Allosterically Activated Protein Self-Assembly for the Construction of Helical Microfilaments with Tunable Helicity.

Protein allostery, a chemical-to-mechanical effect that can precisely regulate protein structure, exists in many proteins. Herein, we demonstrate that protein allostery can be used to drive self-assembly for the construction of tunable protein architectures. Calmodulin (CaM) was chosen as a model allosteric protein. Ca2+ -mediated contraction of CaM to a closed state can activate CaM and its ligand to self-assemble into a 1D protein helical microfilament. Conversely, relaxation of CaM to the open state can unwind and further dissociate the helical assemblies. Fine regulation of the protein conformation by tuning the external Ca2+ level allows us to obtain various protein helical nanostructures with tunable helicity. This study offers a new approach toward chemomechanically controlled protein self-assembly.

Selective Double-Labeling of Cell-Free Synthesized Proteins for More Accurate smFRET Studies.

Förster resonance energy transfer (FRET) studies performed at the single molecule level have unique abilities to probe molecular structure, dynamics, and function of biological molecules. This technique requires specimens, like proteins, equipped with two different fluorescent probes attached at specific positions within the molecule of interest. Here, we present an approach of cell-free protein synthesis (CFPS) that provides proteins with two different functional groups for post-translational labeling at the specific amino acid positions. Besides the sulfhydryl group of a cysteine, we make use of an azido group of a p-azido-l-phenylalanine to achieve chemical orthogonality. Herein, we achieve not only a site-specific but, most importantly, also a site-selective, label scheme that permits the highest accuracy of measured data. This is demonstrated in a case study, where we synthesize human calmodulin (CaM) by using a CFPS kit and prove the structural integrity and the full functionality of this protein.

Construction of a Ca(2+)-gated artificial channel by fusing alamethicin with a calmodulin-derived extramembrane segment.

Using native chemical ligation, we constructed a Ca(2+)-gated fusion channel protein consisting of alamethicin and the C-terminal domain of calmodulin. At pH 5.4 and in the absence of Ca(2+), this fusion protein yielded a burst-like channel current with no discrete channel conductance levels. However, Ca(2+) significantly lengthened the specific channel open state and increased the mean channel current, while Mg(2+) produced no significant changes in the channel current. On the basis of 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescent measurement, Ca(2+)-stimulated gating may be related to an increased surface hydrophobicity of the extramembrane segment of the fusion protein.

Development of the fluorescent biosensor hCalmodulin (hCaM)L39C-monobromobimane(mBBr)/V91C-mBBr, a novel tool for discovering new calmodulin inhibitors and detecting calcium.

A novel, sensible, and specific fluorescent biosensor of human calmodulin (hCaM), namely hCaM L39C-mBBr/V91C-mBBr, was constructed. The biosensor was useful for detecting ligands with opposing fluorescent signals, calcium ions (Ca(2+)) and CaM inhibitors in solution. Thus, the device was successfully applied to analyze the allosteric effect of Ca(2+) on trifluoroperazine (TFP) binding to CaM (Ca(2+)K(d) = 0.24 µM ± 0.03 with a stoichiometry 4.10 ± 0.15; TFPK(d) ∼ 5.74-0.53 µM depending on the degree of saturation of Ca(2+), with a stoichiometry of 2:1). In addition, it was suitable for discovering additional xanthones (5, 6, and 8) with anti-CaM properties from the fungus Emericella 25379. The affinity of 1-5, 7, and 8 for the complex (Ca(2+))(4)-CaM was excellent because their experimental K(d)s were in the nM range (4-498 nM). Docking analysis predicted that 1-8 bind to CaM at sites I, III, and IV as does TFP.

Activación de la calcio calmodulina quinasa II, CaMKII, por el uridin nucleósido difosfato, UDP, en neuronas granulares de cerebelo / Calcium Calmoduline Kinase II, CaMKII, activation by the uridine nucleoside diphosphate, UPD, in cerebellar granule neurons

Las neuronas granulares de cerebelo en cultivo presentan receptores metabotrópicos de nucleótidos de tipo P2Y6, cuyo agonista fisiológico específico es el nucleótido, uridina difosfato, UDP. Estudios de PCR muestran la presencia de este receptor y el incremento de la expresión con el tiempo. Los estudios de respuesta en célula individual mediante microfluorimetría muestran un incremento del calcio citosólico al estimular con UDP, siendo los incrementos mas significativos en el soma. El incremento del calcio citosólico produce la activación de diversos enzimas dependientes de calcio y concretamente de la calcio-calmodulina quinasa II, CAMKII, enzima que se encuentra ampliamente distribuida en toda la topografía de la neurona granular. Este enzima al activarse se auto-fosforila y mediante anticuerpos contra la forma fosforilada se pueden detectar las zonas mas activas en la célula. Cuando se estimula con UDP, la CaMKII fosforilada aparece fundamentalmente asociada al soma neural, con mucha menor actividad en las prolongaciones axodendríticas, lo que se corresponde con la distribución de los receptores P2Y6 funcionales

