Biomedical subjects

C M Kay

Publications and source records attributed to C M Kay.

At least 73 records · Page 4Linked to original sources

PubMed1995

Calcium binding to the regulatory N-domain of skeletal muscle troponin C occurs in a stepwise manner.

Ca2+ binding to a recombinant regulatory N-domain (residues 1-90) of chicken troponin C (NTnC) has been investigated with the use of heteronuclear multidimensional NMR spectroscopy. The protein has been cloned in pET3a vector and expressed in minimal media in Escherichia coli to allow uniform 15N and 13C labeling. The NMR spectra have been resolved and completely assigned [Gagné et al. (1994) Protein Sci. 3, 1961-1974]. Ca2+ titration monitored by 2D (1H, 15N)-HMQC NMR spectral changes revealed that Ca2+ binding to sites I and II of NTnC is a stepwise process and that chemical shift changes occur throughout the N-domain upon the binding of each Ca2+. The Ca2+ dissociation constants for the binding of the first and second Ca2+ were determined to be 0.8 microM < or = Kd1 < or = 3 microM and 5 microM < or = Kd2 < or = 23 microM, respectively. This mechanism is believed to represent that of the N-domain in intact TnC since we have shown earlier that the properties of the N-domain (1-90) were identical to those of the N-domain in intact TnC [Li et al. (1994) Biochemistry 33, 917-925]. In contrast, however, our previous Ca2+ fluorescence and far-UV CD studies on F29W NTnC and F29W TnC indicated cooperative Ca2+ binding to sites I/II and no detectable differences in their affinities. To rationalize these observations, a direct comparison was made of the Ca2+ titration of NTnC and F29W NTnC as monitored by far-UV CD spectroscopy. Unlike F29W NTnC, NTnC gave a biphasic curve with binding constants in reasonable agreement with the NMR data. Although the far-UV CD spectra of NTnC and the F29W NTnC domain were the same in the absence of Ca2+, the Ca(2+)-induced negative ellipticity increase for NTnC is significantly smaller than for F29W NTnC. These observations indicate that the F29W mutation has perturbed the Ca2+ binding properties of the N-domain and its CD spectroscopic properties in the Ca(2+)-saturated state.

Amino Acid Sequence↗

PubMed1995

Calcium-induced dimerization of troponin C: mode of interaction and use of trifluoroethanol as a denaturant of quaternary structure.

Protein aggregation can be a problem, especially as a large number of proteins become available for structural studies at fairly high concentrations using solution techniques such as NMR spectroscopy. The muscle regulatory protein troponin C (TnC) undergoes a calcium-induced dimerization at neutral pH with a dissociation constant for the dimerization of 0.4 mM at 20 degrees C. The present study indicates that the mode of dimerization involves the N-domain of one monomer interacting with the N-domain of another monomer. Addition of the solvent trifluoroethanol (TFE) to a concentration of 15%, v/v, results in a 10-fold increase in the dimer dissociation constant of calcium-saturated TnC (4 mM at 20 degrees C), making TnC predominantly a monomer for spectroscopic studies. Further, TFE, at the concentrations used herein, acts to perturb the quaternary structure of TnC without adversely affecting the secondary or tertiary structure as evidenced by minimal changes to its CD spectra and 1H, 13C, and 15N NMR chemical shifts.

Animals↗

PubMed1995

Interaction between caltropin and the C-terminal region of smooth muscle caldesmon.

Caltropin (CaT) binds caldesmon (CaD) in a Ca(2+)-dependent manner with an affinity higher than that of calmodulin (CaM). Photo-crosslinking between CaT and a benzophenone-labeled C-terminal CaD fragment (27K) results in a 35-kDa protein that corresponds to the 1:1 adduct between CaT and 27K. In the absence of Ca2+, no crosslinking is obtained. This result is similar to that obtained with CaM and 27K. The apparent affinity of CaM for GS17C, a CaM-binding peptide of CaD, is weakened by CaT, suggesting CaT competes with CaM for the peptide. In contrast to CaM, CaT does not induce changes in the tryptophan fluorescence of GS17C. Thus although the two Ca(2+)-binding proteins behave similarly, there are differences in their interactions with CaD.

