Characterization of the biologically important interaction between troponin C and the N-terminal region of troponin I.

The N-terminal regulatory region of Troponin I, residues 1-40 (TnI 1-40, regulatory peptide) has been shown to have a biologically important function in the interactions of troponin I and troponin C. Truncated analogs corresponding to shorter versions of the N-terminal region (1-30, 1-28, 1-26) were synthesized by solid-phase methodology. Our results indicate that residues 1-30 of TnI comprises the minimum sequence to retain full biological activity as measured in the acto-S1-TM ATPase assay. Binding of the TnI N-terminal regulatory peptides (TnI 1-30 and the N-terminal regulatory peptide (residues 1-40) labeled with the photoprobe benzoylbenzoyl group, BBRp) were studied by gel electrophoresis and photochemical cross-linking experiments under various conditions. Fluorescence titrations of TnI 1-30 were carried out with TnC mutants that carry a single tryptophan fluorescence probe in either the N- or C-domain (F105W, F105W/C domain (88-162), F29W and F29W/N domain (1-90)) (Fig. 1). Low Kd values (Kd < 10(-7) M) were obtained for the interaction of F105W and F105W/C domain (88-162) with TnI 1-30. However, there was no observable change in fluorescence when the fluorescence probe was located at the N-domain of the TnC mutant (F29W and F29W/N domain (1-90)). These results show that the regulatory peptide binds strongly to the C-terminal domain of TnC.

Structural and functional studies on Troponin I and Troponin C interactions.

Troponin I (TnI) peptides (TnI inhibitory peptide residues 104-115, Ip; TnI regulatory peptide resides 1-30, TnI1-30), recombinant Troponin C (TnC) and Troponin I mutants were used to study the structural and functional relationship between TnI and TnC. Our results reveal that an intact central D/E helix in TnC is required to maintain the ability of TnC to release the TnI inhibition of the acto-S1-TM ATPase activity. Ca(2+)-titration of the TnC-TnI1-30 complex was monitored by circular dichroism. The results show that binding of TnI1-30 to TnC caused a three-folded increase in Ca(2+) affinity in the high affinity sites (III and IV) of TnC. Gel electrophoresis and high performance liquid chromatography (HPLC) studies demonstrate that the sequences of the N- and C-terminal regions of TnI interact in an anti-parallel fashion with the corresponding N- and C-domain of TnC. Our results also indicate that the N- and C-terminal domains of TnI which flank the TnI inhibitory region (residues 104 to 115) play a vital role in modulating the Ca(2+)- sensitive release of the TnI inhibitory region by TnC within the muscle filament. A modified schematic diagram of the TnC/TnI interaction is proposed.

Biological function and site II Ca2+-induced opening of the regulatory domain of skeletal troponin C are impaired by invariant site I or II Glu mutations.

To investigate the roles of site I and II invariant Glu residues 41 and 77 in the functional properties and calcium-induced structural opening of skeletal muscle troponin C (TnC) regulatory domain, we have replaced them by Ala in intact F29W TnC and in wild-type and F29W N domains (TnC residues 1-90). Reconstitution of intact E41A/F29W and E77A/F29W mutants into TnC-depleted muscle skinned fibers showed that Ca(2+)-induced tension is greatly reduced compared with the F29W control. Circular dichroism measurements of wild-type N domain as a function of pCa (= -log[Ca(2+)]) demonstrated that approximately 90% of the total change in molar ellipticity at 222 nm ([theta](222 nm)) could be assigned to site II Ca(2+) binding. With E41A, E77A, and cardiac TnC N domains this [theta](222 nm) change attributable to site II was reduced to < or =40% of that seen with wild type, consistent with their structures remaining closed in +Ca(2+). Furthermore, the Ca(2+)-induced changes in fluorescence, near UV CD, and UV difference spectra observed with intact F29W are largely abolished with E41A/F29W and E77A/F29W TnCs. Taken together, the data indicate that the major structural change in N domain, including the closed to open transition, is triggered by site II Ca(2+) binding, an interpretation relevant to the energetics of the skeletal muscle TnC and cardiac TnC systems.

