De Novo Design of Peptides Targeted to the EF Hands of Calmodulin
2000; Elsevier BV; Volume: 275; Issue: 4 Linguagem: Inglês
10.1074/jbc.275.4.2676
ISSN1083-351X
AutoresMatteo Villain, Patricia L. Jackson, Michael K. Manion, Wen‐Ji Dong, Zhengchang Su, Giorgio Fassina, Tonny M. Johnson, Ted T. Sakai, N. Rama Krishna, J. Edwin Blalock,
Tópico(s)Protein Structure and Dynamics
ResumoThis report describes the use of the concept of inversion of hydropathy patterns to the de novo design of peptides targeted to a predetermined site on a protein. Eight- and 12-residue peptides were constructed with the EF hands or Ca2+-coordinating sites of calmodulin as their anticipated points of interaction. These peptides, but not unrelated peptides nor those with the same amino acid composition but a scrambled sequence, interacted with the two carboxyl-terminal Ca2+-binding sites of calmodulin as well as the EF hands of troponin C. The interactions resulted in a conformational change whereby the 8-mer peptide-calmodulin complex could activate phosphodiesterase in the absence of Ca2+. In contrast, the 12-mer peptide-calmodulin complex did not activate phosphodiesterase but rather inhibited activation by Ca2+. This inhibition could be overcome by high levels of Ca2+. Thus, it would appear that the aforementioned concept can be used to make peptide agonists and antagonists that are targeted to predetermined sites on proteins such as calmodulin. This report describes the use of the concept of inversion of hydropathy patterns to the de novo design of peptides targeted to a predetermined site on a protein. Eight- and 12-residue peptides were constructed with the EF hands or Ca2+-coordinating sites of calmodulin as their anticipated points of interaction. These peptides, but not unrelated peptides nor those with the same amino acid composition but a scrambled sequence, interacted with the two carboxyl-terminal Ca2+-binding sites of calmodulin as well as the EF hands of troponin C. The interactions resulted in a conformational change whereby the 8-mer peptide-calmodulin complex could activate phosphodiesterase in the absence of Ca2+. In contrast, the 12-mer peptide-calmodulin complex did not activate phosphodiesterase but rather inhibited activation by Ca2+. This inhibition could be overcome by high levels of Ca2+. Thus, it would appear that the aforementioned concept can be used to make peptide agonists and antagonists that are targeted to predetermined sites on proteins such as calmodulin. calmodulin 5-dimethylaminonaphthalene-1-sulfonyl-calmodulin methylene chloride methylanthraniloyl cyclic GMP 3-(N-morpholine)propanesulfonic acid 3′:5′-cyclic nucleotide 5′-nucleotidohydrolase activator-deficient from bovine brain reverse phase-high performance liquid chromatography 1-ethyl-2-[3- (ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl]naphtho[1,2-d]thiazolium bromide tryptic digested CaM fragment 1 (Ala1-Arg74) tryptic digested CaM fragment 2 (Asp78-Lys148) slow muscle troponin C Mastoparan 7 Accumulating evidence suggests that a simple binary code of polar and nonpolar amino acids arranged in the appropriate order is sufficient to build helical bundle structures and artificial peptides with rudimentary function (for review see Ref. 1.Blalock J.E. Nat. Med. 1995; 1: 876-878Crossref PubMed Scopus (80) Google Scholar). Therefore, only the sequence location, not the identity, of the polar and nonpolar amino acids must be explicitly specified for the formation of a stable helical structure or biologically active peptide. Such binary coding has been successfully employed to produce biologically active analogs of corticotrophin and growth hormone-releasing hormone (2.Clarke B.L. Blalock J.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9708-9711Crossref PubMed Scopus (17) Google Scholar, 3.Weigent D.A. Clarke B.L. Blalock J.E. ImmunoMethods. 1994; 5: 91-97Crossref PubMed Scopus (23) Google Scholar), to design proteins that fold into compact α-helical bundles (4.Kamtekar S. Schiffer J.M. Xiong H. Babik J.M. Hecht M. Science. 