Influence of Zn2+ and water on the transport properties of a pyrrolidinium dicyanamide ionic liquid.

In order to expand our understanding of a potential zinc-based battery electrolyte, we have characterized the physical and transport properties of the ionic liquid (IL) 1-butyl-1-methylpyrrolidinium dicyanamide ([C4mpyr][dca]) containing various levels of both Zn(2+) and H2O. Detailed measurements of density, viscosity, conductivity, and individual anion and cation diffusion coefficients using pulsed-field-gradient (PFG) NMR combined with NMR chemical shifts and spin-lattice relaxation (T1) NMR experiments provide insights into the motion and chemical environment of all molecular species. We find that the various techniques for probing ion transport and dynamics form a coherent picture as a function of electrolyte composition. Zn(2+) addition causes a moderate reduction in the self-diffusion of the IL anion and cation, whereas the addition of H2O increases ion mobility by increasing the liquid's overall fluidity. Temperature-dependent (13)C T1 experiments of the dca carbon analyzed using Bloembergen-Purcell-Pound fits show monotonic slowing of anion dynamics with Zn(2+) addition, suggesting increased Zn(2+)/dca(-) association. T1 experiments show minimal change in the spin-lattice relaxation of cation or anion upon H2O addition, suggesting that H2O is playing no significant role in Zn(2+) speciation. Finally, we employ a novel electrophoretic NMR technique to directly determine the electrophoretic mobility of the C4mpyr cation, which we discuss in the context of impedance-based conductivity measurements.

The crystal structure of the globular domain of sheep prion protein.

The prion protein PrP is a naturally occurring polypeptide that becomes transformed from a normal conformation to that of an aggregated form, characteristic of pathological states in fatal transmissible spongiform conditions such as Creutzfeld-Jacob Disease and Bovine Spongiform Encephalopathy. We report the crystal structure, at 2 A resolution, of residues 123-230 of the C-terminal globular domain of the ARQ allele of sheep prion protein (PrP). The asymmetric unit contains a single molecule whose secondary structure and overall organisation correspond to those structures of PrPs from various mammalian species determined by NMR. The globular domain shows a close association of helix-1, the C-terminal portion of helix-2 and the N-terminal portion of helix-3, bounded by the intramolecular disulphide bond, 179-214. The loop 164-177, between beta2 and helix-2 is relatively well structured compared to the human PrP NMR structure. Analysis of the sheep PrP structure identifies two possible loci for the initiation of beta-sheet mediated polymerisation. One of these comprises the beta-strand, residues 129-131 that forms an intra-molecular beta-sheet with residues 161-163. This strand is involved in lattice contacts about a crystal dyad to generate a four-stranded intermolecular beta-sheet between neighbouring molecules. The second locus involves the region 188-204, which modelling suggests is able to undergo a partial alpha-->beta switch within the monomer. These loci provide sites within the PrPc monomer that could readily give rise to early intermediate species on the pathway to the formation of aggregated PrPSc containing additional intermolecular beta-structure.

Calmodulin tagging provides a general method of using lanthanide induced magnetic field orientation to observe residual dipolar couplings in proteins in solution.

A general method is presented for magnetic field alignment of proteins in solution. By tagging a target protein with calmodulin saturated with paramagnetic lanthanide ions it is possible to measure substantial residual dipolar couplings (RDC) whilst minimising the effects of pseudocontact shifts on the target protein. A construct was made consisting of a calmodulin-binding peptide (M13 from sk-MLCK) attached to a target protein, dihydrofolate reductase in this case. The engineered protein binds tightly to calmodulin saturated with terbium, a paramagnetic lanthanide ion. By using only a short linker region between the M13 and the target protein, some of the magnetic field alignment induced in the CaM(Tb3+)4 is effectively transmitted to the target protein (DHFR). 1H-15N HSQC IPAP experiments on the tagged complex containing 15N-labelled DHFR-M13 protein and unlabelled CaM(Tb3+)4 allow one to measure RDC contributions in the aligned complex. RDC values in the range +4.0 to -7.4 Hz were measured at 600 MHz. Comparisons of 1H-15N HSQC spectra of 15N-DHFR-M13 alone and its complexes with CaM(Ca2+)4 and CaM(Tb3+)4 indicated that (i) the structure of the target protein is not affected by the complex formation and (ii) the spectra of the target protein are not seriously perturbed by pseudocontact shifts. The use of a relatively large tagging group (CaM) allows us to use a lanthanide ion with a very high magnetic susceptibility anisotropy (such as Tb3+) to give large alignments while maintaining relatively long distances from the target protein nuclei (and hence giving only small pseudocontact shift contributions).