Cultured granule cells from cerebellum exhibit nucleotide metabotropic receptors such as the P2Y6 subtype, which physiological agonist is the uridine diphosphate, UDP. The PCR analysis show the presence of P2Y6 messenger RNA, increasing with the days in culture. Single cell microfluorimetric studies show citosolic calcium increase in response to UDP, this being more significant at the soma level. The cytosolic calcium increase triggers cellular responses mediated by calcium dependent enzymes. This is the case for calcium-calmodulin kinase II, CaMKII, which is extensively distributed through the granule neuron, according the immunocytochemical studies. This enzyme once activated is able to autophosphorylate and by using antibodies against the phosphorylated form the active zones can be detected. After UDP stimulation, the location of the phosphorylated form of CaMKII appears to be mainly at the neural soma, with lower presence at the axodendritic prolongations, which correlates with the functional P2Y6 subtype receptor distribution

Phosphorylation of calmodulin fragments by protein kinase CK2. Mechanistic aspects and structural consequences.

Calmodulin is phosphorylated in vivo and in vitro by protein kinase CK2 in a manner that is unique among CK2 substrates for being inhibited by the regulatory beta-subunit of the kinase and dramatically enhanced by polybasic peptides. Using synthetic fragments of calmodulin variably encompassing the CK2 phosphorylation sites here we show that individual phosphorylation of Thr79, Ser81, Ser101, and Thr117 is critically influenced by the size and composition of the peptides and that the C-terminal domain of calmodulin is implicated both in down-regulation of calmodulin phosphorylation by the beta-subunit and in its abnormal responsiveness to polylysine. A far-Western blot analysis discloses polylysine-dependent interaction between calmodulin and the N-terminal domain of the beta-subunit. We also show that phosphorylation of Ser81 hampers subsequent phosphorylation of Thr79 and by itself promotes the unfolding of the central helix, whose flexibility is instrumental to the interaction with calmodulin-dependent enzymes. Collectively taken, our data are consistent with a multifaceted regulation of calmodulin phosphorylation through the concerted action of distinct CaM domains, the catalytic and regulatory subunits of CK2, and polycationic effectors mimicking in vivo the effect of polylysine.

Multiplexed sorting of libraries on libraries: a novel method for empirical protein design by affinity-driven phage enrichment on synthetic peptide arrays.

Chemically synthesized peptide arrays on planar cellulose carriers are proposed as libraries of ligands suitable for the multiplexed simultaneous capture of peptide-specific acceptor proteins from a large randomly mutagenized library of acceptor proteins presented on bacteriophage M13 particles. This experimental set-up can be exploited to rapidly screen for individual new, distinct binding partners from two complementary libraries (two-dimensional screening). The technical feasibility of this empirical protein design approach was demonstrated with calmodulin as an aceptor protein using an array of mastoparan variants for multiplexed phage affinity enrichment.

Modifying Mg2+ binding and exchange with the N-terminal of calmodulin.

To follow Mg2+ binding to the N-terminal of calmodulin (CaM), we substituted Phe in position 19, which immediately precedes the first Ca2+/Mg2+ binding loop, with Trp, thus making F19WCaM (W-Z). W-Z has four acidic residues in chelating positions, two of which form a native Z-acid pair. We then generated seven additional N-terminal CaM mutants to examine the role of chelating acidic residues in Mg2+ binding and exchange with the first EF-hand of CaM. A CaM mutant with acidic residues in all of the chelating positions exhibited Mg2+ affinity similar to that of W-Z. Only CaM mutants that had a Z-acid pair were able to bind Mg2+ with physiologically relevant affinities. Removal of the Z-acid pair from the first EF-hand produced a dramatic 58-fold decrease in its Mg2+ affinity. Additionally, removal of the Z-acid pair led to a 1.8-fold increase in the rate of Mg2+ dissociation. Addition of an X- or Y-acid pair could not restore the high Mg2+ binding lost with removal of the Z-acid pair. Therefore, the Z-acid pair in the first EF-hand of CaM supports high Mg2+ binding primarily by increasing the rate of Mg2+ association.

Thermodynamic analysis of calcium and magnesium binding to calmodulin.

