Biomedical subjects

C M Kay

Publications and source records attributed to C M Kay.

At least 127 records · Page 7Linked to original sources

PubMed1992

Human apolipoprotein A-I liberated from high-density lipoprotein without denaturation.

Apolipoprotein A-I (apoA-I) was liberated from human high-density lipoprotein (HDL) without exposure to organic solvents or chaotropic salts by the action of isolated insect hemolymph lipid transfer particle (LTP). LTP-catalyzed lipid redistribution results in transformation of HDL into larger, less dense particles accompanied by an overall decrease in HDL particle surface area:core volume ratio, giving rise to an excess of amphiphilic surface components. Preferential dissociation of apolipoprotein versus phospholipid and unesterified cholesterol from the particle surface results in apolipoprotein recovery in the bottom fraction following ultracentrifugation at a density = 1.23 g/mL. ApoA-I was then isolated from other contaminating HDL apolipoproteins by incubation with additional HDL in the absence of LTP, whereupon apolipoprotein A-II and the C apolipoproteins reassociate with the HDL surface by displacement of apoA-I. After a second density gradient ultracentrifugation, electrophoretically pure apoA-I was obtained. Sedimentation equilibrium experiments revealed that apoA-I isolated via this method exhibits a tendency to self-associate in an aqueous solution while its circular dichroism spectrum was indicative of a significant amount of alpha-helix. Both measurements are consistent with that observed on material prepared by denaturation/renaturation. The ability of apoA-I to activate lecithin:cholesterol acyltransferase was found to be similar to that of apoA-I isolated by conventional methods. The present results illustrate that LTP-mediated alteration in lipoprotein particle surface area leads to dissociation of substantial amounts of surface active apoprotein components, thus providing the opportunity to isolate apoA-I without the denaturation/renaturation steps common to all previous isolation procedures.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Conformation and dynamics of bovine brain S-100a protein determined by fluorescence spectroscopy.

We have used time-resolved laser fluorescence spectroscopy to investigate the intensity and anisotropy decays of the single tryptophan residue in bovine brain S-100a (alpha beta) protein. The steady-state and acrylamide quenching results indicated that the Trp 90 of the alpha-subunit was partially buried in a relatively nonpolar environment at pH 7.5. Both Ca2+ and pH 8.5 slightly enhanced the exposure of the residue to the solvent, but the residue remained partially buried in the calcium complex at both pH values. The best representation of the intensity decays was a linear combination of three exponential terms, regardless of solvent condition and temperature. The three lifetimes (tau i) were in the range of 0.4-5 ns and insensitive to emission wavelength, but their fractional amplitudes (alpha i) shifted in favor of the shortest component (alpha 1) when the decays were measured at the blue end of the emission spectrum. These results suggest that an excited-state interaction between the indole ring and the side chain of an adjacent residue may be responsible for the observed shortest lifetime. In the presence of Ca2+, the three lifetimes remained relatively unaltered, but the values of alpha 1 decreased by a factor of 2.3 at pH 7.2 and a factor of 1.8 at pH 8.2. This Ca(2+)-induced decrease may be attributed to disruption of the putative excited-state interaction resulting from reorientations of the alpha-helical segments flanking a Ca(2+)-binding loop (residues 62-73). At both pH 7.2 and 8.4, the anisotropy decays of the apoprotein followed a biexponential decay law.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Synthetic model proteins. Positional effects of interchain hydrophobic interactions on stability of two-stranded alpha-helical coiled-coils.

We have designed a model protein that consists of two identical 35-residue polypeptide chains, parallel and in-register arranged in a two-stranded alpha-helical coiled-coil structure. This structure is stabilized by interchain hydrophobic interactions between leucine residues at positions "a" and "d" of a repeating heptad sequence. To determine the positional effects of interchain hydrophobic interactions on the stability of the coiled-coil, a single leucine residue in each chain at position "a" (9, 16, 23, 30) and "d" (5, 12, 19, 26, 33) was systematically replaced by an alanine. All these proteins formed two-stranded alpha-helical coiled-coils in benign conditions (0.05 M phosphate, 0.1 M KCl, pH 7). The stability of each mutant protein was determined by guanidine hydrochloride denaturation experiments, where the decrease in ellipticity at 220 nm was monitored by circular dichroism. The single alanine replacements of a leucine residue at hydrophobic positions a and/or d are all shown to destabilize the coiled-coil structure. The non-equivalent hydrophobic positions a and d make an equivalent contribution to protein stability along the majority of the coiled-coil structure (positions 9-30). The small decrease in coiled-coil stability caused by Leu----Ala substitution at either ends of the coiled-coil suggested that the Leu-Leu hydrophobic interactions are less important at the ends of the coiled-coil and the ends of the coiled-coil are more flexible. Analysis of the difference between the ellipticity in benign buffer and in 50% trifluoroethanol (delta theta 220) and the slope term from a plot of the free energy of unfolding versus guanidine hydrochloride concentration also supported the conclusion that the leucine residues at the ends of the coiled-coil are much less buried than in the middle section of the coiled-coil.