Calcium-induced refolding of the calmodulin V136G mutant studied by NMR spectroscopy: evidence for interaction between the two globular domains.

The Ca(2+) titration of the (15)N-labeled mutant V136G calmodulin has been monitored using (1)H-(15)N HSQC NMR spectra. Up to a [Ca(2+)]/[CaM] ratio of 2, the Ca(2+) ions bind predominantly to sites I and II on the N-domain in contrast with the behavior of the wild-type calmodulin where the C-terminal domain has the higher affinity for Ca(2+). Surprisingly, the Ca(2+)-binding affinity for the N-domain in the mutant calmodulin is greater than that for the N-domain in the wild-type protein. The mutated C-domain is observed as a mixture of unfolded, partially folded (site III occupied), and native-like folded (sites III and IV occupied) conformations, with relative populations dependent on the [Ca(2+)]/[CaM] ratio. The occupancy of site III independently of site IV in this mutant shows that the cooperativity of Ca(2+) binding in the C-domain is mediated by the integrity of the domain structure. Several NH signals from residues in the Ca(2+)-bound N-domain appear as two signals during the Ca(2+) titration indicating separate species in slow exchange, and it can be deduced that these result from the presence and absence of interdomain interactions in the mutant. It is proposed that an unfolded part of the mutated C-domain interacts with sites on the N-domain that normally bind to target proteins. This would also account for the increase in the Ca(2+) affinity for the N-domain in the mutant compared with the wild-type calmodulin. The results therefore show the wide-ranging effects of a point mutation in a single Ca(2+)-binding site, providing details of the involvement of individual residues in the calcium-induced folding reactions.

Ligand binding and thermodynamic stability of a multidomain protein, calmodulin.

Chemical and thermal denaturation of calmodulin has been monitored spectroscopically to determine the stability for the intact protein and its two isolated domains as a function of binding of Ca2+ or Mg2+. The reversible urea unfolding of either isolated apo-domain follows a two-state mechanism with relatively low deltaG(o)20 values of approximately 2.7 (N-domain) and approximately 1.9 kcal/mol (C-domain). The apo-C-domain is significantly unfolded at normal temperatures (20-25 degrees C). The greater affinity of the C-domain for Ca2+ causes it to be more stable than the N-domain at [Ca2+] > or = 0.3 mM. By contrast, Mg2+ causes a greater stabilization of the N- rather than the C-domain, consistent with measured Mg2+ affinities. For the intact protein (+/-Ca2+), the bimodal denaturation profiles can be analyzed to give two deltaG(o)20 values, which differ significantly from those of the isolated domains, with one domain being less stable and one domain more stable. The observed stability of the domains is strongly dependent on solution conditions such as ionic strength, as well as specific effects due to metal ion binding. In the intact protein, different folding intermediates are observed, depending on the ionic composition. The results illustrate that a protein of low intrinsic stability is liable to major perturbation of its unfolding properties by environmental conditions and liganding processes and, by extension, mutation. Hence, the observed stability of an isolated domain may differ significantly from the stability of the same structure in a multidomain protein. These results address questions involved in manipulating the stability of a protein or its domains by site directed mutagenesis and protein engineering.

Enhancement by Mg2+ of domain specificity in Ca2+-dependent interactions of calmodulin with target sequences.

