Interaction of terminal transferase with single-stranded DNA

A 58-kDa monomer of terminal transferase was isolated from calf thymus using a monoclonal antibody affinity column. The enzymatic activity was comparable to that of the 44-kDa alpha beta dimer isolated by conventional methods. Binding of the two enzyme forms to single-stranded DNA was monitored by fluorescence. The site size of both forms was approximately 11 +/- 2 nucleotides. Binding of the 44-kDa alpha beta dimer to polydeoxyadenosine was examined under several conditions. The cooperativity parameter increased from about 90 in the presence of Mg2+ to 300-400 in the absence of Mg2+. The observed dissociation constant of 3-5 microM was essentially independent of salt concentration, whereas the intrinsic dissociation constant decreased about 5-fold in the presence of Mg2+. The binding parameters of the 58-kDa monomer were independent of buffer composition and were similar to those of the 44-kDa alpha beta dimer in the presence of Mg2+. These results indicate that the additional 14-kDa peptide sequences present in the high molecular mass monomer form are not part of the DNA-binding site of terminal transferase.     

Calbindin D28k EF-hand ligand binding and oligomerization: four high-affinity sites--three modes of action

Calbindin D28k, a highly conserved protein with Ca2+-sensing and Ca2+-buffering capabilities, is abundant in brain and sensory neurons. This protein contains six EF-hand subdomains, four of which bind Ca2+ with high affinity. Calbindin D28k can be reconstituted from six synthetic peptides corresponding to the six EF-hands, indicating a single-domain structure with multiple interactions between the EF-hand subdomains. In this study, we have undertaken a detailed characterization of the Ca2+-binding and oligomerization properties of each individual EF-hand peptide using CD spectroscopy and analytical ultracentrifugation. Under the conditions tested, EF2 is monomeric and does not bind Ca2+, whereas EF6, which binds Ca2+ weakly, aggregates severely. We have therefore focused this study on the high-affinity binding sites, EF-hands 1, 3, 4, and 5. Our sedimentation equilibrium data show that, in the presence of Ca2+, EF-hands 1, 3, 4, and 5 all form dimers in solution in which the distribution between the monomer, dimer, and higher order oligomers differs. The processes of Ca2+ binding and oligomerization are linked to different degrees, and three main mechanisms emerge. For EF-hands 1 and 5, the dimer binds Ca2+ more strongly than the monomer and Ca2+ binding drives dimerization. For EF-hand 4, dimer formation requires only one of the monomers to be Ca2+-bound. In this case, the Ca2+ affinity is independent of dimerization. For EF-hand 3, dimerization occurs both in the absence and presence of Ca2+, while oligomerization increases in the presence of Ca2+.     

Monomeric state of S100P protein: Experimental and molecular dynamics study

S100 proteins constitute a large subfamily of the EF-hand superfamily of calcium binding proteins. They possess one classical EF-hand Ca²⁺-binding domain and an atypical EF-hand domain. Most of the S100 proteins form stable symmetric homodimers. An analysis of literature data on S100 proteins showed that their physiological concentrations could be much lower than dissociation constants of their dimeric forms. It means that just monomeric forms of these proteins are important for their functioning. In the present work, thermal denaturation of apo-S100P protein monitored by intrinsic tyrosine fluorescence has been studied at various protein concentrations within the region from 0.04–10 μM. A transition from the dimeric to monomeric form results in a decrease in protein thermal stability shifting the mid-transition temperature from 85 to 75 °C. Monomeric S100P immobilized on the surface of a sensor chip of a surface plasmon resonance instrument forms calcium dependent 1 to 1 complexes with human interleukin-11 (equilibrium dissociation constant 1.2 nM). In contrast, immobilized interleukin-11 binds two molecules of dimeric S100P with dissociation constants of 32 nM and 288 nM. Since effective dissociation constant of dimeric S100P protein is very low (0.5 μM as evaluated from our data) the sensitivity of the existing physical methods does not allow carrying out a detailed study of S100P monomer properties. For this reason, we have used molecular dynamics methods to evaluate structural changes in S100P upon its transition from the dimeric to monomeric state. 80-ns molecular dynamics simulations of kinetics of formation of S100P, S100B and S100A11 monomers from the corresponding dimers have been carried out. It was found that during the transition from the homo-dimer to monomer form, the three S100 monomer structures undergo the following changes: (1) the helices in the four-helix bundles within each monomer rotate in order to shield the exposed non-polar residues; (2) almost all lost contacts at the dimer interface are substituted with equivalent and newly formed interactions inside each monomer, and new stabilizing interactions are formed; and (3) all monomers recreate functional hydrophobic cores. The results of the present study show that both dimeric and monomeric forms of S100 proteins can be functional.     

