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• W1658364279 abstract "Thermodynamic parameters of interactions of calcium-saturated calmodulin (Ca2+-CaM) with melittin, C-terminal fragment of melittin, or peptides derived from the CaM binding regions of constitutive (cerebellar) nitric-oxide synthase, cyclic nucleotide phosphodiesterase, calmodulin-dependent protein kinase I, and caldesmon (CaD-A, CaD-A*) have been measured using isothermal titration calorimetry. The peptides could be separated into two groups according to the change in heat capacity upon complex formation, ΔCp. The calmodulin-dependent protein kinase I, constitutive (cerebellar) nitric-oxide synthase, and melittin peptides have ΔCp values clustered around −3.2 kJ·mol−1·K−1, consistent with the formation of a globular CaM-peptide complex in the canonical fashion. In contrast, phosphodiesterase, the C-terminal fragment of melittin, CaD-A, and CaD-A* have ΔCp values clustered around −1.6 kJ·mol−1·K−1, indicative of interactions between the peptide and mostly one lobe of CaM, probably the C-terminal lobe. It is also shown that the interactions for different peptides with Ca2+-CaM can be either enthalpically or entropically driven. The difference in the energetics of peptide/Ca2+-CaM complex formation appears to be due to the coupling of peptide/Ca2+-CaM complex formation to the coil-helix transition of the peptide. The binding of a helical peptide to Ca2+-CaM is dominated by favorable entropic effects, which are probably mostly due to hydrophobic interactions between nonpolar groups of the peptide and Ca2+-CaM. Applications of these findings to the design of potential CaM inhibitors are discussed. Thermodynamic parameters of interactions of calcium-saturated calmodulin (Ca2+-CaM) with melittin, C-terminal fragment of melittin, or peptides derived from the CaM binding regions of constitutive (cerebellar) nitric-oxide synthase, cyclic nucleotide phosphodiesterase, calmodulin-dependent protein kinase I, and caldesmon (CaD-A, CaD-A*) have been measured using isothermal titration calorimetry. The peptides could be separated into two groups according to the change in heat capacity upon complex formation, ΔCp. The calmodulin-dependent protein kinase I, constitutive (cerebellar) nitric-oxide synthase, and melittin peptides have ΔCp values clustered around −3.2 kJ·mol−1·K−1, consistent with the formation of a globular CaM-peptide complex in the canonical fashion. In contrast, phosphodiesterase, the C-terminal fragment of melittin, CaD-A, and CaD-A* have ΔCp values clustered around −1.6 kJ·mol−1·K−1, indicative of interactions between the peptide and mostly one lobe of CaM, probably the C-terminal lobe. It is also shown that the interactions for different peptides with Ca2+-CaM can be either enthalpically or entropically driven. The difference in the energetics of peptide/Ca2+-CaM complex formation appears to be due to the coupling of peptide/Ca2+-CaM complex formation to the coil-helix transition of the peptide. The binding of a helical peptide to Ca2+-CaM is dominated by favorable entropic effects, which are probably mostly due to hydrophobic interactions between nonpolar groups of the peptide and Ca2+-CaM. Applications of these findings to the design of potential CaM inhibitors are discussed. Understanding detailed molecular mechanisms that govern macromolecular interactions represents one of the major goals of structural biology. One of the important prerequisites for success in this line of research depends on the choice of an appropriate biological system. Calcium-dependent target recognition by calmodulin might be an ideal model system, because of its well characterized nature and its central role in cellular metabolism. Calmodulin (CaM)1 is a small, acidic, eukaryotic