Defining the region of troponin-I that binds to troponin-C.

The kinetics and energetics of the binding of three troponin-I peptides, corresponding to regions 96-131 (TnI96-131), 96-139 (TnI96-139), and 96-148 (TnI96-148), to skeletal chicken troponin-C were investigated using multinuclear, multidimensional NMR spectroscopy. The kinetic off-rate and dissociation constants for TnI96-131 (400 s-1, 32 microM), TnI96-139 (65 s-1, <1 microM), and TnI96-148 (45 s-1, <1 microM) binding to TnC were determined from simulation and analysis of the behavior of 1H,15N-heteronuclear single quantum correlation NMR spectra taken during titrations of TnC with these peptides. Two-dimensional 15N-edited TOCSY and NOESY spectroscopy were used to identify 11 C-terminal residues from the 15N-labeled TnI96-148 that were unperturbed by TnC binding. TnI96-139 labeled with 13C at four positions (Leu102, Leu111, Met 121, and Met134) was complexed with TnC and revealed single bound species for Leu102 and Leu111 but multiple bound species for Met121 and Met134. These results indicate that residues 97-136 (and 96 or 137) of TnI are involved in binding to the two domains of troponin-C under calcium saturating conditions, and that the interaction with the regulatory domain is complex. Implications of these results in the context of various models of muscle regulation are discussed.

Structure and interaction site of the regulatory domain of troponin-C when complexed with the 96-148 region of troponin-I.

The structure of the regulatory domain of chicken skeletal troponin-C (residues 1-90) when complexed with the major inhibitory region (residues 96-148) of chicken skeletal troponin-I was determined using multinuclear, multidimensional NMR spectroscopy. This complex represents the first interaction formed between the regulatory domain of troponin-C and troponin-I after calcium binding in the regulation of muscle contraction. The stoichiometry of the complex was determined to be 1:1, with a dissociation constant in the 1-40 microM range. The structure of troponin-C in the complex was calculated from 1039 NMR distance and 111 dihedral angle restraints. When compared to the structure of this domain in the calcium saturated "open" form but in the absence of troponin-I, the bound structure appears to be slightly more "closed". The troponin-I peptide-binding site was found to be in the hydrophobic pocket of calcium saturated troponin-C, using edited/filtered NMR experiments and chemical shift mapping of changes induced in the regulatory domain upon peptide binding. The troponin-I peptide (residues 96-148) was found to bind to the regulatory domain of troponin-C very similarly, but not identically, to a shorter troponin-I peptide (region 115-131) thought to represent the major interaction site of troponin-I for this domain of troponin-C.

Interactions of structural C and regulatory N domains of troponin C with repeated sequence motifs in troponin I.

The actomyosin ATPase inhibitory protein troponin I (TnI) plays a central regulatory role in skeletal and cardiac muscle contraction and relaxation through its calcium-dependent interactions with troponin C (TnC) and actin. Previously we have demonstrated the utility of F29W and F105W mutants of TnC for measurement of binding affinities of inhibitory peptide TnI(96-116) to its regulatory N and structural C domains, both in isolation and in the intact TnC molecule [Pearlstone, J. R. & Smillie, L. B. (1995) Biochemistry 34, 6932-6940]. This approach is now extended to fragment TnI(96-148). Curve-fitting analyses of fluorescence changes induced in the intact TnC mutants and the isolated N and C domains by increasing [TnI(96-148)] have permitted the assignments of K(D) values (designated K(D,N) and K(D,C)) to the interaction of TnI(96-148) with the N and C domains, respectively, of intact TnC. Taken together with the previous data for TnI(96-116) binding, it can be concluded that, within TnI(96-148), residues 96-116 are primarily responsible for binding to C domain of intact TnC and residues 117-148 to its N domain. Inspection of the available mammalian and avian skeletal muscle TnI amino acid sequences reveals a previously unrecognized conserved motif repeated 3-fold, once in the inhibitory peptide region (approximately residues 101-114; designated alpha) and twice more in the region of residues approximately 121-132 (beta) and approximately 135-146 (gamma). The number and distribution of these motifs have important structural implications for the TnI x C complex. In the beta motif of cardiac TnI, as compared with skeletal, several changes in charged amino acids are suggested as candidates responsible for the greater sensitivity of cardiac Ca2+-regulated actomyosin to acidic pH as in ischemia.