The crystal structure at 2A resolution of the Ca2+ -binding protein S100P

S100P is a small calcium-binding protein of the S100 EF-hand-containing family of proteins. Elevated levels of its mRNA are reported to be associated with the progression to hormone independence and metastasis of prostate cancer and to be associated with loss of senescence in human breast epithelial cells in vitro. The first structure of human recombinant S100P in calcium-bound form is now reported at 2.0A resolution by X-ray diffraction. A flexible linker connects the two EF-hand motifs. The protein exists as a homodimer formed by non-covalent interactions between large hydrophobic areas on monomeric S100P. Experiments with an optical biosensor to study binding parameters of the S100P monomer interaction showed that the association rate constant was faster in the presence of calcium than in their absence, whereas the dissociation rate constant was independent of calcium. The K(d) values were 64(+/-24)nM and 2.5(+/-0.8) microM in the presence and in the absence of calcium ions, respectively. Dimerization of S100P is demonstrated in vivo using the yeast two-hybrid system. The effect of mutation of specific amino acids suggests that dimerization in vivo can be affected by amino acids on the dimer interface and in the hydrophobic core.     

Conformational change coupling the dimerization and activation of KSHV protease

The mechanism of herpesviral protease activation upon dimerization was studied using two independent spectroscopic assays augmented by directed mutagenesis. Spectroscopic changes, attributable to dimer interface conformational plasticity, were observed upon dimerization of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr). KSHV Pr's dissociation constant of 585 +/- 135 nM at 37 degrees C was measured by a concentration-dependent, 100-fold increase in specific activity to a value of 0.275 +/- 0.023 microM product min(-1) (microM enzyme)(-1). A 4 nm blue-shifted fluorescence emission spectrum and a 25% increase in ellipticity at 222 nm were detected by circular dichroism upon dimer association. This suggested enhanced hydrophobic packing within the dimer interface and/or core, as well as altered secondary structures. To better understand the structure-activity relationship between the monomer and the dimer, KSHV Pr molecules were engineered to remain monomeric via substitution of two separate residues within the dimer interface, L196 and M197. These mutants were proteolytically inactive while exhibiting the spectroscopic signature and thermal stability of wild type, dissociated monomers (T(M) = 75 degrees C). KSHV Pr conformational changes were found to be relevant in vivo, as the autoproteolytic inactivation of KSHV Pr at its dimer disruption site [Pray et al. (1999) J. Mol. Biol. 289, 197-203] was detected in viral particles from KSHV-infected cells. This characterization of structural plasticity suggests that the structure of the KSHV Pr monomer is stable and significantly different from its structure in the dimer. This structural uniqueness should be considered in the development of compounds targeting the dimer interface of KSHV Pr monomers.     