Animals↗

PubMed1995

Zn2+ binding to cardiac calsequestrin.

Zn2+ binding to canine cardiac calsequestrin was investigated using the Zn2+ specific fluorescence dye salicylcarbohydrazone (SACH), 65Zn2+ overlay and Zn(2+)-IDA chromatography. Cardiac calsequestrin binds approximately 200 moles of Zn2+/mole of protein with the Kd = 300 microM. Zn2+ binding to calsequestrin was further confirmed by 65Zn2+ overlay and Zn(2+)-dependent aggregation of the protein. However, calsequestrin did not bind to a Zn(2+)-IDA-agarose column, indicating that histidine residues may not be involved in Zn2+ binding to the protein. Circular dichroism revealed only minor Zn(2+)-dependent conformational changes in calsequestrin. We conclude that calsequestrin is a Ca(2+)- and Zn(2+)-binding protein and that Zn2+ may modulate the structure and function of the protein.

Animals↗

PubMed1995

Direct demonstration of an intramolecular SH2-phosphotyrosine interaction in the Crk protein.

Many signal transduction processes are mediated by the binding of Scr-homology-2 (SH2) domains to phosphotyrosine (pTyr)-containing proteins. Although most SH2-pTyr interactions occur between two different types of molecules, some appear to involve only a single molecular type. It has been proposed that the enzymatic activity and substrate recognition of the Src-family kinases, and the protein-binding and transforming activity of Crk-family adaptor proteins, are regulated by intramolecular SH2-pTyr interactions. In addition, the DNA-binding activity of Stat transcription factors seems to be regulated by SH2-mediated homodimerization. Here we examine the phosphorylated and non-phosphorylated forms of murine Crk II (p-mCrk and mCrk, respectively) using a combination of physical techniques. The Crk protein contains a single SH2 domain and two SH3 domains in the order SH2-SH3-SH3. There is a tyrosine-phosphorylation site between the two SH3 domains at residue 221 which is phosphorylated in vivo by the Abl tyrosine kinase. Using NMR spectroscopic analysis, we show here that the SH2 domain of purified p-mCrk is bound to pTyr, and by hydrodynamic measurements that the phosphorylated protein is monomeric. These results provide direct demonstration of an intramolecular SH2-pTyr interaction in a signalling molecule.

Amino Acid Sequence↗

PubMed1995

Effect of caltropin on caldesmon-actin interaction.

The binding of chicken gizzard caldesmon to actin was studied both in the presence and the absence of caltropin using Airfuge centrifugation experiments, disulfide cross-linking studies, and the fluorescent probe acrylodan (6-acryloyl-2-(dimethylamino)naphthalene). In co-sedimentation studies most of the caldesmon pelleted along with actin. However, when caldesmon in the presence of caltropin was mixed with actin, caldesmon did not pellet along with actin following high speed centrifugation, suggesting that caltropin has significantly weakened its binding to actin. The caltropin effect was noticed even when tropomyosin was included in the reaction mixture. Acrylodan-labeled caldesmon, when excited at 375 nm, had an emission maximum at 515 +/- 2 nm. The addition of actin produced a nearly 70% increase in fluorescent intensity, accompanied by a blue shift in the emission maximum (i.e. lambda em (max) = 505 +/- 2 nm), suggesting that the probe now occupies a more nonpolar environment. Titration of labeled caldesmon with actin indicated a strong affinity (K alpha = approximately 6 x 10(7) M-1). When actin was titrated with labeled caldesmon in the presence of caltropin in a 0.2 mM Ca2+ medium, its affinity for caldesmon was lowered (K alpha = approximately 2 x 10(7) M-1). Caltropin, which is very effective in reversing caldesmon's inhibition of the actin-activated myosin ATPase (Mani, R. S., McCubbin, W. D., and Kay, C. M. (1992) Biochemistry 31, 11896-11901), is shown in the present study to have a pronounced effect on its binding to actin, suggesting a major role for caltropin in regulating caldesmon in smooth muscle.