Distinct regions of troponin I regulate Ca2+-dependent activation and Ca2+ sensitivity of the acto-S1-TM ATPase activity of the thin filament.

The regions of troponin I (TnI) responsible for Ca2+-dependent activation and Ca2+ sensitivity of the actin-myosin subfragment 1-tropomyosin ATPase (acto-S1-TM) activity have been determined. A colorimetric ATPase assay at pH 7.8 has been applied to reconstituted skeletal muscle thin filaments at actin:S1:TM ratios of 6:1:2. Several TnI fragments (TnI-(104-115), TnI-(1-116), and TnI-(96-148)) and TnI mutants with single amino acid substitutions within the inhibitory region (residues 104-115) were assayed to determine their roles on the regulatory function of TnI. TnI-(104-115) is sufficient for achieving maximum inhibition of the acto-S1-TM ATPase activity and its importance was clearly shown by the reduced potency of TnI mutants with single amino acid substitutions within this region. However, the function of the inhibitory region is modulated by other regions of TnI as observed by the poor inhibitory activity of TnI-(1-116) and the increased potency of the inhibitory region by TnI-(96-148). The regulatory complex composed of TnI-(96-148) plus troponin T-troponin C complex (TnT.C) displays the same Ca2+ sensitivity (pCa50) as intact troponin (Tn) or TnI plus TnT.C while those regulatory complexes composed of TnT.C plus either TnI-(104-115) or TnI-(1-116) had an increase in their pCa50 values. This indicates that the Ca2+ sensitivity or responsiveness of the thin filament is controlled by TnI residues 96-148. The ability of Tn to activate the acto-S1-TM ATPase activity in the presence of calcium to the level of the acto-S1 rate was mimicked by the regulatory complex composed of TnI-(1-116) plus TnT.C and was not seen with complexes composed with either TnI-(104-115) or TnI-(96-148). This indicates that the N terminus of TnI in conjunction with TnT controls the degree of activation of the ATPase activity. Although the TnI inhibitory region (104-115) is the Ca2+-sensitive switch which changes binding sites from actin-TM to TnC in the presence of calcium, its function is modulated by both the C-terminal and N-terminal regions of TnI. Thus, distinct regions of TnI control different aspects of Tn's biological function.

Evidence for two-site binding of troponin I inhibitory peptides to the N and C domains of troponin C.

The interactions of two troponin I peptides, Ip1 (residues 96-116) and Ip2 (residues 104-116), with spectral probe mutants F29W and F105W of intact troponin C (TnC) and of isolated N (residues 1-90) and C (residues 88-162) domains of TnC have been examined. Ip-induced fluorescence emission spectral changes were observed with all four proteins in the presence of Ca2+. Different dependencies of these spectral changes on Ip concentration for intact F29W and F105W are interpreted in terms of two binding sites on TnC. The binding of Ip1 to the C domain (KD1 = 0.50 microM) is 20-40-fold stronger than to the N domain. The binding affinity of Ip1 to both the N and C domains is greater than that of Ip2. The binding strengths of Ip1 to the N domain of intact F29W and isolated F29W/ND are the same within experimental error; that to isolated F105W/CD is weakened by 5-6-fold relative to the C domain of intact F105W. Ip-induced fluorescence changes are dependent on the presence of Ca2+ and are not seen in the presence of Mg2+ alone nor in the absence of divalent cations. This is true even though Ip2 binds to TnC under all three conditions, as demonstrated by affinity chromatography. The accumulated evidence indicates that the F-->W mutations have not significantly affected the binding of Ip peptides to TnC.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Properties of isolated recombinant N and C domains of chicken troponin C.