Calcium-binding calmyrin forms stable covalent dimers in vitro, but in vivo is found in monomeric form

The EF-hand Ca(2+)-binding protein calmyrin is expressed in many tissues and can interact with multiple effector proteins, probably as a sensor transferring Ca(2+) signals. As oligomerization may represent one of Ca(2+)-signal transduction mechanisms, we characterised recombinant calmyrin forms using non-reducing SDS/PAGE, analytical ultracentrifugation and gel filtration. We also aimed at identification of biologically active calmyrin forms. Non-reducing SDS/PAGE showed that in vitro apo- and Ca(2+)-bound calmyrin oligomerizes forming stable intermolecular disulfide bridges. Ultracentrifugation indicated that at a 220 microM initial protein concentration apo-calmyrin existed in an equilibrium of a 21.9 kDa monomer and a 43.8 kDa dimer (trimeric or tetrameric species were not detected). The dimerization constant was calculated as Ka = 1.78 x 103 M(-1) at 6oC. Gel filtration of apo- and Ca(2+)-bound calmyrin at a 100 microM protein concentration confirmed an equilibrium of a monomer and a covalent dimer state. Importantly, both monomer and dimer underwent significant conformational changes in response to binding of Ca(2+). However, when calmyrin forms were analyzed under non-reducing conditions in cell extracts by Western blotting, only monomeric calmyrin was detected in human platelets and lymphocytes, and in rat brain. Moreover, in contrast to recombinant calmyrin, crosslinking did not preserve any dimeric species of calmyrin regardless of Ca(2+) concentrations. In summary, our data indicate that although calmyrin forms stable covalent dimers in vitro, it most probably functions as a monomer in vivo.     

CD31 (PECAM-1) exists as a dimer and is heavily N-glycosylated.

CD31 (PECAM-1) is a highly abundant cell surface glycoprotein expressed on hemopoietic and endothelial cells where it functions as a homophilic adhesion and signaling receptor. Since dimerization and appropriate glycosylation are important features in the regulation of cell surface interactions and signal transduction, we studied the pattern of glycosylation as well as the ability of CD31 to undergo dimerization, both in solution and when expressed on cell membranes. CD31 is heavily glycosylated, with an approximate carbohydrate content of 21%. Nineteen neutral and thirteen sialylated glycans were identified. Ultracentrifugation analysis showed that soluble recombinant CD31 exists in equilibrium between a monomer and a dimer with an approximate dissociation constant of 12.5 microM. Chemical cross-linking studies of both soluble and membrane-expressed CD31 confirmed that CD31 exists as a dimer. These studies suggest that, like E-cadherin, PECAM-dimerization is likely to play a role in CD31 adhesion and signaling.     

Investigating site-specific effects of the -X glutamate in a parvalbumin CD site model peptide

The -X glutamate in a 33-residue model peptide comprising the CD site of carp parvalbumin 4.25 (ParvCD) was replaced with aspartate (ParvCD-XD) and the effect on calcium-dependent dimerization and calcium affinity assessed. The peptide ParvCD demonstrates a 10(5)-fold lower calcium affinity than the same site in the native protein. Both the ParvCD and ParvCD-XD model peptides fail to bind magnesium. The low calcium affinity and failure of the model ParvCD site to bind magnesium may be due to higher enthalpic costs of chelation by the -X glutamate. Replacement of the -X glutamate with an aspartate resulted in a twofold increase in the calcium affinity of both the monomer and dimer forms and a twofold increase in the calcium dependent dimerization of the peptide. A -X glutamate to aspartate replacement in 33-residue model peptides corresponding to bovine brain calmodulin site 3 (R. M. Procyshyn and R. E. Reid, Arch. Biochem. Biophys. 311, 425-429, 1994) and in Escherichia coli d-galactose-binding protein (S. K. Drake, K. L. Lee, and J. J. Falke, Biochemistry 35, 6697-6705, 1996) agree with results in the ParvCD site. However, in rat oncomodulin a -X glutamate to aspartate replacement increases calcium affinity (R. C. Hapak, P. J. Lammers, W. A. Palmisano, E. R. Birnbaum, and M. T. Henzl, J. Biol. Chem. 264, 18751-18760, 1989). The different effect of a -X glutamate to aspartate substitution in the different sites suggests site-specific factors dictating the thermodynamic contribution of the -X glutamate to calcium affinity.     