Mg2+ binds to calmodulin without inducing the changes in secondary structure that are characteristic of Ca2+ binding, or the exposure of hydrophobic surfaces that are involved in typical Ca2+-dependent target interactions. The binding of Mg2+ does, however, produce significant spectroscopic changes in residues located in the Ca2+-binding loops, and the Mg-calmodulin complex is significantly different from apo-calmodulin in loop conformation. Direct measurement of Mg2+ binding constants, and the effects of Mg2+ on Ca2+ binding to calmodulin, are consistent with specific binding of Mg2+, in competition with Ca2+. Mg2+ increases the thermodynamic stability of calmodulin, and we conclude that under resting, nonstimulated conditions, cellular Mg2+ has a direct role in conferring stability on both domains of apo-calmodulin. Apo-calmodulin binds typical target sequences from skeletal muscle myosin light chain kinase and neuromodulin with Kd approximately 70-90 nM (at low ionic strength). These affinities are virtually unchanged by 5 mM Mg2+, in marked contrast to the strong enhancement of peptide affinity induced by Ca2+. Under conditions of stimulation and increased [Ca2+], Mg2+ has a role in directing the mode of initial target binding preferentially to the C-domain of calmodulin, due to the opposite relative affinities for binding of Ca2+ and Mg2+ to the two domains. Mg2+ thus amplifies the intrinsic differences of the domains, in a target specific manner. It also contributes to setting the Ca2+ threshold for enzyme activation and increases the importance of a partially Ca2+-saturated calmodulin-target complex that can act as a regulatory kinetic and equilibrium intermediate in Ca2+-dependent target interactions.

Conformational and metal-binding properties of androcam, a testis-specific, calmodulin-related protein from Drosophila.

Androcam is a testis-specific protein of Drosophila melanogaster, with 67% sequence identity to calmodulin and four potential EF-hand calcium-binding sites. Spectroscopic monitoring of the thermal unfolding of recombinant calcium-free androcam shows a biphasic process characteristic of a two-domain protein, with the apo-N-domain less stable than the apo-C-domain. The two EF hands of the C-domain of androcam bind calcium cooperatively with 40-fold higher average affinity than the corresponding calmodulin sites. Magnesium competes with calcium binding [Ka(Mg) approximately 3 x 10(3) M(-1)]. Weak calcium binding is also detected at one or more N-domain sites. Compared to apo-calmodulin, apo-androcam has a smaller conformational response to calcium and a lower alpha-helical content over a range of experimental conditions. Unlike calmodulin, a tryptic cleavage site in the N-domain of apo-androcam remains trypsin sensitive in the presence of calcium, suggesting an altered calcium-dependent conformational change in this domain. The affinity of model target peptides for androcam is 10(3)-10(5) times lower than for calmodulin, and interaction of the N-domain of androcam with these peptides is significantly reduced. Thus, androcam shows calcium-induced conformational responses typical of a calcium sensor, but its properties indicate calcium sensitivity and target interactions significantly different from those of calmodulin. From the sequence differences and the altered calcium-binding properties it is likely that androcam differs from calmodulin in the conformation of residues in the second calcium-binding loop. Molecular modeling supports the deduction that there are significant conformational differences in the N-domain of androcam compared to calmodulin, and that these could affect the surface, conferring a different specificity on androcam in target interactions related to testis-specific calcium signaling functions.

NMR approaches for monitoring domain orientations in calcium-binding proteins in solution using partial replacement of Ca2+ by Tb3+.

This work shows that the partial replacement of diamagnetic Ca2+ by paramagnetic Tb3+ in Ca2+/calmodulin systems in solution allows the measurement of interdomain NMR pseudocontact shifts and leads to magnetic alignment of the molecule such that significant residual dipolar couplings can be measured. Both these parameters can be used to provide structural information. Species in which Tb3+ ions are bound to only one domain of calmodulin (the N-domain) and Ca2+ ions to the other (the C-domain) provide convenient systems for measuring these parameters. The nuclei in the C-domain experience the local magnetic field induced by the paramagnetic Tb3+ ions bound to the other domain at distances of over 40 A from the Tb3+ ion, shifting the resonances for these nuclei. In addition, the Tb3+ ions bound to the N-domain of calmodulin greatly enhance the magnetic susceptibility anisotropy of the molecule so that a certain degree of alignment is produced due to interaction with the external magnetic field. In this way, dipolar couplings between nuclear spins are not averaged to zero due to solution molecular tumbling and yield dipolar coupling contributions to, for example, the one-bond 15N-1H splittings of up to 17 Hz in magnitude. The degree of alignment of the C-domain will also depend on the degree of orientational freedom of this domain with respect to the N-domain containing the Tb3+ ions. Pseudocontact shifts for NH groups and 1H-15N residual dipolar couplings for the directly bonded atoms have been measured for calmodulin itself, where the domains have orientational freedom, and for the complex of calmodulin with a target peptide from skeletal muscle myosin light chain kinase, where the domains have fixed orientations with respect to each other. The simultaneous measurements of these parameters for systems with domains in fixed orientations show great potential for the determination of the relative orientation of the domains.