Actins↗

PubMed1995

Modulation of substrate selectivity in plasma lipid transfer protein reaction over structural variation of lipid particle.

The modulation of substrate selectivity of human plasma LTP reaction is the subject of the present investigation. The moderate selectivity by a factor of 5 to 6 was observed in the LTP-catalyzed transfer of cholesteryl ester over triacylglycerol between plasma lipoproteins. On the other hand, the transfer of cholesteryl ester by LTP was highly selective over the negligible transfer of triacylglycerol, by a factor of 60 to 500, between the microemulsions with LDL size, regardless of the activators such as human and pig apolipoprotein (apo) A-I, human apo C-III and apo E that bound to the surface of the emulsion in equilibrium. The presence of free cholesterol in these microemulsions reduced slightly the rate of cholesteryl ester transfer but had no effect on triacylglycerol transfer. Other surface-active reagents such as cholic acid, Triton X-100 and Tween-20, did not have an effect on the triacylglycerol transfer either. Triacylglycerol transfer by LTP became measurable between such lipid particles as prepared by co-sonication of lipid with pig apo A-I and isolated as the mixed-microemulsions in the density of LDL and HDL. In these conditions, the substrate selectivity for cholesteryl ester over triacylglycerol was a factor of 6 to 16 mimicking the ratio in plasma lipoproteins. The conformation of pig apo A-I estimated by circular dichroism showed that its apparent helical content was further more induced when apo A-I was integrated into the mixed-microemulsion by co-sonication than the lipid-bound apo A-I in equilibrium. Apo A-I, thus integrated into lipid particles, was highly resistant to the denaturation by guanidine hydrochloride while the lipid-bound apo A-I in equilibrium was denatured as readily as the lipid-free protein. Thus, triacylglycerol transfer by LTP was induced by structural modulation of substrate-carrying lipid particles such as higher integration of apolipoproteins.

Animals↗

PubMed1995

Cloning and structure-function analysis of the Leishmania donovani kinetoplastid membrane protein-11.

This report describes the complete translated gene sequence, predicted secondary structure and lipid bilayer association of a novel kinetoplastid membrane protein (KMP-11) from Leishmania donovani promastigotes. KMP-11 was previously referred to as the lipophosphoglycan-associated protein (LPGAP). The isolation, species distribution and chemical characterization, including a partial protein sequence analysis and post-translational modifications, of this major membrane component have been described [Jardim, Funk, Caprioli and Olafson (1995) Biochem. J. 305, 307-313]. C.d. measurements of KMP-11 indicated a very high helical content estimated to be approximately 86% in trifluoroethanol. This was in agreement with computer-based secondary-structure analyses which predicted KMP-11 to be almost exclusively alpha-helical, with the protein adopting a helix-loop-helix motif. Arrangement of the residues located in the putative helical regions on an Edmundson helical wheel showed that this molecule could have a strongly amphipathic conformation and provided an explanation for how such a highly charged protein might be inserted into the plasma membrane. Evidence in support of KMP-11 association with lipid bilayers was provided by showing that KMP-11 could mediate carboxyfluorescein release from liposomes. These findings suggested that KMP-11 may function in part to increase bilayer pressure, stabilizing molecules such as lipophosphoglycan within the parasite pellicular membrane.

Amino Acid Sequence↗

PubMed1995

Protein destabilization by electrostatic repulsions in the two-stranded alpha-helical coiled-coil/leucine zipper.