Herpesvirus protease inhibition by dimer disruption

Kaposi's sarcoma-associated herpesvirus (KSHV), like all herpesviruses, encodes a protease (KSHV Pr), which is necessary for the viral lytic cycle. Herpesvirus proteases function as obligate dimers; however, each monomer has an intact, complete active site which does not interact directly with the other monomer across the dimer interface. Protein grafting of an interfacial KSHV Pr alpha-helix onto a small stable protein, avian pancreatic polypeptide, generated a helical 30-amino-acid peptide designed to disrupt the dimerization of KSHV Pr. The chimeric peptide was optimized through protein modeling of the KSHV Pr-peptide complex. Circular dichroism analysis and gel filtration chromatography revealed that the rationally designed peptide adopts a helical conformation and is capable of disrupting KSHV Pr dimerization, respectively. Additionally, the optimized peptide inhibits KSHV Pr activity by 50% at a approximately 200-fold molar excess of peptide to KSHV Pr, and the dissociation constant was estimated to be 300 microM. Mutagenesis of the interfacial residue M197 to a leucine resulted in an inhibitory concentration which was twofold higher for KSHV Pr M197L than for KSHV Pr, in agreement with the model that the dimer interface is involved in peptide binding. These results indicate that the dimer interface, as well as the active sites, of herpesvirus proteases is a viable target for inhibiting enzyme activity.     

Formation of actin dimers as studied by small angle neutron scattering

Small angle neutron scattering has been used to study the dimensions of G-actin and the formation of low molecular weight actin oligomers under conditions where rapid polymerization does not take place. In the presence of 200 microM Ca2+, actin in solution consists of a single component with a radius of gyration (Rg) of 19.9 +/- 0.4 A, consistent with the known molecular dimensions of the G-actin molecule. In the presence of 50 microM Mg2+, however, formation of an actin species with a larger Rg occurs over a 4-h period. Multicomponent fits were tried and the data were best fit assuming two components, the monomer and a species with an Rg of 29 +/- 1 A. This latter value is consistent with the dimensions expected for certain actin dimers. The apparent dissociation constant for dimer formation is approximately 150 microM with forward and reverse rate constants of 6.0 X 10(-7) microM-1 s-1 and 8.8 X 10(-5) s-1, respectively. Kinetic fluorescence experiments show that the dimer formed in the presence of low levels of Mg2+ is a nonproductive complex which does not participate in the polymerization process. However, the addition of cytochalasin D to actin in the presence of 50 microM Mg2+ rapidly induces the formation of dimers, presumably related to cytochalasin's ability to nucleate actin polymerization.     

Numerical study of the entropy loss of dimerization and the folding thermodynamics of the GCN4 leucine zipper

A lattice-based model of a protein and the Monte Carlo simulation method are used to calculate the entropy loss of dimerization of the GCN4 leucine zipper. In the representation used, a protein is a sequence of interaction centers arranged on a cubic lattice, with effective interaction potentials that are both of physical and statistical nature. The Monte Carlo simulation method is then used to sample the partition functions of both the monomer and dimer forms as a function of temperature. A method is described to estimate the entropy loss upon dimerization, a quantity that enters the free energy difference between monomer and dimer, and the corresponding dimerization reaction constant. As expected, but contrary to previous numerical studies, we find that the entropy loss of dimerization is a strong function of energy (or temperature), except in the limit of large energies in which the motion of the two dimer chains becomes largely uncorrelated. At the monomer-dimer transition temperature we find that the entropy loss of dimerization is approximately five times smaller than the value that would result from ideal gas statistics, a result that is qualitatively consistent with a recent experimental determination of the entropy loss of dimerization of a synthetic peptide that also forms a two-stranded alpha-helical coiled coil.     