Phosphate release during microtubule assembly: what stabilizes growing microtubules?

The molecular mechanism underlying microtubule dynamic instability depends on the relationship between the addition of tubulin-GTP to a growing microtubule and its hydrolysis in the microtubule lattice to tubulin-GDP, with release of inorganic phosphate (Pi). Since this relationship remains controversial, we have re-examined the release of Pi upon microtubule assembly using a fluorometric assay for Pi, based on the phosphate-binding protein of Escherichia coli [Brune M., Hunter, J. L., Corrie, J. E. T., and Webb, M. R. (1994) Biochemistry 33, 8262-8271]. Microtubule assembly and Pi release were monitored simultaneously in a standard fluorimeter as an increase in the turbidity and fluorescence, respectively, in tubulin-GTP solutions assembled under conditions supporting dynamic instability. At the steady state of assembly, Pi release is nonlinear with respect to time, proceeding at a rate determined by the following: (a) the intrinsic GTPase activity of the nonpolymerized tubulin-GTP, and (b) the microtubule number concentration, which decreases progressively. Direct observation of the time course of nucleated microtubule assembly indicates that Pi release is closely coupled to microtubule elongation, even during the initial stages of assembly when uncoupling of tubulin-GTP addition and GTP hydrolysis would be most evident. Studies of the inhibition and reversal of the growth phase by cytostatic drugs show no evidence of a burst of Pi release. We conclude that nucleotide hydrolysis can keep pace with tubulin-GTP addition rates of 200 molecules per second per microtubule and that extended caps of tubulin-GTP or tubulin-GDP-Pi are not generated in normal assembly, nor are they required to stabilize growing microtubules or to support the phenomenon of dynamic instability of microtubules at the steady state.

Ca2+ coordination to backbone carbonyl oxygen atoms in calmodulin and other EF-hand proteins: 15N chemical shifts as probes for monitoring individual-site Ca2+ coordination.

Examination of the NMR 15N chemical shifts of a number of EF-hand proteins shows that the shift value for the amido nitrogen of the residue in position 8 of a canonical EF-hand loop (or position 10 of a pseudo EF-hand loop) provides a good indication of metal occupation of that site. The NH of the residue in position 8 is covalently bonded to the carbonyl of residue 7, the only backbone carbonyl that coordinates to the metal ion in a canonical EF-hand loop. Upon metal coordination to this carbonyl, there is an appreciable deshielding of the 15N nucleus at position 8 (+4 to +8 ppm) due to the polarization of the O(7)=C(7)-N(8) amido group and the corresponding reduction in the electron density of the nitrogen atom. This deshielding effect is effectively independent of the binding of metal to the other site of an EF-hand pair, allowing the 15N shifts to be used as probes for site-specific occupancy of metal binding sites. In addition, a Ca2+-induced change in side-chain Halpha-Calpha-Cbeta-Hbeta torsion angle for isoleucine or valine residues in position 8 can also contribute to the deshielding of the amide 15N nucleus. This conformational effect occurs only in sites I or III and takes place upon binding a Ca2+ ion to the other site of an EF-hand pair (site II or IV) regardless of whether the first site is occupied. The magnitude of this effect is in the range +5 to +7 ppm. A Ca2+ titration of 15N-labeled apo-calmodulin was performed using 2D 1H-15N HSQC NMR spectra. The changes in the 15N chemical shifts and intensities for the peaks corresponding to the NH groups of residues in position 8 of the EF-hand loops allowed the amount of metal bound at sites II, III and IV to be monitored directly at partial degrees of saturation. The peak corresponding to site I could only be monitored at the beginning and end of the titration because of line broadening effects in the intermediate region of the titration. Sites III and IV both titrate preferentially and the results demonstrate clearly that sites in either domain fill effectively in parallel, consistent with a significant positive intradomain cooperativity of calcium binding.