The two globular N and C domains of chicken troponin C (TnC) are connected by an exposed alpha-helix (designated D/E; residues 86-94). Recombinant N (residues 1-90) and C (residues 88-162) domains containing either F29 or W29 and F105 or W105 have been engineered and expressed in Escherichia coli. These termination and initiation sites were chosen to minimize disruption of side-chain interactions between the D/E helix and other residues. W29 and W105 served as useful spectral probes for monitoring Ca(2+)-induced structural transitions of the N and C domains, respectively [Pearlstone et al. (1992) Biochemistry 31, 6545-6553; Trigo-Gonzalez et al. (1992) Biochemistry 31, 7009-7015]. By all criteria tested, the properties of the isolated F29W/N domain (1-90) were identical to those of the N domain in intact F29W. These included fluorescence emission spectra in the absence and presence of Ca2+/Mg2+, far-UV CD spectra, and Ca2+ affinity as monitored by fluorescence and ellipticity at 221 nm. Similar but not identical properties were observed for isolated F105W/C domain (88-162) and intact F105W. A summation of the far-UV CD spectra (+/- Ca2+) of the two domains was virtually superimposable on that of the intact protein. Of the total Ca(2+)-induced ellipticity change at 221 nm, 27% could be assigned to the N domain and 73% to the C domain. The data suggest a significant Ca(2+)-induced transition involving secondary structural elements of the N domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Spectroscopic analysis of a methionine-48 to tyrosine mutant of chicken troponin C.

A mutant (M48Y) of chicken skeletal muscle troponin C was prepared in which Tyr replaced Met-48 of the recombinant protein (rTnC). Since Tyr and Trp are normally absent, spectral properties could be unambiguously assigned to the site of substitution. In the crystal structure, this residue lies at the COOH-terminal end of the B-helix of the N domain in a region postulated to undergo a significant conformational change to a more polar environment upon Ca2+ binding [Herzberg et al. (1986) J. Biol. Chem. 261, 2638-2644]. Comparison of the far-UV CD spectra of M48Y and rTnC in the absence and presence of Ca2+ indicated no overall structural alteration due to the mutation. However, Ca2+ titration of the ellipticity change showed a reduction in Ca2+ affinity and cooperativity of sites I and II. A Ca(2+)-induced increase in the near-UV ellipticity of M48Y at pH 7.12 and a red shift in its UV absorbance spectrum occurred over a range of free [Ca2+] attributable to the N-domain transition only. This was largely abolished at pH 5.3 where Ca2+ no longer binds to sites I and II. That region of the 1H NMR spectrum attributable to Tyr was broadened upon Ca2+ binding. These Ca(2+)-induced changes are consistent with the environment of the Tyr side chain becoming chiral, less polar, and more immobile, all in a direction opposite to that predicted. These observations indicate that while the general features of the postulated model are valid, it is unlikely to be correct in detail.

Construction and characterization of a spectral probe mutant of troponin C: application to analyses of mutants with increased Ca2+ affinity.

