Fluorescence analysis of calmodulin mutants containing tryptophan: conformational changes induced by calmodulin-binding peptides from myosin light chain kinase and protein kinase II.

Peptide-induced conformational changes in five isofunctional mutants of calmodulin (CaM), each bearing a single tryptophan residue either at the seventh position of each of the four calcium-binding loops (i.e., amino acids 26, 62, 99, and 135) or in the central helix (amino acid 81) were studied by using fluorescence spectroscopy. The peptides RS20F and RS20CK correspond to CaM-binding amino acid sequence segments of either nonmuscle myosin light chain kinase (nmMLCK) or calmodulin-dependent protein kinase II (CaMPK-II), respectively. Both steady-state and time-resolved fluorescence data were collected from the various peptide-CaM complexes. Steady-state fluorescence intensity measurements indicated that, in the presence of an excess of calcium, both peptides bind to the calmodulin mutants with a 1:1 stoichiometry. The tryptophans located in loops I and IV exhibited red-shifted emission maxima (356 nm), high quantum yields (0.3), and long average lifetimes (6 ns). They responded in a similar manner to peptide binding, by only slight changes in their fluorescence features. In contrast, the fluorescence intensity of the tryptophans in loops II and III decreased markedly, and their fluorescence spectrum was blue-shifted upon peptide binding. Analysis of the tryptophan fluorescence decay of the last mentioned calmodulins supports a model in which the equilibrium between two (Trp-99) or three (Trp-62) states of these tryptophan residues, each characterized by a different lifetime, was altered toward the blue-shifted short lifetime component upon peptide binding. Taken together, these data provide new evidence that both lobes of calmodulin are involved in peptide binding. Both peptides induced similar changes in the fluorescence properties of the tryptophan residues located in the calcium-binding loops, with the exception of calmodulin with Trp-135. For this last mentioned calmodulin, slight differences were observed. Tryptophan in the central helix responded differently to RS20F and RS20CK binding. RS20F binding induced a red-shift in the emission maximum of Trp-81 while RS20CK induced a blue-shift. The quenching rate of Trp-81 by iodide was slightly reduced upon RS20CK binding, while RS20F induced a 2-fold increase. These results provide evidence that the environment of Trp-81 is different in each case and are, therefore, consistent with the hypothesis that the central helix can play a differential role in the recognition of, or response to, CaM-binding structures.     

Fluorescence study ofEscherichia coli cyclic AMP receptor protein

Time-resolved, steady-state fluorescence and fluorescence-detected circular dichroism (FDCD) have been used to resolve the fluorescence contributions of the two tryptophan residues, Trp-13 and Trp-85, in the cyclic AMP receptor protein (CRP). The iodide and acrylamide quenching data show that in CRP one tryptophan residue, Trp-85, is buried within the protein matrix and the other, Trp-13, is moderately exposed on the surface of the protein. Fluorescence-quenching-resolved spectra show that Trp-13 has emission at about 350 nm and contributes 76–83% to the total fluorescence emission. The Trp-85, unquenchable by iodide and acrylamide, has the fluorescence emission at about 337 nm. The time-resolved fluorescence measurements show that Trp-13 has a longer fluorescence decay time. The Trp-85 exhibits a shorter fluorescence decay time. In the CRP-cAMP complex the Trp-85, previously buried in the apoprotein becomes totally exposed to the iodide and acrylamide quenchers. The FDCD spectra indicate that in the CRP-cAMP complex Trp-85 remains in the same environment as in the protein alone. It has been proposed that the binding of cAMP to CRP is accompanied by a hinge reorientation of two protein domains. This allows for penetration of the quencher molecules into the Trp-85 residue previously buried in the protein matrix.     

The interaction of melittin with calmodulin and its tryptic fragments

Melittin has been found to interact with both the N- and C-terminal half-molecules of calmodulin, as well as the intact molecule, in the presence of Ca2+. The interaction results in a major change in the microenvironment of Trp-19, which is in a more nonpolar, solvent-shielded, and immobilized microenvironment in the complex. The properties of Tyr-99 and Tyr-138 of calmodulin are altered by complex formation. From measurements of the efficiencies of radiationless energy transfer from Trp-19 to the nitro derivatives of Tyr-99 and/or Tyr-138, it is concluded that Trp-19 is located in proximity to the C-terminal lobe of calmodulin in the complex.     