Resolution of Trp near UV CD spectra of calmodulin-domain peptide complexes into the 1La and 1Lb component spectra.

Near uv CD spectra of Trp residues in proteins frequently show a complex line shape deriving from the overlap of 1La and 1Lb electronic transitions. This study presents an original empirical method of resolving these components, based on the near uv CD spectra of well-defined complexes of calmodulin domains with target peptides containing a single Trp residue and derived from the skeletal muscle myosin light chain kinase target sequence. Spectra of 4 complexes were used to obtain the 1La and 1Lb component spectra that were then used to analyze further complexes. The broad and featureless 1La spectrum is centered at 279 nm, the 1Lb spectrum shows vibrational fine structure with maxima at 274.9, 281.5, and 289.8 nm. The CD spectrum of most complexes could successfully be fitted with one 1La and one 1Lb spectrum, the 1Lb spectrum being negative for all complexes but the 1La spectrum showing either positive or negative sign. Spectra of some complexes, however, failed to be adequately represented by only one 1La and one 1Lb spectrum. Instead, they could be fitted with one 1Lb spectrum and two 1La spectra with different sign and position. The method is successful in identifying and quantitating the relative intensities of a two-component system, consistent with a single conformation for tryptophan in a protein, and provides a simple indication of cases where a more complicated explanation is required.

Specificity and symmetry in the interaction of calmodulin domains with the skeletal muscle myosin light chain kinase target sequence.

The specificity of interaction of the isolated N- and C-terminal domains of calmodulin with peptide WFFp (Ac-KRRWKKNFIAVSAANRFK-amide) and variants of the target sequence of skeletal muscle myosin light chain kinase was investigated using CD and fluorescence. Titrations show that two molecules of either domain bind to 18-residue target peptides. For WFFp, the C-domain binds with 4-fold higher affinity to the native compared with the non-native site; the N-domain shows similar affinity for either site. The selectivity of the C-domain suggests that it promotes occupancy of the correct binding site for intact calmodulin on the target sequence. Far UV CD spectra show the extra helicity induced in forming the 2:1 C-domain-peptide or the 1:1:1 C-domain-N-domain-peptide complex is similar to that induced by calmodulin itself; binding of the C-domain to the Trp-4 site is essential for developing the full helicity. Calmodulin-MLCK-peptide complexes show an approximate two-fold rotational relationship between the two highly homologous domains, and the 2:1 C (or N)-domain-peptide complexes evidently have a similar rotational symmetry. This implies that a given domain can bind sequences with opposite peptide polarities, significantly increasing the possible range of conformations of calmodulin in its complexes, and extending the versatility and diversity of calmodulin-target interactions.

The role of beta-sheet interactions in domain stability, folding, and target recognition reactions of calmodulin.

Single-residue mutations have been made of the hydrophobic Ile or Val residue in position 8 of each of the four calcium-binding loop sequences (sites I-IV) of Drosophila calmodulin. These highly conserved residues are part of the hydrophobic core of either calmodulin domain and are involved in the structural link of two calcium-binding sites via a short antiparallel beta-sheet. In the apo-form, the replacement of Ile (or Val) by Gly causes a significant destabilization, shown by the unfolding of the secondary structure of the domain carrying the mutation. In the presence of calcium, the deficiency in alpha-helical structure at 20 degrees C is restored for the mutants at site I, II, or III but not at site IV, which requires the further binding of a high-affinity target peptide to re-establish the native conformation. The extent of the destabilization is seen in the depression of the melting temperature of individual domains, which can be as large as 80 degrees C in the case of Ca4-CaM(V136G). However, because of low values of the unfolding enthalpy for calmodulin domains, only relatively low values of <2 kcal/mol are implied for DeltaDeltaG, the free energy of destabilization due to mutation. Consistent with this, the secondary structure of any unfolded mutant domain is highly sensitive to solvent composition and is largely refolded in the presence of 12.5% (v/v) aqueous trifluoroethanol. Compared to wild-type calmodulin, the affinities of the mutants for calcium and target peptides from sk-MLCK at 20 degrees C are significantly reduced but the effects are relatively small. These results indicate that the conformation of calmodulin can be dramatically altered by mutation of a single highly conserved residue but that changes in solvent or the binding of a target sequence can readily compensate for this, restoring the wild-type properties. The results also suggest that the integrity of both the apo- and holo-forms of calmodulin is important for the maintenance of its biological function and confirm the importance of conserving the structural function of the residues involved in the beta-sheet interactions.