Ca2+-binding protein of 148 amino acid residues that is arranged into two lobes of similar size and structure (1Babu Y.S. Bugg C.E. Cook W.J. J. Mol. Biol... 1988; 204: 191-204Google Scholar, 2Chattopadhyaya R. Meador W.E. Means A.R. Quiocho F.A. J. Mol. Biol... 1992; 228: 1177-1192Google Scholar). Each lobe consists of two EF hand helix-loop-helix Ca2+-binding motifs, and thus CaM is capable of binding two Ca2+ ions per lobe, four in total. NMR solution structures of apo-CaM reveal that Ca2+ binding leads to significant structural rearrangements of the CaM molecule (3Kuboniwa H. Tjandra N. Grzesiek S. Ren H. Klee C.B. Bax A. Nat. Struct. Biol... 1995; 2: 768-776Google Scholar, 4Zhang M. Tanaka T. Ikura M. Nat. Struct. Biol... 1995; 2: 758-767Google Scholar). The alignment of the helices within each lobe changes upon Ca2+ binding, resulting in the exposure of two methionine-rich hydrophobic “patches,” one in each lobe (5Vogel H.J. Zhang M. Mol. Cell Biochem... 1995; 149–150: 3-15Google Scholar, 6Yuan T. Ouyang H. Vogel H.J. J. Biol. Chem... 1999; 274: 8411-8420Google Scholar). These hydrophobic patches enable CaM in the Ca2+-loaded form (Ca2+-CaM) to interact with a number of intracellular proteins and enzymes that are involved in a wide variety of different biochemical processes (7Means A.R. VanBerkum M.F. Bagchi I. Lu K.P. Rasmussen C.D. Pharmacol. Ther... 1991; 50: 255-270Google Scholar, 8Vogel H.J. Biochem. Cell Biol... 1994; 72: 357-376Google Scholar, 9Nelson M.R. Chazin W.J. Protein Sci.. 1998; 7: 270-282Google Scholar). CaM-binding sequences are generally 15–25 amino acids long with little amino acid sequence homology, but the majority do have the propensity to form an amphipathic α-helix with one or two aromatic or bulky hydrophobic “anchor” residues (often tryptophans) located on the hydrophobic face (10Crivici A. Ikura M. Annu. Rev. Biophys. Biomol. Struct... 1995; 24: 85-116Google Scholar, 11O'Neil K.T. DeGrado W.F. Trends Biochem. Sci... 1990; 15: 59-64Google Scholar, 12Rhoads A.R. Friedberg F. FASEB J... 1997; 11: 331-340Google Scholar). Binding of a target peptide to Ca2+-CaM occurs via distinct conformational changes upon which the Ca2+-CaM molecule wraps around the peptide, forming a globular complex (13Ikura M. Clore G.M. Gronenborn A.M. Zhu G. Klee C.B. Bax A. Science.. 1992; 256: 632-638Google Scholar, 14Meador W.E. Means A.R. Quiocho F.A. Science.. 1992; 257: 1251-1255Google Scholar, 15Meador W.E. Means A.R. Quiocho F.A. Science.. 1993; 262: 1718-1721Google Scholar). This is enabled by the unwinding of the central linker region of CaM, which is actually quite flexible in solution (16Heidorn D.B. Trewhella J. Biochemistry.. 1988; 27: 909-915Google Scholar, 17Barbato G. Ikura M. Kay L.E. Pastor R.W. Bax A. Biochemistry.. 1992; 31: 5269-5278Google Scholar). The peptide, which is unstructured in the unbound state, forms an α-helix upon binding to CaM, and residues on the hydrophobic face of the peptide, especially the anchor residues, interact extensively with the hydrophobic patches of CaM. All interactions between CaM and the peptide are through amino acid side chains, which is unique for a protein-protein complex. It is curious how so many different target sequences with relatively little sequence homology can be bound by CaM with such high affinity. A primary reason no doubt has to be hydrophobic effects, because of the well known importance of the hydrophobic patches of CaM and their interaction with the hydrophobic face of target sequences. “Classical” hydrophobic interactions involve the favorable burial of nonpolar groups from the contact with water and are considered to be entropic in nature (18Kauzmann W. Adv. Protein Chem... 1959; 14: 1-63Google Scholar). However, in a recent calorimetric study, it was shown