Fluorescence study ofEscherichia coli cyclic AMP receptor protein

Time-resolved, steady-state fluorescence and fluorescence-detected circular dichroism (FDCD) have been used to resolve the fluorescence contributions of the two tryptophan residues, Trp-13 and Trp-85, in the cyclic AMP receptor protein (CRP). The iodide and acrylamide quenching data show that in CRP one tryptophan residue, Trp-85, is buried within the protein matrix and the other, Trp-13, is moderately exposed on the surface of the protein. Fluorescence-quenching-resolved spectra show that Trp-13 has emission at about 350 nm and contributes 76–83% to the total fluorescence emission. The Trp-85, unquenchable by iodide and acrylamide, has the fluorescence emission at about 337 nm. The time-resolved fluorescence measurements show that Trp-13 has a longer fluorescence decay time. The Trp-85 exhibits a shorter fluorescence decay time. In the CRP-cAMP complex the Trp-85, previously buried in the apoprotein becomes totally exposed to the iodide and acrylamide quenchers. The FDCD spectra indicate that in the CRP-cAMP complex Trp-85 remains in the same environment as in the protein alone. It has been proposed that the binding of cAMP to CRP is accompanied by a hinge reorientation of two protein domains. This allows for penetration of the quencher molecules into the Trp-85 residue previously buried in the protein matrix.Abbreviations CRP cyclic AMP receptor protein - NATA N-acetyltryptophanamide - FQRS fluorescence-quenching-resolved spectra - FDCD fluorescence-detected circular dichroism - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulfate - FPLC fast protein liquid chromatography     

Engineered Tet repressor mutants with single tryptophan residues as fluorescent probes. Solvent accessibilities of DNA and inducer binding sites and interaction with tetracycline

Mutants of the Tn10-encoded Tet repressor containing single or no tryptophan residues were constructed by oligonucleotide-directed mutagenesis. The Trp-75 to Phe exchange reduces the dissociation rate of the complex with the inducer tetracycline by a factor of 2. The Trp-43 to Phe exchange has no effect on inducer binding. The fluorescence emission spectra of both tryptophan residues are quenched to a different extent by binding of tetracycline: Trp-75 is quenched to zero and Trp-43 to only 50%. It is concluded that Trp-75 is in the vicinity of the inducer binding site. The different fluorescence emission spectra of both tryptophan residues depend on the native structure of Tet repressor. Quenching studies with iodide indicate that the DNA binding motif is solvent exposed in free repressor and moves towards the interior of the protein upon inducer binding. The inducer binding site is in the interior of the protein. The fluorescence of tetracycline is enhanced upon binding to Tet repressor. The excitation at 280 nm results mainly from the change in environment and in part from energy transfer from tryptophan to the drug.     

Distribution of distances between the tryptophan and the N-terminal residue of melittin in its complex with calmodulin, troponin C, and phospholipids.

We used frequency-domain measurements of fluorescence resonance energy transfer to measure the distribution of distances between Trp-19 of melittin and a 1-dimethylamino-5-sulfonylnaphthalene (dansyl) residue on the N-terminal-alpha-amino group. Distance distributions were obtained for melittin free in solution and when complexed with calmodulin (CaM), troponin C (TnC), or palmitoyloleoyl-L-alpha-phosphatidylcholine (POPC) vesicles. A wide range of donor (Trp-19)-to-acceptor (dansyl) distances was found for free melittin, which is consistent with that expected for the random coil state, characterized by a Gaussian width (full width at half maxima) of 28.2 A. In contrast, narrow distance distributions were found for melittin complexed with CaM, 8.2 A, or with POPC vesicles, 4.9 A. A somewhat wider distribution was found for the melittin complex with TnC, 12.8 A, suggesting the presence of heterogeneity in the mode of binding between melittin and TnC. For all the complexes the mean Trp-19 to dansyl distance was near 20 A. This value is somewhat smaller than expected for the free alpha-helical state of melittin, suggesting that binding with CaM or TnC results in a modest decrease in the length of the melittin molecule.     