Phosphorylation of tau by glycogen synthase kinase 3beta affects the ability of tau to promote microtubule self-assembly.

To study the effects of phosphorylation by glycogen synthase kinase-3beta (GSK-3beta) on the ability of the microtubule-associated protein tau to promote microtubule self-assembly, tau isoform 1 (foetal tau) and three mutant forms of this tau isoform were investigated. The three mutant forms of tau had the following serine residues, known to be phosphorylated by GSK-3, replaced with alanine residues so as to preclude their phosphorylation: (1) Ser-199 and Ser-202 (Ser-199/202-->Ala), (2) Ser-235 (Ser-235-->Ala) and (3) Ser-396 and Ser-404 (Ser-396/404-->Ala). Wild-type tau and the mutant forms of tau were phosphorylated with GSK-3beta, and their ability to promote microtubule self-assembly was compared with the corresponding non-phosphorylated tau species. In the non-phosphorylated form, wild-type tau and all of the mutants affected the mean microtubule length and number concentrations of assembled microtubules in a manner consistant with enhanced microtubule nucleation. Phosphorylation of these tau species with GSK-3beta consistently reduced the ability of a given tau species to promote microtubule self-assembly, although the affinity of the tau for the microtubules was not greatly affected by phosphorylation since the tau species remained largely associated with the microtubules. This suggests that the regulation of microtubule assembly can be controlled by phosphorylation of tau at sites accessible to GSK-3beta by a mechanism that does not necessarily involve the dissociation of tau from the microtubules.

Kinetic control of the dissociation pathway of calmodulin-peptide complexes.

The mechanism of dissociation reactions induced by calcium chelators has been studied for complexes of Drosophila calmodulin with target peptides, including four derived from the skeletal muscle myosin light chain kinase target sequence. Reactions were monitored by fluorescence stopped-flow techniques using a variety of intrinsic probes and the indicator Quin2. For most of the complexes, apparently biphasic kinetics were observed in several fluorescence parameters. The absence of any obvious relationship between dissociation rates and peptide affinities implies kinetic control of the dissociation pathway. A general mechanism for calcium and peptide dissociation was formulated and used in numerical simulation of the experimental data. Unexpectedly, the rate of the slowest step decreases with increasing [peptide]/[calmodulin] ratio. Numerical simulation shows this step could contain a substantial contribution from a reversible relaxation process (involving the species Ca2-calmodulin-peptide), convolved with the following step (loss of C-terminal calcium ions). The results indicate the potentially key kinetic role of the partially calcium-saturated intermediate species. They show that subtle changes in the peptide sequence can have significant effects on both the dissociation rates and also the dissociation pathway. Both effects could contribute to the variety of regulatory behavior shown by calmodulin with different target enzymes.

Dynamic properties of nucleated microtubules: GTP utilisation in the subcritical concentration regime.

Microtubule assembly kinetics have been studied quantitatively under solution conditions supporting microtubule dynamic instability. Purified GTP-tubulin (Tu-GTP) and covalently cross-linked short microtubule seeds (EGS-seeds; Koshland et al. (1988) Nature 331, 499) were used with and without biotinylation. Under sub-critical concentration conditions ([Tu-GTP] < 5.3 microM), significant microtubule growth of limited length was observed on a proportion of the EGS-seeds by immuno-electron microscopy. A sensitive fluorescence assay for microtubule GDP production was developed for parallel assessment of GTP utilisation. This revealed a correlation between the detected microtubule growth and the production of tubulin-GDP, deriving from the shortening phase of the dynamic microtubules. This correlation was confirmed by the action of nocodazole, a specific inhibitor of microtubule assembly, that was found to abolish the GDP release. The variation of the GDP release with tubulin concentration (Jh(c) plot) was determined below the critical concentration (Cc). The GDP production observed was consistent with the elongation of the observed seeded microtubules with an apparent rate constant of 1.5 x 10(6) M-1 second-1 above a threshold of approximately 1 microM tubulin. The form of this Jh(c) plot for elongation below Cc is reproduced by the Lateral Cap model for microtubule dynamic instability adapted for seeded assembly. The behaviour of the system is contrasted with that previously studied in the absence of detectable microtubule elongation (Caplow and Shanks (1990) J. Biol. Chem. 265, 8935-8941). The approach provides a means of monitoring microtubule dynamics at concentrations inaccessible to optical microscopy, and shows that essentially the same dynamic mechanisms apply at all concentrations. Numerical simulation of the subcritical concentration regime shows dynamic growth features applicable to the initiation of microtubule growth in vivo.