that binding of a CaM target peptide from smooth muscle myosin light chain kinase (smMLCK) is driven by enthalpic not entropic factors (19Wintrode P.L. Privalov P.L. J. Mol. Biol... 1997; 266: 1050-1062Google Scholar). It could be that the increase in solvent entropy is offset by other phenomena, such as the loss of mobility of some amino acid side chains (20Siivari K. Zhang M. Palmer III, A.G. Vogel H.J. FEBS Lett... 1995; 366: 104-108Google Scholar, 21Lee A.L. Kinnear S.A. Wand A.J. Nat. Struct. Biol... 2000; 7: 72-77Google Scholar) or the loss of backbone entropy in the target peptide as it forms an α-helix in the complex (22Yuan T. Walsh M.P. Sutherland C. Fabian H. Vogel H.J. Biochemistry.. 1999; 38: 1446-1455Google Scholar). Enthalpic effects must also play a key role, and these could include van der Waal's interactions among the hydrophobic residues in the complex, as well as salt bridges and hydrogen bonds between acidic residues on CaM and basic residues on the target peptide. Because of the promiscuous nature of CaM in its ability to bind such a wide range of target sequences, it is quite possible that the relative contribution of the various factors involved in binding could be different for different target peptides. Thus, a detailed calorimetric investigation of the binding of many different peptides to CaM is warranted. Because of the vast amount of knowledge and the pivotal importance of CaM/target recognition in eukaryotic cells, a more complete understanding of the detailed mechanism of Ca2+-dependent CaM/peptide interactions can lay down the foundation for design of specific targets and inhibitors for this and other related systems. In this paper we report the results of the direct calorimetric measurements of thermodynamics of complex formation of nine different peptides with Ca2+-CaM. These peptides were derived from the CaM-binding sequences of CaM-dependent protein kinase I (23Gomes A.V. Barnes J.A. Vogel H.J. Arch. Biochem. Biophys... 2000; 379: 28-36Google Scholar), cyclic nucleotide phosphodiesterase (22Yuan T. Walsh M.P. Sutherland C. Fabian H. Vogel H.J. Biochemistry.. 1999; 38: 1446-1455Google Scholar), caldesmon (24Zhan Q.Q. Wong S.S. Wang C.L. J. Biol. Chem... 1991; 266: 21810-21814Google Scholar, 25Zhang M. Vogel H.J. Protein Pept. Lett... 1997; 4: 291-297Google Scholar, 26Zhou N. Yuan T. Mak A.S. Vogel H.J. Biochemistry.. 1997; 36: 2817-2825Google Scholar), constitutive cerebellar nitric-oxide synthase (27Zhang M. Vogel H.J. J. Biol. Chem... 1993; 268: 22420-22428Google Scholar, 28Zhang M. Yuan T. Aramini J.M. Vogel H.J. J. Biol. Chem... 1995; 270: 20901-20907Google Scholar), and bee venom melittin. 2T. Yuan, H. Ouyang, M. E. Huque, K. Siivari, and H. J. Vogel, manuscript in preparation. 2T. Yuan, H. Ouyang, M. E. Huque, K. Siivari, and H. J. Vogel, manuscript in preparation. The peptides studied are illustrated in Fig. 1. Analysis of the thermodynamic data for CaM-peptide complex formation allowed us to shed more light on the nature of physical forces underlying Ca2+-CaM/target interactions. The CaM expression plasmid pCaM, which contains a synthetic mammalian CaM gene, was a gift from Dr. T. Grundström (University of Umeå, Sweden) and has been described elsewhere (27Zhang M. Vogel H.J. J. Biol. Chem... 1993; 268: 22420-22428Google Scholar, 30Waltersson Y. Linse S. Brodin P. Grundstrom T. Biochemistry.. 1993; 32: 7866-7871Google Scholar). CaM was purified fromEscherichia coli cells containing pCaM by published methods (31Gopalakrishna R. Anderson W.B. Biochem. Biophys. Res. Commun... 