Tryptophan fluorescence as a probe of placental ribonuclease inhibitor binding to angiogenin

The binding of human placental ribonuclease inhibitor (PRI) to angiogenin, a human protein that induces neovascularization, occurs with a 1:1 stoichiometry and is accompanied by a 50% increase in tryptophan fluorescence. In contrast, the binding of PRI to bovine pancreatic RNase A or to angiogenin oxidized at its single tryptophan residue results in a quenching of fluorescence. These observations suggest that there is a change in the local environment of Trp-89 of angiogenin. Quenching experiments with acrylamide are consistent with the view that Trp-89 is exposed in the native protein and becomes less accessible upon formation of the complex with PRI. Stopped-flow kinetic measurements monitoring the fluorescence enhancement indicate a two-step mechanism for the binding of PRI to angiogenin. The first step involves rapid formation of an enzyme-inhibitor complex, EI, followed by a slower isomerization of EI to a tight enzyme-inhibitor complex, EI*: (Formula: see text). In 0.1 M NaCl at pH 6 and 25 degrees C, the values of K1 and K2 are 0.53 microM and 97 s-1, respectively. The apparent second-order rate constant of association at protein concentrations much less than K1 is approximated by K2/K1 and equals 1.8 X 10(8) M-1 s-1. The corresponding value for the association of PRI with RNase A is only slightly higher, 3.4 X 10(8) M-1 s-1. The effects of pH and sodium chloride concentration on the association rate of PRI with angiogenin suggest the importance of ionizable groups and ionic interactions, respectively, in the association process.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Fluorescence quenching of Trp-314 of liver alcohol dehydrogenase by oxygen

The quenching of the fluorescence of liver alcohol dehydrogenase (LADH) by molecular oxygen has been studied by both fluorescence lifetime and intensity measurements. This was done in the presence of 1 M acrylamide which selectively quenches the fluorescence of the surface tryptophan residue, Trp-15, thus allowing us to focus on the quenching of the deeply buried tryptophan, Trp-314, by molecular oxygen. Such studies yielded a Stern-Volmer plot of F0/F with a greater slope than the corresponding tau o/tau plot. This indicates that both dynamic and static quenching of Trp-314 occurs. The temperature dependence of the dynamic quenching of LADH by oxygen was also studied at three temperatures, from which we determined the activation enthalpy for the quenching of Trp-314 to be about 10 kcal/mol. The oxygen quenching of a ternary complex of LADH, NAD+ and trifluoroethanol was also studied. The rate constant for dynamic quenching of Trp-314 by oxygen was found to be approximately the same in the ternary complex as that in the unliganded enzyme.     

Spatial relationship between the prodan site,Trp-214, and Cys-34 residues in human serum albumin and loss of structure through incremental unfolding

Prodan (6-propionyl-2-(dimethylamino)-naphthalene), a competitive inhibitor of warfarin binding to human serum albumin (HSA) at drug site I, was used to determine the inter- and intradomain distances of HSA. The fluorescence resonance energy transfer (FRET) distances between prodan and Trp-214, prodan and 7-(diethyl amino)-4-methylcoumarin 3-maleimide (CM)-modified Cys-34, and Trp-214 and CM-Cys-34 were determined to be 25.5 +/- 0.5 A, 33.1 +/- 0.8 A, and 32.4 +/- 1 A, respectively. FRET analysis showed that low concentration of palmitic acid (5 microM) increased the interdomain distance between the Trp-214 in domain II and CM-Cys-34 in domain I by approximately 5 A without perturbing the secondary structure of HSA and the immediate environment of Trp-214. Palmitic acid (5 microM) increased the prodan fluorescence by increasing the quantum yield of bound prodan without altering the tryptophan environment. However, palmitic acid (>10 microM) decreased the prodan fluorescence and increased the tryptophan fluorescence. Our results indicate that the high affinity palmitic acid binding site is located at the interface of domains I and II. On the basis of our measurements, a schematic model representing the drug site-1, Trp-214, and Cys-34 along with the palmitic acid sites has been constructed. In addition, prodan fluorescence, FRET, and ligand binding were used to monitor guanidine hydrochloride-induced denaturation of HSA. An analysis of the equilibrium unfolding data suggests that HSA undergoes a two-state unfolding transition with no detectable intermediate. However, kinetic analysis using multiple probes and thermal denaturation studies showed that the unfolding of the prodan site in HSA preceded the unfolding of tryptophan environment. In addition, the separation of domain I and II occurred before the global unfolding of the protein. The data support the idea that HSA loses its structure incrementally during its unfolding.     