Target recognition by calmodulin: dissecting the kinetics and affinity of interaction using short peptide sequences.

The interaction between calmodulin (CaM) and peptide M13, its target binding sequence from skeletal muscle myosin light chain kinase, involves predominantly two sets of interactions, between the N-terminal target residues and the C-domain of calmodulin, and between the C-terminal target residues and the N-domain of calmodulin (Ikura M et al., 1992, Science 256:632-638). Using short synthetic peptides based on the two halves of the target sequence, the interactions with calmodulin and its separate C-domain have been studied by fluorescence and CD spectroscopy, calcium binding, and kinetic techniques. Peptide WF10 (residues 1-10 of M13) binds to CaM with Kd approximately 1 microM; peptide FW10 (residues 9-18 of M13, with Phe-17-->Trp substitution) binds to CaM with Kd approximately 100 microM. The effect of peptide WF10 on calcium binding to calmodulin produces a biphasic saturation curve, with marked enhancement of affinity for the binding of two calcium ions to the C-domain, forming a stable half-saturated complex, Ca2-CaM-peptide, and confirming the functional importance of the interaction of this sequence with the C-domain. Stopped-flow studies show that the EGTA-induced dissociation of WF10 from Ca4-CaM proceeds by a reversible relaxation mechanism from a kinetic intermediate state, also involving half-saturation of CaM, and the same mechanism is evident for the full target peptide. Interaction of the N-terminal target residues with the C-domain is energetically the most important component, but interaction of calmodulin with the whole target sequence is necessary to induce the full cooperative interaction of the two contiguous elements of the target sequence with both N- and C-domains of calmodulin. Thus, the interaction of calmodulin with the M13 sequence can be dissected on both a structural and kinetic basis into partial reactions involving intermediates comprising distinct regions of the target sequence. We propose a general mechanism for the calcium regulation of calmodulin-dependent enzyme activation, involving an intermediate complex formed by interaction of the calmodulin C-domain and the corresponding part of the target sequence. This intermediate species can function to regulate the overall calcium sensitivity of activation and to determine the affinity of the calmodulin target interaction.

Spectroscopic characterization of a high-affinity calmodulin-target peptide hybrid molecule.

We describe the properties of a hybrid protein comprising the full length of the Xenopus laevis calmodulin sequence, followed by a pentapeptide linker (GGGGS), and residues 3-26 of M13, the calmodulin binding region of skeletal muscle myosin light chain kinase. The properties of the hybrid protein are compared with those of the complex formed between Drosophila calmodulin and a peptide corresponding to residues 1-18 of the M13 sequence. The addition of calcium to the hybrid protein produces pronounced changes in the near- and far-UV CD spectra, in the fluorescence emission spectrum of the single tryptophan residue at position 4 in the M13 sequence, and in the accessibility of this tryptophan residue to acrylamide quenching. These changes are consistent with the tryptophan residue being immobilized in a hydrophobic environment and with the hybrid protein adopting a more alpha-helical structure when calcium is bound. The increased alpha-helicity derives from changes in both the calmodulin and peptide regions of the hybrid protein. Changes in the circular dichroism and fluorescence properties of the hybrid protein as a function of the calcium to hybrid protein ratio are consistent with the fact that these changes parallel the cooperative binding of all four calcium ions. The hybrid protein shows greatly increased affinity (>250-fold) for calcium compared with calmodulin itself. Macroscopic calcium binding constants (K(1)-K(4)) were determined from calcium titrations performed in the presence of the calcium chelator Quin 2. Values for log(K(1)K(2)) and log(K(3)K(4)) were determined to be 15.4 +/- 0.2 and 15.59 +/- 0.22 (20 degrees C). The corresponding values for Drosophila calmodulin alone are 11.65 +/- 0.15 and 9.66 +/- 0.25. Consistent with this increased affinity for calcium stopped-flow kinetic studies suggest that the dissociation rate for the N-terminal calcium ions is reduced to at least 0.77 s(-1), compared with approximately 700 s(-1) for Drosophila calmodulin in the absence of peptide. This hybrid protein illustrates the principle whereby the binding of a peptide sequence covalently attached to calmodulin can enhance the average calcium affinity by more than 2 orders of magnitude. Conversely, the target sequence in the hybrid protein undergoes a calcium-induced conformational change to bind to the calmodulin in a conformation very similar to that of the corresponding dissociable target sequence binding to calmodulin, but with a greatly enhanced affinity due to its physical proximity to the binding site. This avoidance of the energetic penalty of dissociation may be a key contributory factor in determining the high affinity and specificity of the complex multiple interactions involved in recognition of biological targets by calmodulin.