1993; 262: 1680-1685Crossref PubMed Scopus (659) Google Scholar), and to develop computer programs that simulate or predict some aspects of protein folding (5.Dill K.A. Bromberg S. Yue K. Fiebig K.M. Yee D.P. Thomas P.D. Chan H.S. Protein Sci. 1995; 4: 561-602Crossref PubMed Scopus (1353) Google Scholar). Considering that 20 different amino acids are encompassed by the binary code, one would expect a marked degree of sequence degeneracy for a given shape, since any one of a number of specific polar or nonpolar amino acids could occupy a given position in the sequence. Indeed, experimental evidence has confirmed that gross shape is degenerate with regard to sequence in that any number of different primary amino acid sequences with the same binary code can fold into compact α-helical structures (4.Kamtekar S. Schiffer J.M. Xiong H. Babik J.M. Hecht M. Science. 1993; 262: 1680-1685Crossref PubMed Scopus (659) Google Scholar). More recent studies have shown that, unlike simple helical structures, a five-letter amino acid alphabet is minimally required to build a well ordered, β-sheet containing protein architecture (6.Riddle D.S. Santiago J.V. Bray-Hall S.T. Doshi N. Grantcharova V.P. Yi Q. Baker D. Nat. Struct. Biol. 1997; 4: 805-809Crossref PubMed Scopus (277) Google Scholar). In this particular instance, a small β-sheet protein, the SH3 domain, could be constructed with 95% of the residues being Ile, Lys, Glu, Ala, and Gly. Interestingly, the pattern of hydropathy of the wild type SH3 domain was largely maintained in the two sequence-simplified structures. 1M. Villain and J. E. Blalock, unpublished observations. This would seem once again to point to the importance of the pattern of hydropathy in building more complex structures as well as simple helical ones. If, as described above, the gross architecture of a peptide or protein is in part determined by its pattern of hydropathy, then exactly inverting a particular pattern or code may result in a second peptide or protein with a complementary surface contour to the first since the hydrophobic effect is involved yet in reversed orientation (for review see Ref. 1.Blalock J.E. Nat. 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In the later instance, since A and U are complementary and in the second codon position specify hydrophilic and hydrophobic R groups, respectively, and considering that second base G and C generally encode slightly hydrophilic R groups, amino acid sequences deciphered from noncoding strands of DNA will have exactly inverted patterns of hydropathy relative to those of coding strands (for review see Ref. 1.Blalock J.E. Nat. Med. 1995; 1: 876-878Crossref PubMed Scopus (80) Google Scholar). Such peptides specified by complementary nucleotide sequences (11.Bost K.L. Smith E.M. Blalock J.E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1372-1375Crossref PubMed Scopus (179) Google Scholar) or designed by simply inverting the hydropathic pattern (8.Fassina G. Roller P.P. Olson A.D. Thorgeirsson S.S. Omichinski J.G. J. Biol. Chem. 1989; 264: 11252-11257Abstract Full Text PDF PubMed Google Scholar) are termed complementary peptides and have characteristics suggestive of complementary structure. 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ImmunoMethods. 1994; 5: 158-166Crossref PubMed Scopus (21) Google Scholar, 31.Jarpe M.A. Blalock J.E. Basava C. Anantharamiah G.M. Peptides: Design, Synthesis, and Biological Activity. Springer-Verlag Inc., New York1994: 165-179Crossref Google Scholar, 32.Blalock J.E. Trends Biotechnol. 1990; 8: 140-144Abstract Full Text PDF PubMed Scopus (103) Google Scholar, 33.Borovsky D. Powell C.A. Nayar J.K. Blalock J.E. Hayes T.K. FASEB J. 1994; 8: 350-355Crossref PubMed Scopus (57) Google Scholar, 34.Pascual D.W. Bost K.L. Pept. Res. 1989; 2: 207-212PubMed Google Scholar). Most recently, a novel hormone receptor was cloned, and its binding site was localized using this principle (17.Ruiz-Opazo N. Akimoto K. Herrera V.L.M. Nat. Med. 1995; 1: 1074-1081Crossref PubMed Scopus (69) Google Scholar). Not unexpectedly, the aforementioned degeneracy that is observed for the relationship between sequence and structure applies to the complementary sequence since the same codes are operative. Thus a number of complementary peptides that are properly patterned are predicted to bind the appropriate target sequence. This has been previously observed (35.Blalock J.E. Bost K.L. Biochem. J. 1986; 234: 679-683Crossref PubMed Scopus (104) Google Scholar, 36.Torres B.A. Johnson H.M. J. Neuroimmunol. 