Effect of lipids on the binding of calmodulin with cyclic nucleotide phosphodiesterase

The effects of various lipids on calmodulin interaction with Ca-dependent phosphodiesterase were investigated. Palmitic, myristic and stearic acids increased the enzyme activity; the degree of the enzyme activation by calmodulin was decreased thereby. Oleic acid produced a weak activating effect on phosphodiesterase but completely blocked calmodulin action. The effects of the fatty acids under study were reversible, the activation constant was equal to 10(-4)-5 X 10(-4) M. In the presence of Ca2+ phosphoinositides and fatty acids changed the fluorescence intensity of dansyl-labelled calmodulin; in the absence of Ca2+ the lipids did not affect protein fluorescence. The lipids had no influence on the protein affinity for Ca2+. During chromatography of phosphodiesterase on calmodulin-Sepharose the enzyme was eluted from the column both in the presence of EGTA and palmitic acid. It was concluded that fatty acids prevent the formation of the calmodulin - phosphodiesterase complex. This effects may both be due to the lipid binding to the enzyme and to calmodulin.     

Sites of interaction of calmodulin with trifluoperazine and glucagon

The combination of glucagon with calmodulin alters the microenvironment of Tyr-99, but not Tyr-138, and is blocked by the binding of trifluoperazine by calmodulin. Trp-25 of glucagon is probably involved in the zone of interaction, which may also overlap one or more strong binding sites for trifluoperazine. From energy transfer measurements, one strong binding site for trifluoperazine probably involves the N-terminal region of binding domain III. Energy transfer and other evidence suggest that the zone of contact with glucagon involves the N-terminal region of binding domain III.     

Ca2(+)-dependent regulation of the spectrin/actin interaction by calmodulin and protein 4.1

The Ca2(+)-dependent regulation of the erythroid membrane cytoskeleton was investigated. The low-salt extract of erythroid membranes, which is mainly composed of spectrin, protein 4.1, and actin, confers a Ca2+ sensitivity on its interaction with F-actin. This Ca2+ sensitivity is fortified by calmodulin and antagonized by trifluoperazine, a potent calmodulin inhibitor. Additionally, calmodulin is detected in the low-salt extract. These results suggest that calmodulin is the sole Ca2(+)-sensitive factor in the low-salt extract. The main target of calmodulin in the erythroid membrane cytoskeleton was further examined. Under native conditions, calmodulin forms a stable and equivalent complex with protein 4.1 as determined by calmodulin affinity chromatography, cross-linking experiments, and fluorescence binding assays with an apparent Kd of 5.5 x 10(-7) M irrespective of the free Ca2+ concentration. Domain mapping with chymotryptic digestion reveals that the calmodulin-binding site resides within the N-terminal 30-kDa fragment of protein 4.1. In contrast, the interaction of calmodulin with spectrin is unexpectedly weak (Kd = 1.2 x 10(-4) M). Given the content of calmodulin in erythrocytes (2-5 microM), these results imply that the major target for calmodulin in the erythroid membrane cytoskeleton is protein 4.1. Low- and high-shear viscometry and binding assays reveal that an equivalent complex of calmodulin with protein 4.1 regulates the spectrin/actin interaction in a Ca2(+)-dependent manner. At a low Ca2+ concentration, protein 4.1 potentiates the actin cross-linking and the actin binding activities of spectrin. At a high Ca2+ concentration, the protein 4.1-potentiated actin cross-linking activity but not the actin binding activity of spectrin is suppressed by Ca2+/calmodulin. The Ca2(+)-dependent regulation of the spectrin/protein 4.1/calmodulin/actin interaction is discussed.     

Changes in the state of tryptophan residues in T4 phage lysozyme during binding to a competitive inhibitor

The analysis of the intrinsic fluorescence parameters of T4 phage lysozyme in free state and in complex with inhibitor--disaccharide-tetrapeptide from the E. coli cell wall has been carried out. A comparison of the fluorescence changes with the results obtained by difference spectrophotometry and with the data of Elwell and Schellman on the intrinsic fluorescence of wild type WT and mutant eRI T4 phage lysozymes and a consideration of the three dimensional structure of the protein allows to represent the protein fluorescence parameters as a sum of contributions of the individual tryptophan residues. According to the proposed scheme Trp-126 does not emit neither in the free protein nor in the complex; the fluorescence parameters of Trp-158 (lambda m 332 nm, q = 0.27) are not affected by binding of the inhibitor, but all the fluorescence changes are due to the rise of the quantum yield (from 0.135 to 0.315) and the blue shift (from 332 to 328 nm) of the fluorescence of Trp-138.     