1982; 104: 830-836Google Scholar, 32Putkey J.A. Ts'ui K.F. Tanaka T. Lagace L. Stein J.P. Lai E.C. Means A.R. J. Biol. Chem... 1983; 258: 11864-11870Google Scholar, 33Vogel H.J. Lindahl L. Thulin E. FEBS Lett... 1983; 157: 241-246Google Scholar). Melittin (MEL) was purchased from Sigma. The peptides corresponding to the CaM binding sequence of cerebellar nitric-oxide synthase (cNOS), cyclic nucleotide phosphodiesterase (PDE), calmodulin-dependent protein kinase I (CaMKI), caldesmon (CaD-A, CaD-A*, CaD-B1, and CaD-B2), and the C terminus of melittin (MLC) were commercially synthesized at the Core Facility for Protein/DNA Chemistry at Queens University (Kingston, Canada). The sequences of these are shown in Fig. 1. All peptides were additionally purified on Sephasyl-Peptide C18 12 μ (Amersham Pharmacia Biotech) reverse-phase column attached to the AKTA system using 0–100% acetonitrile gradient in the presence of 0.1% trifluoroacetic acid (34Richardson J.M. McMahon K.W. MacDonald C.C. Makhatadze G.I. Biochemistry.. 1999; 38: 12869-12875Google Scholar). The purity of peptides was >95% as judged by analytical high pressure liquid chromatography. For the titrations of Ca2+-CaM with all but PDE and CaD-A* peptides, the stock solutions were dialyzed extensively against at least two changes of corresponding PIPES buffer containing 5 mm PIPES, 100 mm NaCl, 2 mmCaCl2, pH 7.0. In the case of the CaMKI peptide, additional experiments were performed in buffers that contained 5 mmimidazole, sodium cacodylate, or MOPS, pH 7.0, supplemented with 100 mm NaCl and 2 mm CaCl2. The PDE and CaD-A* peptides contain cysteine residues potentially capable of forming intermolecular disulfide bonds. To keep PDE and CaD-A* in the reduced form, these peptides were incubated for 2 h in 50 mm Tris pH 7.5 buffer containing 1% β-mercaptoethanol followed by dialysis against 5 mmPIPES, 100 mm NaCl, 2 mm CaCl2, pH 7.0, buffer containing the reducing agent 1 mm tris-(carboxyethyl)-phosphine. For PDE and CaD-A* titration experiments, CaM was also dialyzed against thetris-(carboxyethyl)-phosphine-containing buffer. Spectrapor CE dialysis membranes with a 1000-Da molecular mass cut-off were used for dialyses. The concentrations of stock solutions of CaM and peptides after dialysis were measured spectrophotometrically. The following molar extinction coefficients have been used: CaM, 2,900 m−1cm−1 at 276 nm; CaMKI, PDE, MEL, MLC, CaD-A, and CaD-A*, 5690 m−1 cm−1 at 280 nm; and cNOS, 386 m−1 cm−1 at 258 nm. The extinction coefficients were calculated according as described (35Gill S.C. von Hippel P.H. Anal. Biochem... 1989; 182: 319-326Google Scholar) taking into account the corrections for light scattering (36Winder A.F. Gent W.L. Biopolymers.. 1971; 10: 1243-1251Google Scholar). Methylation of the cysteine residue of the CaD-A* peptide was performed using methyl-4-nitrobenzenesulfonate, as described (37Torchinski Y.M. Sulphur in Proteins. Pergamon Press, New York1980Google Scholar). The degree of modification was checked by the reaction with the Cys-specific reagent 5,5-dithiobis-2-nitrobenzoic acid by monitoring the changes in absorbance at 412 nm. The reaction mixture was subjected to chromatography on Sephasyl-Peptide C18 12μ (Amersham Pharmacia Biotech) reverse-phase column on an AKTA system using 0–100% acetonitrile gradient in the presence of 0.1% trifluoroacetic acid. The overall procedure for ITC experiments was similar to that described previously (38Lopez M.M. Yutani K. Makhatadze G.I. J. Biol. Chem... 1999; 274: 33601-33608Google Scholar). Briefly, 5 μl of the peptide solution at concentrations between 0.5 and 1.3 mm were injected into the cell containing Ca2+-CaM. The concentration of CaM in the cell varied between 0.01 and 0.06 mm depending on the magnitude of the observed heat effects. In the case of melittin, the titration was reversed, i.e. melittin at concentration 0.016 mm was in the cell, and CaM at concentration 0.6 mm was used in the titration syringe. This was done to avoid the