A spectral probe mutant (F29W) of chicken skeletal muscle troponin C (TnC) has been prepared in which Phe-29 has been substituted by Trp. Residue 29 is at the COOH-terminal end of the A helix immediately adjacent to the Ca2+ binding loop of site I (residues 30-41) of the regulatory N domain. Since this protein is naturally devoid of Tyr and Trp, spectral features can be assigned unambiguously to the single Trp. The fluorescent quantum yield at 336 nm is increased almost 3-fold in going from the Ca(2+)-free state to the 4Ca2+ state with no change in the wavelength of maximum emission. Comparisons of the Ca2+ titration curves of the change in far-UV CD and fluorescence emission indicated that the latter was associated only with the binding of 2Ca2+ to the regulatory sites I and II. No change in fluorescence was detected by titration with Mg2+. The Ca(2+)-induced transitions of both the N and C domains were highly cooperative. Addition of Ca2+ also produced a red shift in the UV absorbance spectrum and a reduction in positive ellipticity as monitored by near-UV CD measurements. The fluorescent properties of F29W were applied to an investigation of five double mutants: F29W/V45T, F29W/M46Q, F29W/M48A, F29W/L49T, and F29W/M82Q. Ca2+ titration of their fluorescent emissions indicated in each case an increased Ca2+ affinity of their N domains. The magnitude of these changes and the decreased cooperativity observed between Ca2+ binding sites I and II for some of the mutants are discussed in terms of the environment of the mutated residues in the 2Ca2+ and modeled 4Ca2+ states.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of and corrections to sequences of turkey and chicken troponins-C. Effects of Thr-130 to Ile mutation on Ca2+ affinity.

Reported differences in the primary structures of chicken muscle troponin C (Wilkinson, J.M. (1976) FEBS Lett. 70, 254-256) and recombinant protein deduced from a chick muscle cDNA (Reinach, F.C. and Karlsson, R. (1988) J. Biol. Chem. 263, 2371-2376) have been reinvestigated. The complete amino acid sequence of turkey muscle troponin C has also been elucidated. Residue 100, originally reported as Asp in the chicken muscle protein, is shown to be Asn in all three structures. The three amino acid sequences are identical except as follows: 1) the blocked NH2-terminal Ala at residue 1 of the chicken protein is replaced by nonblocked Met-Ala in the recombinant protein and by nonblocked Pro in turkey troponin-C; 2) residue 130 is Thr in both avian muscle proteins but Ile in the recombinant protein; 3) Asp-133 in the chicken muscle and recombinant troponins-C is replaced by Glu in the turkey protein; 4) residue 99, originally identified as Glu in the x-ray structure of the turkey protein, is shown to be Ala in all three proteins. Calcium titration of the metal-induced conformational transition of the protein as monitored by far UV CD measurements indicated a significant decrease in Ca2+ affinity of the high-affinity sites in the case of the recombinant protein as compared with the chicken muscle protein. Both pairs of sites showed high cooperativity. That this decreased Ca2+ affinity could be attributed to different amino acid residues at position 130 and not to the differences at the NH2 termini was confirmed by site-specific mutation of Ile-130 to Thr in the recombinant protein. The mutated recombinant protein now titrated identically to the chicken muscle protein. Thr-130, whereas over 21 A from the metal of sites III and IV, is involved in a hydrogen bonding network with structured water and the NH2-terminal region of helix G.

Molecular-mass heterogeneity of Griffonia simplicifolia lectin IV subunits. Differences in the oligosaccharide moieties in the N-terminal region.

Lectin IV of Griffonia simplicifolia (Mr approximately 56,000), which has a strong affinity for both the Lewis b and Y blood-group determinants, is a dimeric protein of two subunits, alpha (29 kDa) and beta (27 kDa), separable by SDS/PAGE and containing covalently linked oligosaccharide. After digestion with N-glycanase, the protein migrates as a single band with a mobility identical with that of the beta-subunit. After cleavage with hydroxylamine of 3H-labelled, but otherwise intact, lectin, the radioactively labelled oligosaccharide was found to be associated with two blocked N-terminal peptides separable by h.p.l.c. and having identical amino acid compositions. One of these had three or four glucosamine residues per molecule, whereas the other had only one or two. Sequence analyses of these, as well as of a 21 kDa hydroxylamine-cleaved fragment and of the intact lectin pretreated with pyroglutamate aminopeptidase, have provided a unique sequence for residues 1-62 of the two subunits. Evidence is presented for two sites of N-linked oligosaccharide attachment at Asn-5 and Asn-18. Whereas the alpha-subunit has oligosaccharide linked to both sites, the beta-subunit has carbohydrate associated with only one (Asn-18). Sugar analyses of the whole lectin reveal a monosaccharide composition of (Xyl)3(Fuc)2(Man)10(GlcNAc)6, representing 6.4% of the mass of the molecule. Taken together with the susceptibility of the Asn-5 linkage (but not of Asn-18) to N-glycanase digestion, the observations indicate that the structures of the oligosaccharides at residues 5 and 18 are different.