The destabilizing effect of electrostatic repulsions on protein stability has been studied by using synthetic two-stranded alpha-helical coiled-coils as a model system. The native coiled-coil consists of two identical 35-residue polypeptide chains with a heptad repeat QgVaGbAcLdQeKf and a Cys residue at position 2 to allow formation of an interchain disulfide bridge. This peptide, designed to contain no intrahelical or interhelical electrostatic interactions, forms a stable coiled-coil structure at 20 degrees C in benign medium (50 mM KCl, 25 mM PO4, pH 7) with a [urea]1/2 value of 6.1 M. Four mutant coiled-coils were designed to contain one or two Glu substitutions for Gln per polypeptide chain. The resulting coiled-coils contained potential i to i' + 5 Glu-Glu interchain repulsions (denoted as peptide E2(15,20)), i to i' + 2 Glu-Glu interchain repulsions (denoted E2(20,22)), or no interchain ionic interactions (denoted E2(13,22) and E1(20)). The stabilities of the coiled-coils were determined by measuring the ellipticities at 222 nm as a function of urea or guanidine hydrochloride concentration at 20 degrees C in the presence and absence of an interchain disulfide bridge. At pH 7, in the presence of urea, the stabilities of E2(13,22) and E2(20,22) were identical suggesting that the potential i to i' + 2 interchain Glu-Glu repulsion in the E2(20,22) coiled-coil does not occur. In contrast, the mutant E2(15,20) is substantially less stable than E2(13,22) or E2(15,20) by 0.9 kcal/mol due to the presence of two i to i' + 5 interchain Glu-Glu repulsions, which destabilize the coiled-coil by 0.45 kcal/mol each. At pH 3 the coiled-coils were found to increase in stability as the number of Glu substitutions were increased. This, combined with reversed-phase HPLC results at pH 7 and pH 2, supports the conclusion that the protonated Glu side chains present at low pH are significantly more hydrophobic than Gln side chains which are in turn more hydrophobic than the ionized Glu side chains present at neutral pH. The protonated Glu residues increase the hydrophobicity of the coiled-coil interface leading to higher coiled-coil stability. The guanidine hydrochloride results at pH 7 show similar stabilities between the native and mutant coiled-coils indicating that guanidine hydrochloride masks electrostatic repulsions due to its ionic nature and that Glu and Gln in the e and g positions of the heptad repeat have very similar effects on coiled-coil stability in the presence of GdnHCl.

Amino Acid Sequence↗

PubMed1995

Human S100b protein: formation of a tetramer from synthetic calcium-binding site peptides.

Human brain S100b protein is a unique calcium-binding protein comprised of two identical 91-amino acid polypeptide chains that each contain two proposed helix-loop-helix (EF-hand) calcium-binding sites. In order to probe the assembly of the four calcium-binding sites in S100b, a peptide comprised of the N-terminal 46 residues of S100b protein was synthesized and studied by CD and 1H NMR spectroscopies as a function of concentration and temperature. At relatively high peptide concentrations and in the absence of calcium, the peptide exhibited a significant proportion of alpha-helix (45%). Decreasing the peptide concentration led to a loss of alpha-helix as monitored by CD spectroscopy and coincident changes in the 1H NMR spectrum. These changes were also observed by 1H NMR spectroscopy as a function of temperature where it was observed that the Tm of the peptide was lowered approximately 14 degrees C with a 17-fold decrease in peptide concentration. Sedimentation equilibrium studies were used to determine that the peptide formed a tetramer in solution in the absence of calcium. It is proposed that this tetrameric fold also occurs in S100b and is a result of the interaction of portions of all four calcium-binding sites.

Amino Acid Sequence↗

PubMed1995

Bacterial expression and site-directed mutagenesis of a functional recombinant apolipoprotein.