Oligomerization-dependent changes in the thermodynamic properties of the TPR-MET receptor tyrosine kinase

Although oligomerization of receptor tyrosine kinases (RTKs) is necessary for receptor activation and signaling, a quantitative understanding of how oligomerization mediates these critical processes does not exist. We present a comparative thermodynamic analysis of functionally active dimeric and functionally inactive monomeric soluble analogues of the c-MET RTK, which clearly reveal that oligomerization regulates the binding affinity and binding kinetics of the kinase toward ATP and tyrosine-containing peptide substrates. Thermodynamic binding data for oligomeric c-MET were obtained from the dimeric TPR-MET oncoprotein, a functionally active fusion derivative of the c-MET RTK. This naturally occurring oncoprotein contains the cytoplasmic domain of c-MET fused to a coiled coil dimerization domain from the nuclear pore complex. Comparative data were obtained from a soluble monomeric kinase compromising the c-MET cytoplasmic domain (cytoMET). Significantly, under equilibrium binding conditions, the oligomeric phosphorylated kinase showed a significantly lower dissociation constant (K(d,dimer) = 11 microM) for a tyrosine-containing peptide derived from the C-terminal tail of the c-MET RTK when compared to the phosphorylated monomeric kinase cytoMET (K(d,monomer) = 140 microM). Surprisingly, equilibrium dissociation constants measured for the kinase and ATP were independent of the oligomerization state of the kinase (approximately 10 microM). Stopped-flow analysis of peptide substrate binding showed that the association rate constants (k(2)) differed 2-fold and dissociation rate constants (k(-2)) differed 10-fold when phosphorylated TPR-MET was compared to phosphorylated cytoMET. ATP binding abrogated the differences in k(2) rates observed between the two oligomeric states of the c-MET cytoplasmic domain. These results clearly imply that oligomerization induces important thermodynamic and conformational changes in the substrate binding regions of the c-MET protein and provide quantitative mechanistic insights into the necessary role of oligomerization in RTK activation.     

Coupling of ligand binding and dimerization of helix-loop-helix peptides: spectroscopic and sedimentation analyses of calbindin D9k EF-hands

Isolated Ca2+-binding EF-hand peptides have a tendency to dimerize. This study is an attempt to account for the coupled equilibria of Ca2+-binding and peptide association for two EF-hands with strikingly different loop sequence and net charge. We have studied each of the two separate EF-hand fragments from calbindin D9k. A series of Ca2+-titrations at different peptide concentrations were monitored by CD and fluorescence spectroscopy. All data were fitted simultaneously to both a complete model of all possible equilibrium intermediates and a reduced model not including dimerization in the absence of Ca2+. Analytical ultracentrifugation shows that the peptides may occur as monomers or dimers depending on the solution conditions. Our results show strikingly different behavior for the two EF-hands. The fragment containing the N-terminal EF-hand shows a strong tendency to dimerize in the Ca2+-bound state. The average Ca2+-affinity is 3.5 orders of magnitude lower than for the intact protein. We observe a large apparent cooperativity of Ca2+ binding for the overall process from Ca2+-free monomer to fully loaded dimer, showing that a Ca2+-free EF-hand folds upon dimerization to a Ca2+-bound EF-hand, thereby presenting a preformed binding site to the second Ca2+-ion. The C-terminal EF-hand shows a much smaller tendency to dimerize, which may be related to its larger net negative charge. In spite of the differences in dimerization behavior, the Ca2+ affinities of both EF-hand fragments are similar and in the range lgK = 4.6-5.3.     

