This minireview makes an initial assessment of the progress made using anisotropy decay measurements for investigating the conformational changes and molecular dynamics in soluble systems. A critical analysis of available data is presented. The anisotropy decays of the tryptophan fluorescence of staphylococcal nuclease, adrenocorticotropin, melittin and of labeled transfer RNA were studied for investigating the functional conformational changes of these systems. The emissions of variously labeled immunoglobulins have been used to elucidate the conformations of these proteins before and after the binding of specific antibodies. Labeled myosin and its fragments have given information on the functional motions of the protein domains. The anisotropy decays of labeled and natural hemoglobin systems have been utilized for exploring the allosteric behavior of these molecules. The data suggest a wide applicability of this technique to the study of protein dynamics and conformational changes of macromolecules.