1990; 27: 191-199Abstract Full Text PDF PubMed Scopus (12) Google Scholar, 37.Johnson H.M. Torres B.A. Langone J.J. ImmunoMethods. Academic Press, Orlando1994: 167-171Google Scholar). The above results suggested that one could specifically target a complementary peptide to interact with a particular site(s) on a given protein. This was initially tested with an eight-residue complementary peptide (termed calcium-like peptide, CALP1) designed to interact with an EF hand motif based on the primordial Ca2+-binding site of the troponin C superfamily (38.Dillon J. Woods W.T. Guarcello V. LeBoeuf R.D. Blalock J.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9726-9729Crossref PubMed Scopus (17) Google Scholar). Micromolar concentrations of CALP1 together with CaM,2 but neither CALP1 nor CaM alone, were found to activate phosphodiesterase in the absence of Ca2+. This demonstrated that the combination of CALP1 and CaM functioned similarly to a combination of Ca2+ and CaM. These results implied but did not prove that CALP1 directly interacted with CaM. Thus in the present studies we have sought evidence for direct binding of CALP1 to CaM, and we tested whether this occurs at or near the EF hands of CaM. Also, as a consequence of the CALP1 design being based on the primordial EF hand motif (38.Dillon J. Woods W.T. Guarcello V. LeBoeuf R.D. Blalock J.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9726-9729Crossref PubMed Scopus (17) Google Scholar, 39.Demaille J.G. Calcium Cell Funct. 1982; 2: 111-144Crossref Google Scholar), the hydropathic profile of CALP1 is not perfectly inverted relative to the EF hand motifs of CaM. In the present study, we have designed a complementary peptide to invert more perfectly the pattern of hydropathy and to extend the sequence length to determine the role of these two factors on the interaction with CaM. Specifically, we synthesized a 12-mer peptide (CALP2; Table I) with an optimized and extended inverted hydropathic profile to EF hand motif 4 of human CaM using AMINOMAT® (Tecnogen, Italy) (40.Fassina G. Cassani G. Corti A. Arch. Biochem. Biophys. 1992; 296: 137-143Crossref PubMed Scopus (27) Google Scholar), a computer program able to determine the optimal complementary peptide sequence to a target. In order to distinguish if any increased affinity was due to the optimal hydropathic pattern or to the increased length or both, we also synthesized CALP3, which represents the first eight residues of CALP2, and CALP4, a 12-mer peptide identical to CALP1 in the first eight residues and CALP2 in the last four (Table I). We tested the binding characteristics of these peptides for CaM using three different approaches as follows: inhibition of complex formation between CaM and the EF hand-binding dye, Stains-all; changes in the emission spectra of dansylated CaM to monitor conformational changes; and real time measurement of the interaction with a biosensor based on surface plasmon resonance detection. We also characterized certain of the interactions with nuclear magnetic resonance spectroscopy and tested for CaM activation with a CaM-stimulated phosphodiesterase (PDE) assay. Finally, we tested the generality of the interaction by assessing the ability of CALP2 and -3 to bind the EF hands of another Ca2+-binding protein, troponin C.Table ICALP series and hydropathy plots* All peptides were synthesized as amino-terminal free amine and carboxyl-terminal free carboxylic acid. Open table in a new tab * All peptides were synthesized as amino-terminal free amine and carboxyl-terminal free carboxylic acid. In all the experiments, buffers were treated with the chelating resin Chelex 100. Stock solution of the peptides were checked for Ca2+ contamination by atomic absorption. Levels were always below 2 μm Ca2+/mm CALP. Thus, Ca2+ contamination was at most 200 nm and was usually 2–20 nm. These levels are below those required to affect any of the assays. Preloaded polyethylene glycol graft polystyrene,O-pentafluorophenyl ester amino acid, and 1-hydroxy-7-azabenzotriazole were purchased from Perspective Biosystems (Framingham, MA). High purity CaM from bovine brain and biotin-labeled calmodulin were purchased from Calbiochem. Its purity was checked by SDS-polyacrylamide gel electrophoresis and the identity verified by electrospray ionization-mass spectroscopy (University of Alabama at Birmingham Core Facility). Neutravidin was from Pierce. D-CaM, PDE, MOPS, Chelex 100, and Stains-all were from Sigma.N,N-Dimethylformamide, DCM, and acetonitrile were purchased from Mallinckrodt (Paris, KY). Mant-cGMP was from Molecular Probes (Eugene, OR). All other reagents were high purity grade obtained from Fisher or Sigma. Selection of the complementary peptide targeted to CaM EF hand 4 motif was carried out as described previously (40.Fassina G. Cassani G. Corti A. Arch. Biochem. Biophys. 