Sequence analysis of twin ATP binding cassette proteins involved in translational control, antibiotic resistance, and ribonuclease L inhibition

The urea-induced unfolding of 'N' isomer (occurring at pH 7.0) and 'B' isomer (occurring at pH 9.0) of human serum albumin was studied by fluorescence and circular dichroism spectroscopic measurements. Urea-induced destabilization in different domains of both the isomers was monitored by using domain specific ligands, hemin (domain-I), chloroform, bilirubin (domain-II), and diazepam (domain-III). Urea-induced denaturation of N and B isomers of HSA showed a two-step, three-state transition with accumulation of intermediates around 4.8-5.2M and 3.0-3.4M urea concentrations, respectively. During first transition (0-4.8M urea for N isomer and 0-3.0M urea for B isomer) a continuous decrease in diazepam binding suggested major conformational changes in domain-III prior to intermediate formation. On the other hand, binding of hemin, a ligand for domain-IB and chloroform, whose binding site is located in domain-IIA remains unchanged up to 5.0M urea for N isomer and 3.0M urea for B isomer. Similarly, fluorescence intensity of Trp-214 that resides in domain-IIA remained unchanged up to the above-said urea concentrations and decreased thereafter. Absence of any decrease in hemin binding, chloroform binding, and Trp-214 fluorescence suggested the non-involvement of domain-IB and domain-IIA in intermediate formation. A significant increase in bilirubin binding prior to intermediate formation showed favorable conformational rearrangement in bilirubin binding cavity formed by loop 4 of domain-IB and loop 3 of domain-IIA. Further, a nearly complete abolishment of bilirubin binding to both isomers around 7.0M and 6.0M urea concentrations, respectively, indicated complete separation of domain-I from domain-II from each other. From these observations it can be concluded that N to B transition of human serum albumin shifted the intermediate formation towards lower urea concentration (3.0-3.4M urea for B isomer as against 4.8-5.2M urea for N isomer). Further both the intermediates were found to possess similar alpha-helical (approximately 39%) content and ligand binding properties.     

Static and kinetic studies of calmodulin and melittin complex.

Ca2+ binding to calmodulin triggers conformational change of the protein which induces exposure of hydrophobic surfaces. Melittin has been believed to bind to Ca(2+)-bound calmodulin through the exposed hydrophobic surfaces. However, tryptophan fluorescence measurements and gel chromatography experiments with the melittin-calmodulin system revealed that melittin bound to calmodulin at zero salt concentration even in the absence of Ca2+; addition of salt removed melittin from Ca(2+)-free calmodulin. This means not only the hydrophobic interaction but also the electrostatic interaction contributes to the melittin-calmodulin binding. The fluorescence stopped-flow studies of the dissociation reaction of melittin-calmodulin complex revealed that Ca2+ removal from the complex induced a conformational change of calmodulin, resulting in reduction of the hydrophobic interaction between melittin and calmodulin, but the electrostatic interaction kept melittin still bound to calmodulin for a subsecond lag period, after which melittin dissociated from calmodulin. The fluorescence stopped-flow experiments on the dissociation reaction of complex of melittin and tryptic fragment(s) of calmodulin revealed that the lag period of the melittin dissociation reaction was attributable to the interaction between the C-terminal half of calmodulin and the C-terminal region of melittin.     

Rapid kinetics of Na+ binding to thrombin

The kinetic mechanism of Na(+) binding to thrombin was resolved by stopped-flow measurements of intrinsic fluorescence. Na(+) binds to thrombin in a two-step mechanism with a rapid phase occurring within the dead time of the spectrometer (<0.5 ms) followed by a single-exponential slow phase whose k(obs) decreases hyperbolically with increasing [Na(+)]. The rapid phase is due to Na(+) binding to the enzyme E to generate the E:Na(+) form. The slow phase is due to the interconversion between E(*) and E, where E(*) is a form that cannot bind Na(+). Temperature studies in the range from 5 to 35 degrees C show significant enthalpy, entropy, and heat capacity changes associated with both Na(+) binding and the E to E(*) transition. As a result, under conditions of physiologic temperature and salt concentrations, the E(*) form is negligibly populated (<1%) and thrombin is almost equally partitioned between the E (40%) and E:Na(+) (60%) forms. Single-site Phe mutations of all nine Trp residues of thrombin enabled assignment of the fluorescence changes induced by Na(+) binding mainly to Trp-141 and Trp-215, and to a lesser extent to Trp-148, Trp-207, and Trp-237. However, the fast phase of fluorescence increase is influenced to different extents by all Trp residues. The distribution of these residues over the entire thrombin surface demonstrates that Na(+) binding induces long-range effects on the structure of the enzyme as a whole, contrary to the conclusions drawn from recent structural studies. These findings elucidate the mechanism of Na(+) binding to thrombin and are relevant to other clotting factors and enzymes allosterically activated by monovalent cations.     