Amino acid sequence of chicken gizzard smooth muscle SM22 alpha.

The complete amino acid sequence of SM22 alpha, a novel and abundant 22-kDa protein from chicken gizzard smooth muscle, was determined by a combination of automated and manual Edman degradation methods on fragments produced by suitable chemical and proteolytic cleavages. The protein consists of a single polypeptide chain of 197 residues, has a Mr of 21, 978, and a net charge of +4.5 at neutral pH. The pattern of alternating hydrophilic and hydrophobic regions throughout the length of SM23 alpha is typical of a globular protein. The overall secondary structural analysis, using several algorithms based on the sequence, predicts approximately 31% alpha-helix, 24% beta-sheet, 18% beta-turn, and 27% random coil. A search against the National Biomedical Research Foundation Protein Sequence Databank (Washington) and GenBank (Los Alamos) failed to demonstrate significant similarity with any other protein of known sequence.

Amino acid sequence of rabbit cardiac troponin T.

The complete amino acid sequence of the major isoform of rabbit cardiac troponin T was determined by the application of manual and automated Edman degradation procedures to fragments generated by suitable chemical or proteolytic cleavages. The protein has a polypeptide chain length of 276 amino acid residues, a Mr of 32,881, is negatively charged at neutral pH, and must be encoded by a different structural gene than rabbit skeletal troponin T. A more basic isoform differs in the NH2-terminal region by the replacement of 7 glutamic acid residues by neutral amino acids. Comparison of the sequence with that of rabbit skeletal troponin T shows close homology in those structural regions (residues 47-151 and 170-236 of rabbit skeletal troponin T) previously implicated in interactions with tropomyosin, troponin I and troponin C and predicts similar secondary structural features. In addition, the NH2- (16 residues) and COOH-terminal (10 residues) segments are homologous. In the cardiac protein, the regions of residues 17-46, 152-169, and 237-249 (rabbit skeletal troponin T numbering scheme) show little similarity with the skeletal protein and include multiple amino acid differences as well as insertions and/or deletions. Within these nonhomologous segments, however, there are regions of high similarity or identity with the amino acid sequence of chicken cardiac troponin T deduced from DNA sequencing (Cooper, T.A., and Ordahl, C.P. (1985) J. Biol. Chem. 260, 11140-11148). These include residues 36-46, 152-161, and 237-242 and may represent regions of functional importance for cardiac troponin T as compared with the skeletal protein.

Comparative studies of the amino acid and nucleotide sequences of pilin derived from Pseudomonas aeruginosa PAK and PAO.

The entire amino acid sequence for Pseudomonas aeruginosa PAO pilin was determined through peptide sequencing and from the complete nucleotide sequence encoding the pilin gene. The precursor PAO pilin is 149 amino acids in length which includes a 6-amino-acid positively charged leader sequence. Comparison of the amino acid sequences of pilin produced by P. aeruginosa PAO and PAK reveals a region of high homology corresponding to the leader peptide and residues 1 to 54 of the mature pilin. The amino acid sequence of the peptide encompassing the major antigenic determinant of PAK differs greatly from that of the equivalent region in PAO. The C-terminal regions of these proteins are semiconserved. Few major differences were found when the predicted secondary structures for PAO and PAK pilins were compared. Major nucleotide sequence variation between the equivalent restriction fragments from PAO and PAK occurred within the areas coding for the peptides containing the immunodominant site for PAK pilin and the C termini.