1992; 296: 137-143Crossref PubMed Scopus (27) Google Scholar) using the computer program AMINOMAT® (Tecnogen ScpA, Italy), with an averaging window r = 9, a range of inverted hydropathy of 0.8 and considering also the eight amino acids of the flanking regions. The program generated 1,417,176 possible sequences, and we chose the one with the lowest Q value (0.0068). This process resulted in a 12-residue peptide defined CALP2. CALP3 was generated by eliminating the four carboxyl-terminal amino acids of CALP2. CALP4 is a chimeric sequence carrying the eight amino acids of CALP1 with the addition at the carboxyl terminus of the four amino acids from the corresponding region of CALP2 (Table I). A scrambled version of CALP2 and peptides with sequences not related to the CALP series were used as negative controls. All the peptides were synthesized in our laboratory using continuous flow solid phase peptide synthesis with Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry, on a PerSeptive Biosystems 9050 Peptide synthesizer. Pre-activatedO-pentafluorophenyl ester amino acids with 1-hydroxy-7-azabenzotriazole and preloaded polyethylene glycol graft polystyrene resin was used. All the peptides were purified by preparative RP-HPLC. The purity of the product was checked by analytical RP-HPLC. The identity of the peptides was confirmed by time of flight matrix-assisted laser desorption ionization mass spectrometry (University of Alabama at Birmingham Core Facility). CaM was suspended in water (300 μm final concentration) and aliquoted. All the peptides were suspended in a 3.3 mm MOPS solution, pH 7.2. Stains-all dye (60 μm) in ethyl glycerol was prepared fresh before each experiment, starting from a stock solution of 500 μm. All experiments were conducted using final concentrations of 1 μm CaM and 20 μm dye. Plastic tubes and the cuvette were washed with EGTA (0.2 m) and rinsed with Chelex water to remove any traces of Ca2+. For the assay 1.4 μl of CaM and different quantities of the peptides were added to 3.3 mm MOPS buffer to reach a final volume of 350 μl. The solutions were incubated at room temperature for 15 min. Ten minutes before reading, 150 μl of the dye solution was added and gently mixed. The spectra of the Stains-all·CaM complexes were recorded on a Shimadzu UV160 spectrophotometer between 400 and 700 nm. Data are presented as (A [ligand]/A [0] × 100) at 639 nm. D-CaM was solubilized in buffer containing 20 mm Tris-HCl, 250 mm NaCl, 5 mm MgCl2, pH 8.0. Measurements were done in duplicate on a PTI fluorescence instrument equipped with a Delta ScanTM dual monochrometer illuminator (South Brunswick, NJ) with a fixed excitation wavelength of 340 nm and scanning the emission between 400 and 600 nm. EGTA (0.5 mm final concentration) was added to the D-CaM (200 nm) solution to reverse any Ca2+ contamination. Increasing amounts of the peptides or Ca2+ were added to the solution, and the increase in emission at 494 nm was measured. Intensity data were corrected for dilution. The data are presented as (Emission[ligand]/Emission[0]) × 100 at 490 nm. Direct measure of the affinities of the peptides for CaM were obtained using a surface plasmon resonance detector (IAsys, Affinity Sensor, Cambridge, UK). A carboxymethylated dextran cuvette was prepared immobilizing Neutravidin using an NHS/EDC activation procedure according to the manufacturer's protocols (IAsys protocol 1.1) This was followed by the attachment of biotin-labeled CaM to the Neutravidin on the cuvette. Binding buffer was 20 mm MOPS, 150 mm NaCl, pH 7.4, containing 0.05% Tween 20 treated with Chelex 100. K d values were calculated using the software FASTfit, supplied by the manufacturers for the analysis of the binding curves. For the association, the curves were fitted to a single rate constant. For the dissociation we also used a single rate constant. The rabbit slow muscle troponin C mutants (C84S/Y111W, C84/R147W, and C84S/Y111W/R147W) were a gift of Dr. Herbert Cheung, and their preparation and characteristics are discussed elsewhere. 