heat of melittin dilution because melittin forms tetramers at high concentrations (39Wilcox W. Eisenberg D. Protein Sci... 1992; 1: 641-653Google Scholar). The blank injections of titrant into corresponding buffer were used to account for the heat of mixing and dilutions. It was found that in all cases the heat effects upon blank injections were less than 1% of the heat effect of CaM-peptide interactions. The heat of the reaction, Q, was obtained by integrating the peak after each injection of peptide ligand using ORIGIN software provided by the manufacturer. The heat of the reaction at each injection is related to the calorimetric enthalpy of binding, ΔHcal, and the other thermodynamic parameters as follows (40Wiseman T. Williston S. Brandts J.F. Lin L.N. Anal. Biochem... 1989; 179: 131-137Google Scholar). Q=n·[CaM]t·ΔHcal·Va2·A−A2−4·[pep]tn·[CaM]tEquation 1 where A=1+[pep]tn·[CaM]t+[pep]tn·Ka·[CaM]t,Equation 2 n is the stoichiometry of the peptide/CaM complex,Ka is the association constant, [CaM]tis the amount of calmodulin in the ITC cell with the volumeVo, and [pep]t is the total concentration of a peptide. ASA values were computed using the modeled three-dimensional structures of CaM complex with the studied peptides as described (41Makhatadze G.I. Privalov P.L. Adv. Protein Chem... 1995; 47: 307-425Google Scholar). The changes in the surface area were divided into four types (41Makhatadze G.I. Privalov P.L. Adv. Protein Chem... 1995; 47: 307-425Google Scholar): 1)ASAbb, the surface area of the backbone atoms C, O, N; 2) ASAarm, the aromatic surface area that is defined as all carbon atoms for Phe, Tyr, Trp except CA, CB; 3)ASAalp, the surface area for aliphatic group that includes all carbon atoms except those that are defined as backbone or aromatic; and 4) ASApol, the surface area for the polar groups that include all side chain oxygen, nitrogen, and sulfur atoms. Two different models for the calculations of the changes in ASA upon peptide/Ca2+-CaM complex formation have been used. For Type I binding, the changes in the ASA upon complex formation, ΔASA, were calculated as follows. ΔASA=ASACaM−pepti−ASACaMi−ASApepiEquation 3 whereASA CaM−pepti is the water-accessible surface of CaM-peptide complex,ASA CaMi is the water-accessible surface area of free CaM, andASA pepi is the water-accessible surface area of an unstructured peptide; superscript “i” represents one of the four type of the surface areas: backbone, polar side chains, aliphatic, and aromatic. The CaM-peptide complex was modeled according to one of the several known x-ray and NMR structures, 1CDL, which represents an x-ray structure of the smMLCK/Ca2+-CaM complex (14Meador W.E. Means A.R. Quiocho F.A. Science.. 1992; 257: 1251-1255Google Scholar). The rest of the peptides were threaded into this structure using sequence alignment of Yap et al. (42Yap K.L. Ames J.B. Swindells M.B. Ikura M. Proteins.. 1999; 37: 499-507Google Scholar) performed in the environment of Swiss/Protein Data Bank viewer as described (43Gribenko A.V. Makhatadze G.I. J. Mol. Biol... 1998; 283: 679-694Google Scholar). Several control calculations have been performed to ensure that the threading procedure is reasonably reliable in terms of ASA calculations. The most compelling evidence for the correctness of our threading is comparison of the surface area of smMLCK/Ca2+-CaM complex calculated using Protein Data Bank entry 1CDL with the area of the complex after smMLCK was threaded into the Protein Data Bank entry 1CDM instead of the original sequence corresponding to calmodulin-dependent protein kinase II. The difference is very small particularly in terms of predicted heat capacity change upon complex formation, ΔCp (see Table II). This is mostly due to the fact that the peptides become largely buried