To facilitate structure-function studies of Manduca sexta apolipophorin III (apoLp-III), its nucleotide coding sequence was cloned from a fat body cDNA library by in vitro DNA amplification. The amplification product was cloned in the pET expression vector and introduced into E. coli. After induction, cultures were screened for apoLp-III protein production by immunoblotting with anti-apoLp-III serum. Data obtained indicated the presence of apoLp-III in both cell lysates and media of cell cultures harboring the apoLp-III-pET construct but not in cells containing the parent vector. The protein was isolated from the cell-free supernatant of cultures grown in minimal media 4 h after induction. Verification that the recombinant protein produced was indeed apoLp-III was obtained by electrospray mass spectrometric analysis. Circular dichroism (CD) spectroscopy of the isolated recombinant protein revealed a characteristic content of alpha-helical secondary structure with a further induction of helix upon addition of 50% trifluoroethanol. In urea denaturation studies, monitored by CD, evidence was obtained that recombinant and natural apoLp-III possess indistinguishable thermodynamic properties. In addition, lipid binding assays revealed that recombinant apoLp-III formed stable complexes with phospholipids and was capable of associating with lipoprotein surfaces. Examination of the fluorescence properties of recombinant apoLp-III revealed the presence of a noncovalently associated fluorescent contaminant that was effectively removed by reverse phase HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Effect of chain length on the formation and stability of synthetic alpha-helical coiled coils.

A series of polypeptides containing 9, 12, 16, 19, 23, 26, 30, 33, and 35 amino acid residues was designed to investigate the effects of peptide chain length on the formation and stability of two-stranded alpha-helical dimers or coiled coils. These peptides were synthesized by the solid-phase method, purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and characterized by RP-HPLC, amino acid composition analysis, and mass spectrometry. The amphipathic alpha-helical peptides were designed to dimerize by interchain hydrophobic interactions at positions a and d and interchain salt bridges between lysine and glutamic acid residues at positions e and g of the repeating heptad sequence of Glu-Ile-Glu-Ala-Leu-Lys-Ala (g-a-b-c-d-e-f). The ability of these peptides to form alpha-helical structures in the presence and absence of a helix-inducing reagent (trifluoroethanol) was monitored by circular dichroism spectroscopy. The helicity of the peptides increased with increasing chain length in a cooperative manner. A minimum of three heptads corresponding to six helical turns was required for a peptide to adopt the two-stranded alpha-helical coiled coil conformation in aqueous medium. The increased stability of the peptides as a result of an increase in hydrophobic interactions (chain length) was demonstrated by the shift in the transitions of the guanidine hydrochloride (Gdn.HCl) denaturation and thermal unfolding profiles. The concentrations of denaturant (Gdn.HCl) required to achieve 50% denaturation are 3.2, 4.9, 6.9, and 7.5 M for peptides 23r, 26r, 30r, and 33r, respectively, in aqueous medium. However, the effect of a chain length increase on coiled-coil stability was not additive. The melting temperature, Tm, at which 50% of the helicity is lost, increased by 34 degrees C in changing the peptide chain length from 23 to 26; however, that shift was only 14 degrees C when the chain length was increased from 30 to 33 residues. These results are consistent with a chain length dependent cooperative folding of the peptides into coiled coils.

Amino Acid Sequence↗

PubMed1994

Structural and binding characteristics of the carboxyl terminal fragment of apolipophorin III from Manduca sexta.

The molecular basis of the interaction of apolipophorin III (apoLp-III), an exchangeable apolipoprotein from hemolymph of the sphinx moth. Manduca sexta, with lipoprotein surfaces and phospholipids was studied by investigating the structural and binding properties of the C-terminal fragment of the native protein. A 4K peptide, corresponding to the terminal helical segment of the native protein, was generated by cyanogen bromide treatment, purified by gel filtration and reverse-phase HPLC, and characterized by N-terminal sequencing and amino acid and mass spectrometric analysis. Circular dichroism (CD) spectroscopy of the peptide in buffer indicated a predominantly unstructured state while addition of trifluoroethanol (TFE), a helix-inducing agent, resulted in an alpha-helical structure. Sedimentation equilibrium studies revealed that the 4K peptide was monomeric in buffer. The 4K peptide assumed an alpha-helical conformation in the presence of sodium dodecyl sulfate (SDS) and lysolecithin, but was unstructured in the presence of dimyristoylphosphatidylcholine, either when added to preformed vesicles or upon cosonication, indicating an ability to bind to detergent micelles but not to phospholipid bilayers. Unlike native apoLp-III, the 4K peptide did not confer protection against turbidity development to human low density lipoprotein upon incubation with phospholipase C, indicating an inability to interact with the surface of lipoproteins. Upon interaction with SDS micelles, both the 4K peptide and apoLp-III were resistant to urea-induced denaturation when compared to free apoLp-III, as evaluated by CD spectroscopy. The structural stability conferred upon interaction with detergents was similar for both the peptide and the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Functional and physical characterization of the cell cycle ubiquitin-conjugating enzyme CDC34 (UBC3). Identification of a functional determinant within the tail that facilitates CDC34 self-association.