3W. J. Dong, J. M. Robinson, J. Xing, P. K. Umeda, and H. C. Cheung, submitted for publication. Steady state fluorescence for peptide-protein interaction were conducted using a ISS PC1 photo-counting fluorometer using a 0.1-mm slit. Protein was solubilized at 1 μm in 50 mm MOPS, 100 mm KCl, 50 μm EDTA, pH 7.2. Peptides were solubilized at a 3 mm stock concentration in this protein containing solution. By using 295 nm as the excitation wavelength, we measured the emission spectra between 300 and 400 nm with increasing concentration of the peptides. Spectra were recorded 5 min after addition of the peptide stocks. Acrylamide quenching of CALP2·TnC complex (75 and 1 μm, respectively) and of TnC (1 μm) were conducted in the same buffer, by the addition of aliquots of an 8 m acrylamide solution with λex = 295 nm and λem at the peak of the emission spectrum. In the range of acrylamide concentrations used (0–400 mm), we observed a linear relationship between tryptophan emission decay and quencher concentration. The quenching data were fitted to a modified Stern-Volmer equation,F 0/F = (1 +K SV [Q]); whereF 0 and F are the fluorescence intensities in the absence of and presence of quencher, respectively; [Q] is the molar quencher concentration; andK SV is the Stern-Volmer dynamic quenching constant. Time-resolved fluorescence measurements were conducted as previously reported (42.Dong W.J. Wang C.K. Gordon A.M. Cheung H.C. Biophys. J. 1997; 72: 850-857Abstract Full Text PDF PubMed Scopus (29) Google Scholar). Transient kinetic measurements were performed on a Hi-Tech Scientific PQ/SF-53 stopped-flow spectrometer equipped as described (43.Dong W.J. Wang C.K. Gordon A.M. Rosenfeld S.S. Cheung H.C. J. Biol. Chem. 1997; 272: 19229-19235Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). In a typical binding experiment, one syringe contained 10 μm C84S/R147W TnC or C84S/R147W TnC together with 50 μm CALP2 in 30 mm MOPS, 100 mmKCl, and 50 μm EGTA, pH 7.2. The protein was dialyzed against Chelex 100 resin before use. The other syringe contained 10 mm Ca2+ in the same buffer. CD studies were conducted on a 62 DS VCD spectrometer AVIV (Lakewood, NJ) using a 0.01-cm quartz cuvette, with a scanning range of 195–310 nm, a time constant of 2 s, and a scanning rate of 0.5 nm/s. The buffer used was 20 mmphosphate, pH 7.0 Measurements were obtained using a continuous fluorimetric assay based on Mant-cGMP. Mant-cGMP, a fluorescent derivative of cGMP, shows maximal fluorescence emission at 450 nm. Hydrolysis of Mant-cGMP by PDE in the presence of CaM and Ca2+ causes a decrease in the emission spectra of the compound. The enzymatic reaction can be followed in real time using 280 nm as excitation wavelength and monitoring the emitted light at 450 nm. Experiments were conducted using an excess of the substrate at fixed concentrations of CaM and PDE with constant mixing. By using these conditions the V max of the reaction can be calculated by the slope of the rectilinear part of the enzymatic reaction curve. Mant-cGMP was dissolved in MeOH (1.6 mm) and kept on ice. The reaction was conducted in 0.01 m MOPS, 0.09 m KCl, 5 mm MgCl2, 0.1 mm EGTA, pH 7, at 37 ± 2 °C. Mant-cGMP was diluted in the reaction buffer (8 μm final concentration) with CaM (5 nm) and the different peptides or free Ca2+. The system was equilibrated for 5 min. The reaction was followed using a PTI fluorescence instrument. The rate of the reaction was calculated with a linear regression of 150 data points after addition of PDE. Excitation wavelength was 280 nm, and emitted light was monitored at 450 nm, collecting 30 data points/min. The reaction was started by addition of PDE (0.05 units/ml). For Fig. 10, the percent inhibition was calculated using the formula: 100 − ((V max(pep) −V max(no Cam))/(V max(no peptide)− V max(no Cam))·100), whereV max(pep) is the rate of degradation of cGMP for a given Ca2+ concentration in presence of the peptides,V max(no peptide) is