Studies on the primary structure and antigenic determinants of pilin isolated from Pseudomonas aeruginosa K.

The complete amino acid sequence of Pseudomonas aeruginosa K (PAK) pilin was determined using a combination of automated and manual Edman degradation techniques. Suitable peptides were derived from cyanogen bromide, tryptic, chymotryptic, peptic, thermolytic, and citraconylated tryptic cleavages of unmodified or carboxymethylated pilin. The protein, a single polypeptide chain, has N-methylphenylalanine at the NH2-terminus, a total of 144 residues, a molecular weight of 15013, and an equal number of acid and basic amino acids. The NH2-terminal region (residues 1-43) is very hydrophobic with only three charged residues, suggesting a possible role in subunit-subunit interaction. The two half-cystines, residues 129 and 142, are shown to be linked through a disulfide bridge in the native protein. To delineate the antigenic regions of pilin, the protein was cleaved at Arg-30, Arg-53, and Arg-120 to produce peptide fragments cTI (residues 1-30), cTII (residues 31-53), cTIII (residues 54-120), and cTIV (residues 121-144). cTIII and cTIV were further degraded into several subfragments. The purified peptides were subjected to immunological analysis using direct and competitive enzyme-linked immunosorbent assay procedures. A major antigenic determinant was delineated in a region of the protein encompassing residues 82-101. Three other epitopes were also identified, but reacted with only minor amounts of antibody in the rabbit polyclonal antiserum.

The interaction of rabbit skeletal muscle troponin-T fragments with troponin-I.

The interactions of troponin-I (Tn-I) with a variety of fragments spanning the length of the troponin-T (Tn-T) polypeptide chain have been reinvestigated at physiological ionic strength by affinity chromatographic, gel filtration, and circular dichroism methodologies. Strong binding was observed with fragment T2 (residues 159-259) mimicking that observed with whole Tn-T and Tn-I. Partial binding was seen with the shorter cyanogen bromide (CB) fragments of Tn-T in the order CB4 (residues 176-230) greater than CB6 (residues 239-259) or CB5 (residues 152-175). No interaction with Tn-I was observed with fragments (CB2, CB3, T1) encompassing residues 1-158 of Tn-T. Based on the present results and the work of others, the binding region for Tn-I includes residues 159-259 and perhaps extends into the highly helical CB2 region (residues 71-151) of Tn-T. No evidence has been obtained by ourselves or others for the interaction of the CB3 region (1-70) with Tn-I. A significant increase (11.6%) in alpha-helical content was observed when an equimolar amount of fragment T2 (residues 159-259) was mixed with Tn-I, a result similar to that seen with whole Tn-T and Tn-I.

Pseudomonas pili. Studies on antigenic determinants and mammalian cell receptors.

P. aeruginosa PAK pili are thin 5.2 nm diameter filaments containing a single 15-kd polypeptide subunit which is 144 amino acid residues in length. Studies on pili binding to a variety of synthetic sugars representing many di- tri- and tetra-saccharide structures found in mammalian glycoproteins and glycolipids failed to reveal any significant binding activity. On the other hand, a wide spectrum of binding activities was observed when a variety of structural proteins and enzymes were used as binding substrates. Of 30 proteins tested, phosphorylase b, pyruvate kinase and aldolase showed highest pilus binding activity. It was concluded that the PAK pilus receptor is probably a polypeptide rather than an oligosaccharide. Using arginine-specific cleavage to produce four large peptides, several proteases to produce subfragments of the large peptides, and antipilus rabbit antiserum, PAK pilin was found to contain four antigenic determinants. Epitopes near the NH2- and COOH-termini were only weakly immunogenic, whereas two epitopes near the center of the pilus protein titrated about 85% of the antipilus antibodies. Cleavage of the pilus protein into smaller peptides resulted in marked decreases in the affinity of antigenic peptides for their specific antibodies, suggesting that the immunodominant epitopes of PAK pilin are conformation-specific.