Analytical high-performance affinity chromatography: evaluation by studies of neurophysin self-association and neurophysin-peptide hormone interaction using glass matrices

Bovine neurophysin II (BNP II) was covalently immobilized on both nonporous and porous (200-nm pore diameter) glass beads and incorporated in a high-performance liquid chromatograph to evaluate analytical high-performance affinity chromatography as a microscale method for characterizing biomolecular interactions. By extension of the theoretical treatment of analytical affinity chromatography, both the self-association of neurophysin and its binding of the peptide hormone vasopressin were characterized by using a single chromatographic column containing immobilized neurophysin predominantly in the monomer form. Both [3H] [Arg8]vasopressin (AVP) and 125I-BNP II were rapidly eluted (less than 25 min). The relatively symmetrical elution peaks obtained allowed calculation of both equilibrium dissociation constants and kinetic dissociation rate constants. The dissociation constant measured chromatographically for the AVP-immobilized neurophysin complex, KM/L = 11 microM with porous glass beads and 75 microM with nonporous glass (NPG) beads, was in reasonable agreement with those previously obtained by curve fitting of Scatchard plots (16-20 microM) and from binding to [BNP II]Sepharose (50 microM). The values obtained are larger than that for dissociation of AVP from BNP II dimer, by a factor consistent with the intended nature of immobilized BNP II as monomers. Chromatography of BNP II on the [BNP II]NPG gave a dimer dissociation constant of 166 microM, a value in excellent agreement with that derived from equilibrium sedimentation studies (172 microM). In contrast to the agreement of chromatographic equilibrium binding constants with those measured in solution, the dissociation rate, k-3, determined from the variance of the affinity chromatographic elution profile with nonporous beads, was several orders of magnitude smaller than the solution counterpart.(ABSTRACT TRUNCATED AT 250 WORDS)     

Engineering new metal specificity in EF-hand peptides

Peptides (33-34 amino acids long) corresponding to the helix-turn-helix (EF-hand) motif of the calcium binding site I of Paramecium tetraurelia calmodulin have been synthesized. The linear sequence was unable to acquire a native-like conformation and calcium binding. However, incorporation of a well-positioned disulfide bond bridging the two putative helical regions greatly improved the ordered structure and binding properties. Analyzed by electrospray mass spectrometry, circular dichroism and time-resolved laser-induced fluorescence, such a disulfide-stabilized peptide is shown to acquire a calcium-dependent helical conformation and exhibits native-like affinity for calcium, terbium and europium ions with 30+/-1, 3.5+/-0.6 and 0.6+/-0.1 microM dissociation constants, respectively. Comparable affinities were calculated within the biological construct comprising the entire domain I of Arabidopsis taliana calmodulin. Single sequence mutation (Glu25Asp) in the binding loop of the peptide abolishes calcium affinity, but preserves lanthanide affinity, showing that metal selectivity can be modulated by specific mutations. Such disulfide-stabilized peptides represent useful models to engineer metal specificity in new calmodulin proteins, facilitating the development of new systems to monitor metal pollution in biosensors and to increase metal binding capability of bacterial and plant cells in bioremediation techniques.     

Conformational species of gramicidin A in non-polar solvent. A kinetic and thermodynamic treatment in the absence and presence of phosphatidylcholine as studied by high-performance liquid chromatography

A kinetic and thermodynamic study has been carried out to characterize quantitatively the conformational equilibrium of gramicidin A (GA) in tetrahydrofuran at different peptide concentrations in the absence and presence of egg yolk phosphatidylcholine by using size-exclusion high-performance liquid chromatographic analysis. In the absence of lipid, the experimental data fit a simple dimer-monomer equilibrium, the rate and equilibrium constants for the dissociation process being (1.6 +/- 0.7) X 10(-7) s-1 and (8.5 +/- 0.3) X 10(-6) M, respectively. A higher extent of monomerization and a decrease in the time required for reaching equilibrium are detected in the presence of phospholipid, the kinetic and thermodynamic effects depending on both lipid and GA concentrations. In order to account for these observations a cyclic equilibrium mechanism is proposed which is analysed in terms of four conformational species, namely, free monomer, free dimer, lipid-bound monomer and lipid-bound dimer. The results obtained are discussed in relation to recent literature data on lipid-protein interactions.     

The effect of C-terminal helix stabilization on specific DNA binding by monomeric GCN4 peptides.