upon binding to CaM.Table IIExperimental and calculated values of the heat capacity changes upon peptide/Ca2+-CaM complex formationPeptideΔASAbbΔASApolΔASAalpΔASAarmΔCp,expΔCp,calcÅ2Å2Å2Å2kJ/(mol K) 2-aThe estimated experimental error in ΔCp, exp is at least 10%.kJ/(mol K) 2-bCalculated according to the Equation 8. The first row of values is for Type I interactions; the second row of values is for Type II, modeled as complex with N and C termini of CaM, respectively; and the third row of values is for Type II, modeled according to Protein Data Bank structure 1CFF. See text for more details.smMLCK−870 (−690)−940 (−1000)−2170 (−2010)−240 (−240)−2.72-cData from Ref. 19.−2.6 (−2.5)−670/−660−420/−580−1150/−910−20/−160−0.9/−0.5−540−260−880−150−0.9CaMKI−820−960−2360−380−3.5−3.3−650/−650−550/−480−1260/−1160−100/−310−1.2/−1.4cNOS−790−650−2450−340−3.7−3.8−670/−650−260/−240−1290/−1180−180/−150−1.6/−1.4−570−390−1130−70−1.2MEL−840−540−2240−210−2.8−3.1−710/−710−290/−190−1220/−1180−210/−50−1.4/−1.2MLC−510−700−1520−250−1.4−2.1−350/−350−250/−340−610/−830−50/−210−0.5/−1.2−310−290−940−210−1.5PDE−700−890−2350−270−2.0−3.4−520/−570−470/−500−1200/−1140−60/−210−1.3/−1.3−480−340−1000−190−1.3CaD-A−650−780−1820−380−1.6−2.6−490/−480−340 /−320−1090/−850−240/−210−1.5/−1.0−430−380−850−200−1.0CaD-A*−650−780−1820−380−1.3−2.6−490/−480−340/−320−1090/−850−240/−210−1.5/−1.0−470−400−860−220−1.02-a The estimated experimental error in ΔCp, exp is at least 10%.2-b Calculated according to the Equation 8. The first row of values is for Type I interactions; the second row of values is for Type II, modeled as complex with N and C termini of CaM, respectively; and the third row of values is for Type II, modeled according to Protein Data Bank structure 1CFF. See text for more details.2-c Data from Ref. 19Wintrode P.L. Privalov P.L. J. Mol. Biol... 1997; 266: 1050-1062Google Scholar. Open table in a new tab The surface area of the free CaM was calculated as follows. We decided not to use the reported structural coordinates of CaM based on the fact that even small perturbations in the conformation because of the refinement procedure, crystal symmetry, and resolution significantly affect the absolute values of ASA. This might lead to erroneous results because we are looking at small difference, ΔASA, between two large numbers, ASACaM−pept andASACaM. Thus, free CaM for the purpose of ASA calculations was modeled as a sum of surfaces. ASACaM=ASACaM1–76+ASACaM76–148Equation 4 where ASACaM1–76 andASACaM76–148 are the water-accessible surfaces of the N-terminal (amino acid residues 1–76) and the C-terminal (amino acid residues 76–148) domains of CaM.ASACaM1–76 andASACaM76–148 were calculated using the coordinates of the CaM-peptide complex by simply removing the coordinates of the peptide and using only the coordinates for the residues 1–76 or 76–148, respectively. The change in the surface area calculated using the structure of unligated CaM or as a sum of isolated domains did not have significant effect on the absolute values of the ΔASA change (data not shown). The Type II binding model describes binding of the peptide to only one of the N- or C-terminal lobes of CaM. For the purpose of surface area calculations for Type II binding, hypothetical complexes were modeled as follows. The coordinates of one of the domains in the threaded structure were removed, and surface area was calculated using coordinates for the remaining peptide and another domain plus a hinge region (amino acid residues 72–86). The surface area change upon complex formation was estimated as follows. ΔASATIIN=ASACaMN−pept−ASACaMN−ASApepEquation 5 ΔASATIIC=ASACaMC−pept−ASACaMC−ASApepEquation 6 where the subscripts TIIN and TIICreflect Type II binding of peptides to N- and C-terminal lobes of CaM, respectively; ASACaMN−pept