Like several other ubiquitin-conjugating enzymes, the yeast cell cycle enzyme CDC34 (UBC3) has a carboxyl-terminal extension or tail. These tails appear to carry out unique functions that can vary from one ubiquitin-conjugating enzyme to the next. Using biophysical techniques we have determined that the tail of CDC34 constitutes a highly structured and extended domain. Although the tail of CDC34 is the largest tail identified to date (125 residues), we have found that only 39 residues lying adjacent to the catalytic domain are necessary and sufficient for full cell cycle function and that this region fulfills a novel function that may be common to the tails of other ubiquitin-conjugating enzymes. Cross-linking studies demonstrate that this region facilitates a physical interaction between CDC34 monomers in vitro. Furthermore, phenotypic analysis of various CDC34 derivatives expressed in different cdc34 mutant strains indicates that this region facilitates the same interaction in vivo. Based on these findings, it appears that the cell cycle function of CDC34 is dependent upon the ability of CDC34 monomers to interact with one another and that this interaction is mediated by a small region of the CDC34 tail. The similarity of this region with sequences contained within the tails of the UBC1 and UBC6 enzymes suggests that these tails may function in a similar manner.

Anaphase-Promoting Complex-Cyclosome↗

PubMed1994

The effects of N helix deletion and mutant F29W on the Ca2+ binding and functional properties of chicken skeletal muscle troponin.

To assess the structural and functional significance of the N helix (residues 3-13) of avian recombinant troponin C (rTnC), we have constructed NHdel, in which residues 1-11 have been deleted, both in rTnC and in the spectral probe mutant F29W (Pearlstone, J. R., Borgford, T., Chandra, M., Oikawa, K., Kay, C. M., Herzberg, O., Moult, J., Herklotz, A., Reinach, F. C., and Smillie, L. B. (1992) Biochemistry 31, 6545-6553). Comparison of the far- and near-UV CD spectra (+/- Ca2+) of F29W and F29W/NHdel and titration of the Ca(2+)-induced ellipticity and fluorescence changes indicates that the deletion has little effect on the global fold of the molecule but reduces the Ca2+ affinity of the N domain, but not the C domain, by 1.6-1.8-fold. Comparisons of the mutants NHdel, F29W, and F29W/NHdel with rTnC have been made using several functional assays. In reconstituted troponin-tropomyosin actomyosin subfragment 1 and myofibrillar ATPase systems, both F29W and NHdel have significantly reduced Ca(2+)-activated enzymatic activities. These effects are cumulative in the double mutant F29W/NHdel. On the other hand, maximal isometric tension development in Ca(2+)-activated reconstituted skinned fibers is not affected with F29W and NHdel, although the Ca2+ sensitivity of NHdel in this system is markedly reduced. We conclude that both mutations, NHdel and F29W, are functionally deleterious, possibly affecting interactions of the N domain with troponin I and/or T.

Animals↗

PubMed1994

Properties of human plasma lipid transfer protein in aqueous solution and at interfaces.