Interaction of 7-hydroxy-8-(phenylazo)1,3-naphthalenedisulfonate with bovine plasma albumin. Spectroscopic studies.

Interaction of Orange G (OG) with bovine plasma albumin (BPA) has been investigated using NMR, UV-visible absorption, CD, and fluorescence techniques. The bound conformation of OG is a compact structure with N9-N10 bond in a non-planar syn conformation. The binding causes a decrease in the 478-nm absorption band of OG. The analysis of the binding isotherm generated from UV-visible absorption measurements gives a dissociation constant of 10 microM and stoichiometry 1:1 for BPA.OG complex. Dissociation constant is invariant in the pH range 5.0-8.0 and is approximately 20 times higher at pH 4.0 than its value at pH 7.0. Near and far UV-CD studies indicate alterations in the helical content and in the tertiary structure of the protein on complexation. The binding induces (-) and (+) CD at 335 nm and 465 nm, respectively. The binding also results into an increase in the steady state fluorescence anisotropy of OG without affecting emission maximum and quantum yield. Fluorescence data indicate that quenching of Trp fluorescence by OG is static in nature and OG selectively binds near Trp-135. Observation of similar rotational correlation time for BPA and BPA.OG complex indicates that the overall globular structure of BPA remains unaltered on binding despite certain internal rearrangement in the protein structure.     

Alternative Pathways for Association and Dissociation of the Calmodulin-binding Domain of Plasma Membrane Ca2+-ATPase Isoform 4b (PMCA4b)

The calmodulin (CaM)-binding domain of isoform 4b of the plasma membrane Ca(2+) -ATPase (PMCA) pump is represented by peptide C28. CaM binds to either PMCA or C28 by a mechanism in which the primary anchor residue Trp-1093 binds to the C-terminal lobe of the extended CaM molecule, followed by collapse of CaM with the N-terminal lobe binding to the secondary anchor Phe-1110 (Juranic, N., Atanasova, E., Filoteo, A. G., Macura, S., Prendergast, F. G., Penniston, J. T., and Strehler, E. E. (2010) J. Biol. Chem. 285, 4015-4024). This is a relatively rapid reaction, with an apparent half-time of ～1 s. The dissociation of CaM from PMCA4b or C28 is much slower, with an overall half-time of ～10 min. Using targeted molecular dynamics, we now show that dissociation of Ca(2+)-CaM from C28 may occur by a pathway in which Trp-1093, although deeply embedded in a pocket in the C-terminal lobe of CaM, leaves first. The dissociation begins by relatively rapid release of Trp-1093, followed by very slow release of Phe-1110, removal of C28, and return of CaM to its conformation in the free state. Fluorescence measurements and molecular dynamics calculations concur in showing that this alternative path of release of the PMCA4b CaM-binding domain is quite different from that of binding. The intermediate of dissociation with exposed Trp-1093 has a long lifetime (minutes) and may keep the PMCA primed for activation.     

Photoaffinity labeling of the nucleotide binding site of actin

Rabbit skeletal muscle actin was photoaffinity-labeled by the nucleotide analogue 8-azidoadenosine 5'-triphosphate. In both G-actin and F-actin about 25% covalent incorporation was achieved. The labeled actins were digested with cyanogen bromide, and the labeled peptides were isolated and sequenced. In F-actin the label was bound primarily to Lys-336, while in G-actin the label was bound to Lys-336 or to Trp-356. The results indicate that the nucleotide binding site is near the phalloidin binding site of actin [Vanderkerckhove, J., Deboben, A., Nassal, M., & Wieland, T. (1985) EMBO J. 4, 2815-2818]. The binding of the azido group to Trp-356 in G-actin but not in F-actin may indicate that a change in the conformation of actin occurs in this region.