To elucidate some aspects still debated concerning the interaction of Ca2+ and Mg2+ with CaM, the thermodynamic binding parameters of Ca2+-CaM and Mg2+-CaM complexes were characterized by flow dialysis and isothermal microcalorimetry under different experimental conditions. In particular, the enthalpy and entropy changes associated with Ca2+ and Mg2+ binding to their sites were determined, allowing a better understanding of the mechanism underlying cation-CaM interactions. Ca2+-CaM interaction follows an enthalpy-entropy compensation relationship, suggesting that CaM explores a subspace of isoenergetical conformations which is modified by Ca2+ binding. This Ca2+-induced change in CaM dynamics is proposed to play a key role in CaM function, i.e. in its interaction with and/or activation of target proteins. Furthermore, data show that Mg2+ does not act as a direct competitor for Ca2+ binding on the four main Ca2+ binding sites, but rather as an allosteric effector. This implies that the four main Mg2+ binding sites are distinct from the EF-hand Ca2+ binding sites. Finally, Ca2+ is shown to interact with auxiliary binding sites on CaM. These weak affinity sites were thermodynamically characterized. The results presented here challenge the current accepted view of CaM ion binding.

A structure/activity study of calcium affinity and selectivity using a synthetic peptide model of the helix-loop-helix calcium-binding motif.

The acid pair hypothesis predicts the calcium affinity of the helix-loop-helix calcium-binding motif based on the number and location of acidic amino acid residues in chelating positions of the calcium-binding loop region. This study investigates the effects of the number and position of acidic residues in the loop region on calcium affinity and selectivity using 33-residue synthetic models of single helix-loop-helix calcium-binding motifs. Increasing the number of acidic residues in the octahedrally arranged chelating positions of the loop region from 3 to 4 by replacing an asparagine in the +y position with an aspartic acid increases the calcium affinity of the models between 2- and 38-fold. Differences in affinities are more pronounced in the models containing an x axis acid pair. The calcium affinities of peptide models containing 3 or 4 acidic residues in chelating positions of the loop region and an x axis acid pair are reduced when the residue in the +z position is changed from asparagine to serine. A similar reduction in calcium affinity occurs in the z axis acid paired peptides when the -x chelating residue is changed from serine to asparagine. Models with 3 acidic residues in chelating positions containing a z axis acid pair have greater calcium affinity than comparable peptide models with an x axis acid pair. The presence of x or z axis acid pairs in comparable peptides containing 4 acidic residues in chelating positions does not greatly alter calcium affinity. Calcium selectivity resides in x axis acid paired peptides, whereas z axis acid paired peptides exhibit both magnesium- and calcium-induced structural changes. This ion selectivity may be explained by postulating that the z axis residue side chains produce the initial, rate-limiting interactions with the cation, causing hydration shell destabilization and initiating the subsequent ligand interactions.

Synthetic fragments of calmodulin calcium-binding site III. A test of the acid pair hypothesis.

The acid pair hypothesis describing the interaction of calcium with the helix-loop-helix conformation of EF hands in calmodulin and related proteins predicts that these calcium-binding sites will have increased affinity for calcium if the anionic amino acid dentates in the loop region which interact directly with the cation are paired on the axial vertices of the resulting octahedral arrangement of chelating residues about the cation. As a test of this hypothesis, synthetic 33 residue analogs of bovine brain calmodulin calcium-binding site III have been prepared by the solid-phase method and analyzed for calcium affinity. The native sequence has a Kd of 735 microM for calcium and contains three anionic ligands which assume the +x, +y, and -z coordinates of the octahedral arrangement about the cation, thus precluding any pairing of the anionic ligands. This dissociation constant is 26 times weaker than that obtained from a synthetic analog of the sequentially homologous calcium-binding site III of rabbit skeletal TnC (Kd = 28 microM) which has four anionic ligands paired on the x and z axes. An analog of calmodulin site III with substitutions in the chelating residues at positions 1, 3, 5, 7, 9, and 12 of the 12-residue loop region to make these positions identical to those of rabbit skeletal troponin C site III decreased the calcium dissociation constant of the calmodulin peptide to 19 microM, similar to the troponin C peptide. Two synthetic analogs of calmodulin site III which contain three anionic ligands with two ligands paired on the x axis and two on the z axis have a Kd for calcium of 524 and 59 microM, respectively. This study provides strong support for and a better definition of the acid pair hypothesis and further demonstrates the usefulness of synthetic calcium-binding fragments in delineating the mechanism of calcium regulation of calmodulin and related proteins.

Synthesis of the dodecapeptide corresponding to domain III of bovine brain calmodulin: alpha-beta shift side reactions during the synthesis by the classical method in solution.

We report details of the chemical synthesis of the dodecapeptide corresponding to the calcium binding loop III of bovine brain calmodulin (sequence 93-104) and its fragments 96-04, 93-98, and 99-104. The preparation of the peptides employed classical solution methods and a fragment-condensation strategy. The major difficulties were encountered during the synthesis of the peptides containing the N-terminal sequences -Gly-Asn-Gly- and -Asp-Lys-Asp-Gly-Ans-Gly-, in which alpha-beta shift side reactions were observed.