and ASACaMC−pept are the surface areas of the peptide/N-terminal domain and peptide/C-terminal domain complexes; ASACaMN andASACaMC are the surface areas of N- or C-terminal domains of CaM in the absence of the other domain; andASApep is the surface area of the unbound peptide in the fully extended conformation (see above). As a test of correctness of this model, we also performed calculations based on the NMR structure of the complex of Ca2+-CaM with a target peptide from a Ca2+ pump using Protein Data Bank entry 1CFF(44Elshorst B. Hennig M. Forsterling H. Diener A. Maurer M. Schulte P. Schwalbe H. Griesinger C. Krebs J. Schmid H. Vorherr T. Carafoli E. Biochemistry.. 1999; 38: 12320-12332Google Scholar). The peptide in this structure binds to the C-terminal domain. The surface area change upon complex formation was calculated as follows. ΔASATII=ASA1CFF−ASACaM1CFF−ASApepEquation 7 where ASA1CFF is the surface area of the CaM-peptide complex and ASACaM1CFFis the surface area of the CaM only. The results of the calculations using Equations 6 and 7 are very similar (Table II). Fig. 2 A shows representative results of calorimetric titrations of Ca2+-CaM with two different peptides, CaMKI and PDE. The interaction of CaMKI with Ca2+-CaM is exothermic. In contrast, the reaction of PDE with Ca2+-CaM is endothermic. The sum of the areas of the peaks after each injection normalized per amount of CaM in the cell represents the enthalpy of binding of a given peptide to Ca2+-CaM, ΔHcal. The experimentally measured enthalpy, ΔHcal, consists of two parts. ΔHcal=ΔHb+Δn+·ΔHionEquation 8 where ΔHb is the enthalpy of bindingper se and Δn+·ΔHion is the enthalpy of linked protonation effects (LPE). The latter arises from the fact that the pKa value(s) of one or several groups on the ligand (peptide) or macromolecule (Ca2+-CaM) can change as a result of complex formation, as has been observed in a variety of other systems (45Wyman J. Gill S.J. Binding and Linkage. University Science Books, Mill Valey, CA1990Google Scholar, 46Ferrari M.E. Lohman T.M. Biochemistry.. 1994; 33: 12896-12910Google Scholar, 47Baker B.M. Murphy K.P. Biophys. J... 1996; 71: 2049-2055Google Scholar, 48Makhatadze G.I. Lopez M.M. Richardson III, J.M. Thomas S.T. Protein Sci... 1998; 7: 689-697Google Scholar, 49McCrary B.S. Bedell J. Edmondson S.P. Shriver J.W. J. Mol. Biol... 1998; 276: 203-224Google Scholar, 50Petrosian S.A. Makhatadze G.I. Protein Sci... 2000; 9: 387-394Google Scholar). Hence, the change in the protonation upon binding can be characterized as the difference in the number of protons bound before and after complex formation, Δn+. Equation 8 provides an experimental test for LPE by measuring the binding reaction in the presence of buffers with very different enthalpies of ionization, ΔHion; a plot of ΔHcal versusΔHion will allow the estimate of Δn+. Fig. 2 B compares the experimental enthalpies, ΔHcal, of the CaMKI-Ca2+-CaM complex formation in four different buffers with different enthalpies of ionizations (51Fukada H. Takahashi K. Proteins.. 1998; 33: 159-166Google Scholar): sodium cacodylate (ΔHion = −4 kJ/mol), PIPES (ΔHion = 12 kJ/mol), MOPS (ΔHion = 23 kJ/mol), and imidazole (ΔHion = 36 kJ/mol). The absence of the dependence of ΔHcal on the ΔHion of the buffers shown in Fig.2 B indicates that the interactions of Ca2+-CaM with CaMKI are devoid of LPE (Δn+ is only -0.03). This conclusion is in accord with the results obtained for other peptide/Ca2+-CaM complexes (19Wintrode P.L. Privalov P.L. J. Mol. Biol... 1997; 266: 1050-1062Google Scholar, 52Milos M. Schaer J.J. Comte M. Cox J.A. J. Biol. Chem... 1987; 262: 2746-2749Google Scholar). Because peptide binding to Ca2+-CaM is not accompanied by LPE, most of the experiments were performed in PIPES buffers, and the