Differences in the regulation of microtubule dynamics by microtubule-associated proteins MAP1B and MAP2.

The regulation of microtubule dynamics in vitro by microtubule-associated proteins (MAPs) was examined, using purified porcine MAP1B and MAP2. MAP1B has a significantly smaller effect on the observed critical concentration for microtubule assembly than MAP2. Assembly is faster in the presence of either MAP, and the resulting microtubules are shorter, indicating that nucleation is substantially promoted by the MAPs. Both MAPs stabilise the microtubule lattice as observed from podophyllotoxin-induced disassembly, but the effect of MAP1B is weaker than the effect of MAP2. At steady-state of assembly MAP1B still allows microtubule dynamic instability to occur as inferred from microtubule length changes. The comparison of the effects of MAP1B and MAP2 indicates that the reduction of the observed critical concentration is attributable to the reduction of the depolymerisation rate and correlates with the extent of suppression of dynamic instability. Numerical simulations illustrate that microtubule dynamics are strongly influenced by relatively small changes in the strength of a limited subset of subunit interactions in the lattice. The observed characteristic differences between the MAPs may be important for the regulation of distinct populations of microtubules which coexist in the same cell, where differences in stability and dynamics may be essential for their different spatial roles as, for example, in developing neurons.

Role of the N-terminal region of the skeletal muscle myosin light chain kinase target sequence in its interaction with calmodulin.

The binding of calmodulin (CaM) to four synthetic peptide analogues of the skeletal muscle myosin light chain kinase (sk-MLCK) target sequence has been studied using 1H-NMR. The 18-residue peptide WFF is anchored to CaM via the interaction of the Trp 4 side chain with the C-domain and the Phe 17 side chain with the N-domain of the protein. A peptide corresponding to the first 10 residues (WF10) does not provide the second anchoring residue and is not long enough to span both domains of CaM. 1H-NMR spectroscopy indicates that the WF10 peptide interacts specifically with the C-domain of CaM, and the chemical shifts of the bound Trp side chain are very similar in the CaM:WF10 and CaM:WFF complexes. Binding of the C-domain of CaM to the strongly basic region around Trp 4 of this MLCK sequence may be an important step in target recognition. Comparison of 1H-NMR spectra of CaM bound to WFF, a Trp 4-->Phe analogue (FFF), or a Trp 4-->Phe/Phe 17-->Trp analogue (FFW) suggests that all three peptides bind to CaM in the same orientation, i.e., with the peptide side chain in position 4 interacting with the C-domain and the side chain in position 17 interacting with the N-domain. This indicates that a Trp residue in position 4 is not an absolute requirement for binding this target sequence and that interchanging the Trp 4 and Phe 17 residues does not reverse the orientation of the bound peptide, in confirmation of the deduction from previous indirect studies using circular dichroism (Findlay WA, Martin SR, Beckingham K, Bayley PM, 1995, Biochemistry 34:2087-2094). Molecular modeling/energy minimization studies indicate that only minor local changes in the protein structure are required to accommodate binding of the bulkier Trp 17 side chain of the FFW peptide to the N-domain of CaM.