A synthetic 33-residue analogue of bovine brain calmodulin calcium binding site III: synthesis, purification, and calcium binding.

The sequential solid-phase synthesis of a peptide analogue of bovine brain calmodulin calcium binding site III covering residues 81-113 of the natural sequence is described. Methionine-109 is replaced by a leucine residue to avoid complications in the synthesis and purification. In an attempt to relate the structure of the calcium binding sites in the naturally occurring calcium binding protein to the calcium affinity of these sites, the synthetic analogue is examined for calcium binding by circular dichroism spectroscopy. The calcium binding characteristics are compared to those of a synthetic analogue of the homologous calcium binding site III in rabbit skeletal troponin C. The Kd of the calmodulin site III fragment for Ca2+ is determined as 878 microM whereas the Kd of the troponin C fragment is 30 times smaller at 28 microM. Structural changes induced in the peptides by Ca2+ and trifluoroethanol are similar. This study supports our contention that the single synthetic calcium binding site is a reasonable model for the study of the structure-activity relationships of the calcium binding sites in calcium-regulated proteins such as calmodulin and troponin C.

Azidotyrosylcalmodulin derivatives. Specific probes for protein-binding domains.

Azidocalmodulin has been shown to be a useful probe for calmodulin-binding proteins in a variety of systems (Andreasen, T. J., Keller, C. H. LaPorte, D. C., Edelman, A. M., and Storm, D. R. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 2782-2785). In previous work, this calmodulin derivative was generated by the modification of lysyl residues. We report here that, on the basis of tryptic peptide mapping by reverse-phase high-pressure liquid chromatography, azidolysylcalmodulin prepared by these procedures is modified at 4 of the 7 lysines. We also report the preparation of azidocalmodulins in which the photolabile moiety is incorporated into a single known residue of the molecule by modifying one or the other of the two tyrosyl side chains. This yields azido-Tyr 99-calmodulin, with the photoaffinity label in Ca2+-binding loop III, and azido-Tyr 138-calmodulin, with the photoaffinity label in Ca2+-binding loop IV. The cross-linking characteristics of these two calmodulin derivatives show that the formation of a covalent adduct upon photolysis of a mixture of azidocalmodulin and a target protein is dependent on the location of the nitrene generated by the irradiation. Azido-Tyr 138-calmodulin shows a significant decrease in cross-linking efficiency to targets such as troponin I, troponin T, and myosin light-chain kinase, relative to azido-Tyr 99-calmodulin and the azidolysyl derivatives.

Modified calmodulin calcium binding domain III. Solid phase synthesis, purification and 1H n.m.r. characterization.

The dodecapeptide Ac-Asp-Lys-Asp-Gly-Asn-Gly-Tyr-Ile-Ser-Ala-Ala-Gaba-OH is a modified calmodulin calcium binding domain III. The synthesis of the peptide by the solid phase method with a total protection scheme using PAM-resin is reported. The purified compound has been characterized by 1H n.m.r. spectroscopy, both in the presence and in the absence of calcium ions, at various pHs. No strong specific interaction seems to occur between the peptide and Ca++ ions in water solutions.

Azidocalmodulin derivatives. Activation of, and binding to, three target proteins: aorta myosin light-chain kinase, erythrocyte (Mg2+ + Ca2+)-dependent ATPase and cardiac sarcoplasmic-reticulum kinase.

Different azidocalmodulin derivatives were synthesized by modification of either one carboxylic acid group or one or several arginine residues and their binding and activation capacity investigated in three target enzyme systems. The systems studied were smooth-muscle myosin light-chain kinase, cardiac sarcoplasmic-reticulum kinase and erythrocyte (Mg2+ + Ca2+)-dependent ATPase. The results indicated that the activation ability of each calmodulin derivative was different depending on the system studied. Binding studies carried out by the displacement of 125I-calmodulin indicated that the monosubstitutions did not greatly alter the apparent Kd of calmodulin for the enzymes but that the modification of four arginine residues caused a 4-8-fold increase in the apparent Kd in all systems. These results have shown that azidocalmodulin derivatives may have different degrees of usefulness in the study of calmodulin target proteins in different systems, with the behaviour of the derivatives not predictable on the basis of the nature (soluble or membrane-bound) or the type (ATPase or kinase) of enzyme system to be investigated. However, the monosubstituted calmodulin and, in particular, the carboxylic acid-group-modified derivative (where the modification was statistically dispersed over the protein chain) are good candidates for photolabelling calmodulin-binding proteins.