the rate of degradation for the same Ca2+ concentration without the peptides, andV max(no CaM) is the basal rate without CaM, defined as the basal activity of PDE. Escherichia coli strain BL21 (DE3) harboring plasmid pRK7a for Drosophila melanogaster CaM was obtained from Dr. K. Beckingham (Rice University). Unlabeled CaM was expressed in cells grown in Terrific Broth, and 15N-labeled CaM was purified from cells grown in M9 medium using15NH4Cl as the sole nitrogen source. CaM (labeled and unlabeled) was purified by repeated chromatography on phenyl-Sepharose CL4B followed by ion exchange chromatography on DEAE-cellulose using a linear gradient (0–0.5 m) of sodium chloride in 0.05 m sodium phosphate, pH 5.7. Under these conditions, CaM is obtained as a single peak, eluting at ∼0.35m NaCl. 15N-Labeled CaM tryptic fragments, TRC1 (amino-terminal domain) and TRC2 (carboxyl-terminal domain), were prepared as described previously (44.Andersson A. Forsen S. Thulin E. Vogel H.J. Biochemistry. 1983; 22: 2309-2313Crossref PubMed Scopus (97) Google Scholar, 45.Vogel H.J. Lindahl L. Thulin E. FEBS Lett. 1983; 157: 241-246Crossref Scopus (66) Google Scholar) except the purification of the two fragments was done by RP-HPLC on a Waters Delta Pack C18 300 A (300 × 39 mm inner diameter) column. The fragment identity was check by time of flight matrix-assisted laser desorption ionization mass spectrometry. NMR chemicals shift were consistent with the literature (46.Thulin E. Andersson A. Drakenberg T. Forsen S. Vogel H.J. Proc. Natl. Acad. Sci. U. S. A. 1970; 71: 1862-1870Google Scholar). Apocalmodulin samples were prepared by dissolving the sample in 3 ml of water and dialyzed against 2 changes of 1 m EDTA, 1 mm urea, pH 7.8, (2 times) followed by 0.5 m KCl (2 times), 0.25 m KCl (2 times), and deionized, distilled water (3 times). NMR samples were prepared by dissolving the protein sample in either 100% D2O (Cambridge Isotopes, MA) or 90% H2O, 10% D2O. The pH was adjusted using 0.1 mm DCl or 0.1 mm NaOD solutions. Concentrated peptide sample (2.0 mm CALP1 or 4.0 mm CALP3) solutions were prepared, the pH was adjusted and then titrated into the protein samples. One-dimensional 1H NMR spectra were acquired using a 12 ppm spectral width and presaturation of the HOD resonance. These spectra were acquired on either a Bruker WH400 or a Bruker AM600 instrument. The spectra were processing by Felix (MSI, San Diego, CA). Two-dimensional 1H-15N HSQC (47.Bodenhausen G. Ruben D.J. Chem. Phys. Lett. 1980; 69: 185-189Crossref Scopus (2435) Google Scholar) data were acquired on the Bruker AM600 instrument. The spectra were acquired with 12 ppm spectral width, 2048 complex t 2 points, and 512 t 1 points. All measurements were made at 298 K. Spectra were processed using Felix 95.0 (MSI, San Diego, CA), by zero filling to 2 by 2K data sets. Solvent suppression and first point correction were used for each spectra. A 45° shifted sinebell apodization function was applied in each dimension. By using a biosensor based on surface plasmon resonance detection, we were able to measure the real time interaction of CALP1 and CALP2 with CaM (Fig.1). This binding was specific in that free CaM (25 μm) in the binding buffer completely inhibited the interaction (data not shown). When the binding curves were analyzed with the computer program Fast-Fit, using a first order kinetic equation, CALP1 and CALP2 were found to have dissociation constant (K d) for CaM of 88 and 7.9 μm, respectively (Fig. 1). Thus, optimizing the pattern of inverted hydropathy and increasing the complementary peptide length from 8 to 12 residues resulted in a 11-fold increase in affinity. The binding that was observed by surface plasmon resonance detection could be due to CALP interaction with the EF hands of CaM or its hydrophobic core or both. Based on the following, it is unlikely that CALP is simply a binding peptide for the hydrophobic pocket of CaM. First, our CALP peptides don't match any of the Block consensus patterns for CaM-binding motifs (48.Henikoff J.G. Henikoff S. Pietrokovski S. Nucleic Acids Res. 1999; 27: 226-228Crossref PubMed Scopus (84) Google Scholar, 49.Henikoff S. Henikoff J.G. Genomics. 1994; 19: 97-107Crossref PubMed Scopus (347)
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