DNA binding by a 29-residue, monomeric, GCN4 basic region peptide, GCN4br, as well as by peptide br-C, a monomeric basic-region analogue that is helix stabilized at its C-terminal end by a Lys25. Asp29 side-chain lactam-bridged alanine-rich sequence, was studied at 25 C in an aqueous buffer containing 100 mm NaCl. Mixing of both peptides with duplex DNA containing the cAMP-responsive element (CRE) was accompanied by significant helix stabilization in the peptides, whereas mixing of the peptides with duplex DNA containing a scrambled CRE site was not. Peptide NBD-br-C was synthesized as a fluorescent probe to evaluate these peptide-DNA interactions further. Quantitative analysis of the fluorescence quenching of peptide NBD-br-C by CRE half-site DNA indicated the formation of a 1:1 complex with a dissociation constant of 1.41 +/- 0.22 microm. Competitive displacement fluorescence assays of CRE half-site binding gave dissociation constants of 0.65 +/- 0.09 microm for peptide br-C and 3.9 +/- 0.5 microM for GCN4br, which corresponds to a free energy difference of 1.1 kcal/mol that is attributed to the helix stabilization achieved in peptide br-C. This result indicates that helix initiation by the alpha-helical leucine zipper dimerization motif in native bzip proteins, such as GCN4, contributes significantly to the affinity of basic region peptides for their recognition sites on DNA. Our fluorescence assay should also prove useful for determining dissociation constants for CRE binding by other GCN4 basic region analogues under equilibrium conditions and physiological salt concentrations.     

The dimerization of ferrihaems. II. Equilibrium and kinetic studies of mesoferrihaem dimerization.

Spectrophotometric data have been determined for mesoferrihaem at several pH values and over a range of concentration covering four orders of magnitude. The data reveal a dimerization process according to the equation 2 monomer in equilibrium dimer + H+, analogous to earlier findings for deuteroferrihaem and protoferrihaem. The value of K (defined as K = [dimer] [H+]/[monomer]2) was found to be 6.92.10(-2). This is close to the value for deuteroferrihaem but much less than that for protoferrihaem. This is interpreted in terms of possible additional bonding between the delocalized electron systems in protoferrihaem dimers relative to those of mesoferrihaem and deuteroferrihaem. Rate constants for dimerization were determined by temperature-jump spectrophotometry. The pH dependence of the rate constants is explained in terms of two distinct pathways for the dimerization process. These involve either direct reaction between two undissociated monomer molecules or alternatively an initial acid dissociation of a monomer molecule followed by reaction between an undissociated and dissociated molecule.     

Interaction of troponin I and troponin C: 19F NMR studies of the binding of the inhibitory troponin I peptide to turkey skeletal troponin C.

We have used 19F nuclear magnetic resonance spectroscopy to study the interaction of the inhibitory region of troponin (TnI) with apo- and calcium(II)-saturated turkey skeletal troponin C (TnC), using the synthetic TnI analogue N alpha-acetyl[19FPhe106]TnI(104-115)amide. Dissociation constants of Kd = (3.7 +/- 3.1) x 10(-5) M for the apo interaction and Kd = (4.8 +/- 1.8) x 10(-5) M for the calcium(II)-saturated interaction were obtained using a 1:1 binding model of peptide to protein. The 19F NMR chemical shifts for the F-phenylalanine of the bound peptide are different from the apo- and calcium-saturated protein, indicating a different environment for the bound peptide. The possibility of 2:1 binding of the peptide to Ca(II)-saturated TnC was tested by calculating the fit of the experimental titration data to a series of theoretical binding curves in which the dissociation constants for the two hypothetical binding sites were varied. We obtained the best fit for 0.056 mM less than or equal to Kd1 less than or equal to 0.071 mM and 0.5 mM less than or equal to Kd2 less than or equal to 2.0 mM. These results allow the possibility of a second peptide binding site on calcium(II)-saturated TnC with an affinity 10- to 20-fold weaker than that of the first site.