Human plasma lipid transfer protein (cholesteryl ester transfer protein) has been characterized for its solution and surface properties. The protein is monomeric in aqueous solution up to 0.62 g/L (11.7 microM) as demonstrated by sedimentation equilibrium. It binds to the surface of a lipid microemulsion having an average diameter of 26 nm made from triolein and egg yolk phosphatidylcholine, with an estimated dissociation constant 1.2 x 10(-8) M, and the maximum saturation binding level is 8 protein molecules per particle regardless of the presence of apolipoprotein A-I. Circular dichroism measurement indicated that the protein in solution is predominantly in the beta-sheet/beta-turn conformation with some alpha-helix, and this profile does not undergo drastic change by its binding to the lipid surface. The analysis of the behavior of the protein in its monomolecular layer at the air-buffer interface indicated that it is also monomeric at the interface. LTP molecules occupied the same area per amino acid as other apolipoproteins in the monolayer but had a higher collapse pressure of its monolayer (18 dyn/cm), and the protein stayed at the interface even after the overcompressing monolayer far beyond the collapsing pressure up to 40 dyn/cm.

Apolipoprotein A-I↗

PubMed1994

Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation.

The backbone dynamics of the C-terminal SH2 domain of phospholipase C gamma 1 have been investigated. Two forms of the domain were studied, one in complex with a high-affinity binding peptide derived from the platelet-derived growth factor receptor and the other in the absence of this peptide. 2-D 1H-15N NMR methods, employing pulsed field gradients, were used to determine steady-state 1H-15N NOE values and T1 and T2 15N relaxation times. Backbone dynamics were characterized by the overall correlation time (tau m), order parameters (S2), effective correlation times for internal motions (tau e), and, if required, terms to account for motions on a microsecond-to-millisecond-time scale. An extended two-time-scale formalism was used for residues having relaxation data and that could not be fit adequately using a single-time-scale formalism. The overall correlation times of the uncomplexed and complexed forms of SH2 were found to be 9.2 and 6.5 ns, respectively, suggesting that the uncomplexed form is in a monomer-dimer equilibrium. This was subsequently confirmed by hydrodynamic measurements. Analysis of order parameters reveals that residues in the so-called phosphotyrosine-binding loop exhibited higher than average disorder in both forms of SH2. Although localized differences in order parameters were observed between the uncomplexed and complexed forms of SH2, overall, higher order parameters were not found in the peptide-bound form, indicating that on average, picosecond-time-scale disorder is not reduced upon binding peptide. The relaxation data of the SH2-phosphopeptide complex were fit with fewer exchange terms than the uncomplexed form. This may reflect the monomer-dimer equilibrium that exists in the uncomplexed form or may indicate that the complexed form has lower conformational flexibility on a microsecond-to-millisecond-time scale.

Amino Acid Sequence↗

PubMed1994

Smooth muscle calponin-caltropin interaction: effect on biological activity and stability of calponin.

Calponin inhibits actomyosin Mg2+ ATPase and is proposed to regulate smooth muscle contraction; however, the mechanism by which it exerts its effect and the regulation of its behavior is still under investigation. The proposed methods by which calponin regulation is effected include reversible phosphorylation of calponin which would allow contraction to occur and regulation by interaction with calcium-calmodulin. However, several investigators have been unable to find evidence of in vivo phosphorylation of calponin, and the affinity between calponin and calmodulin is not high enough to suggest that this interaction is biologically significant. In this paper, we present an alternative method of calponin regulation via calcium-caltropin and describe the calponin-caltropin complex for the first time. Caltropin, a calcium-binding protein isolated from smooth muscle, is a dimer under native conditions and interacts with calponin in a calcium-dependent fashion in the ratio of 2 mol of dimer: 1 mol of calponin. The formation of this complex can be monitored by following the fluorescence of an acrylodan label on cysteine 273 of calponin, which undergoes a 35-nm blue shift in wavelength peak from 505 to 470 nm when calponin becomes complexed with caltropin. This fluorescence change when titrated with calcium indicates that the concentration of calcium required for complex formation is approximately 10(-5) M, corresponding to the low-affinity calcium-binding sites of caltropin. This complex was further characterized by circular dichroism (CD).(ABSTRACT TRUNCATED AT 250 WORDS)

2-Naphthylamine↗