measured enthalpies were considered to be entirely the enthalpies of binding,i.e. ΔHcal = ΔHb. The enthalpies of binding of eight different peptides to Ca2+-CaM measured at different temperatures are presented in the TableI. It is remarkable that there is a great variation in the enthalpies of binding ranging from +90 kJ/mol to −66 kJ/mol.Table IThermodynamics of CaM-peptide interactionsPeptideTemperatureBufferNbΔHcalKa°CkJ/molm−1CaMKI5.3PIPES0.9018.8(8.0 ± 4.0) × 10710.0PIPES0.903.3(1.6 ± 1.4) × 10715.0PIPES1.00−15.1(2.8 ± 0.6) × 10720.0PIPES1.10−33.1(2.3 ± 0.3) × 10725.01-aKa = 5.0 × 108m−1 (29).PIPES0.93−50.2(3.4 ± 0.8) × 10725.0Imidazole1.03−49.4(7.0 ± 1.0) × 10725.0Imidazole1.01−52.3(2.4 ± 0.3) × 10725.0NaCacodylate0.80−51.0(6.0 ± 1.0) × 10725.0NaCacodylate0.75−49.0(4.0 ± 0.9) × 10725.0MOPS0.93−50.6(3.2 ± 0.7) × 107cNOS5.2PIPES0.9560.7∼2.4 × 10810.0PIPES0.9243.9∼1.8 × 10815.0PIPES0.9924.3∼1.7 × 10820.0PIPES0.986.7ND25.01-bKa = 5 × 108m−1 (55, 56).PIPES1.09−11.7∼1.3 × 108PDE5.2PIPES0.9051.9∼1.6 × 10811.0PIPES0.9642.7∼1.2 × 10820.0PIPES1.0024.7∼0.9 × 10824.01-cKa = 4.5 × 106m−1 (22).PIPES0.9015.9∼1.1 × 108MEL10.1PIPES1.0090.0(3.0 ± 1.0) × 10815.0PIPES1.0877.8(1.9 ± 0.5) × 10820.01-dKa = 3.3 × 108m−1 (85).PIPES1.0962.3(3.0 ± 1.0) × 108MLC5.0PIPES0.9731.4(2.3 ± 0.4) × 10615.1PIPES0.9616.7(3.8 ± 0.5) × 10625.1PIPESND2.3NDCaD-A5.0PIPES0.93−33.1(4.1 ± 0.4) × 10610.1PIPES0.90−41.8(3.5 ± 0.2) × 10615.0PIPES0.97−49.4(3.3 ± 0.3) × 10620.0PIPES0.92−57.7(2.2 ± 0.1) × 10625.01-eKa = 1.3 × 106m−1 (24).PIPES0.90−66.1(1.4 ± 0.1) × 106CaD-A*5.0PIPES1.03−24.3(0.8 ± 0.4) × 10610.0PIPES1.03−36.0(1.0 ± 0.5) × 10617.0PIPES0.97−39.3(1.7 ± 0.8) × 10625.0PIPES0.96−49.0(1.4 ± 0.7) × 106CaD-A*-CH325.0PIPES0.90−50.6(0.4 ± 0.2) × 1061-a Ka = 5.0 × 108m−1 (29Blumenthal D.K. Takio K. Edelman A.M. Charbonneau H. Titani K. Walsh K.A. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A... 1985; 82: 3187-3191Google Scholar).1-b Ka = 5 × 108m−1 (55Vorherr T. Knopfel L. Hofmann F. Mollner S. Pfeuffer T. Carafoli E. Biochemistry.. 1993; 32: 6081-6088Google Scholar, 56Zhang M. Vogel H.J. J. Biol. Chem... 1994; 269: 981-985Google Scholar).1-c Ka = 4.5 × 106m−1 (22Yuan T. Walsh M.P. Sutherland C. Fabian H. Vogel H.J. Biochemistry.. 1999; 38: 1446-1455Google Scholar).1-d Ka = 3.3 × 108m−1 (85Comte M. Maulet Y. Cox J.A. Biochem. J... 1983; 209: 269-272Google Scholar).1-e Ka = 1.3 × 106m−1 (24Zhan Q.Q. Wong S.S. Wang C.L. J. Biol. Chem... 1991; 266: 21810-21814Google Scholar). Open table in a new tab Under the conditions of the ITC experiment, the binding of most peptides to Ca2+-CaM is close to stoichiometric. This can be seen in Fig. 2 A, which shows that the heat effects during initial injections are comparable and are followed by a quick disappearance of heat effects in later injections, indicating that saturation of Ca2+-CaM with the peptide has been achieved. This means that the molar concentration ratio of peptide to Ca2+-CaM at which the heat of binding on the titration curve reaches 50% of total heat corresponds to the stoichiometry of the binding. For the studied peptides this stoichiometry is essentially one molecule of peptide per one molecule of Ca2+-CaM (TableI). This includes the PDE peptide, which in a previous study had been observed to bind Ca2+-CaM with a stoichiometry of" @default.
• W1658364279 created "2016-06-24" @default.
• W1658364279 creator A5017849319 @default.
• W1658364279 creator A5042190192 @default.
• W1658364279 creator A5043212744 @default.
• W1658364279 creator A5056229406 @default.
• W1658364279 date "2001-04-01" @default.
• W1658364279 modified "2023-10-16" @default.
• W1658364279 title "Energetics of Target Peptide Binding by Calmodulin Reveals Different Modes of Binding" @default.
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