Amino Acid Sequence↗

PubMed1992

The two-stranded alpha-helical coiled-coil is an ideal model for studying protein stability and subunit interactions.

We have designed de novo a two-stranded alpha-helical coiled-coil which consists of two identical 35-residue polypeptide chains arranged in a parallel and in-register alignment. Their structure is stabilized by interchain hydrophobic interactions from hydrophobes at positions "a" and "d" of a repeating heptad sequence. The formation and stability of the coiled-coil is dependent on peptide concentration due to the monomer-dimer equilibrium. In contrast, that coiled-coil containing an inter-helical disulfide bond does not show any concentration dependence in the guanidine hydrochloride denaturation experiments as expected. Replacement of one large hydrophobic Leu residue in each chain with Ala significantly decreases coiled-coil stability in both the reduced and oxidized coiled-coils [decreases in transition midpoint of 1.6M (2.3-0.7) and 2.4M (5.3-2.9), respectively]. A large pH dependence on coiled-coil stability is observed over the pH range 4 to 7 (transition midpoints at pH 4, 5, 5.5, 6 and 7 were 3.8, 3.2, 2.0, 1.2 and 0.7M, respectively). The increasing stability with decreasing pH correlates with the protonation of the Glu acid side-chains and reduction of intrachain repulsions between Glu-Glu side-chains in positions i, i + 3 or i, i + 4 along each alpha-helix of the coiled-coil. In addition, coiled-coil stability increases with increasing ionic strength.

Amino Acid Sequence↗

PubMed1992

Role of interchain alpha-helical hydrophobic interactions in Ca2+ affinity, formation, and stability of a two-site domain in troponin C.

We have previously shown that a 34-residue synthetic peptide representing the calcium-binding site III of troponin C formed a symmetric two-site dimer consisting of two helix-loop-helix motifs arranged in a head-to-tail fashion (Shaw, G.S., Hodges, R.S., & Sykes, B.D., 1990, Science 249, 280-283). In this study the hydrophobicities of the alpha-helices were altered by replacing L-98 and F-102 in the N-terminal region and/or I-121 and L-122 in the C-terminal region with alanine residues. Our results showed that substitution of hydrophobic residues either in the N- or C-terminal region have little effect on alpha-helix formation but resulted in a 100- and 300-fold decrease in Ca2+ affinity, respectively. Simultaneous substitution of both hydrophobes in the N- and C-terminal region resulted in a 1,000-fold decrease in Ca2+ affinity. Data from guanidine hydrochloride denaturation studies suggested that intermolecular interactions occur and that the less hydrophobic analogs had a lower overall conformational stability. These data support the contention that the hydrophobic residues are important in the formation of the two-site domain in troponin C, and this hydrophobic association stabilizes Ca2+ affinity.

Amino Acid Sequence↗

PubMed1992

Conformational changes in mouse MHC class II proteins at acidic pH.

MHC class II molecules bind peptide antigens and present them to T cells. Recent evidence suggests that peptide--MHC class II interaction is regulated by pH. Both peptide binding to and dissociation from MHC class II molecules is enhanced at acidic pH. In this report we use circular dichroism to investigate the possibility that the modulation of peptide association with MHC class II molecules at acidic pH is associated with changes in MHC class II structure. Our results show that a change in the structure of the MHC class II protein A(d) occurs between pH 4 and 5, close to the pH optimum for peptide binding. More drastic changes in A(d) structure occur at lower pH levels, where peptide dissociation is enhanced. The structural changes at pH 4-5 are fully reversible upon neutralization, while changes at pH 3 are not. The pH stability of purified E(d) molecules is somewhat different, with changes observed only at pH less than 4. This may reflect a differential occupancy of the purified molecules with endogenous peptides.

Animals↗

PubMed1992

Single amino acid substitutions can further increase the stability of a thermophilic L-lactate dehydrogenase.

Lactate dehydrogenases are of considerable interest as stereospecific catalysts in the chemical preparation of enantiomerically pure alpha-hydroxyacid synthons. For such applications in synthetic organic chemistry it would be desirable to have enzymes which tolerate elevated temperatures for prolonged reaction times, to increase productivity and to extend their applicability to poor substrates. Here, two examples are reported of significant thermostabilizations, induced by site-directed mutagenesis, of an already thermostable protein, the L-lactate dehydrogenase (EC 1.1.1.27, 35 kDa per monomer subunit) from Bacillus stearothermophilus. Thermal inactivation of this enzyme is accompanied by irreversible unfolding of the native protein structure. The replacement of Arg171 by Tyr stabilizes the enzyme against thermal inactivation and unfolding. This stabilizing effect appears to be based on improved interactions between the subunits in the core of the active dimeric or tetrameric forms of the enzyme. The thermal stability of L-lactate dehydrogenase variants with an active site Arg residue, either in the 171 (wild-type) or in the 102 position, is further increased by sulfate ions. The two stabilizing effects are additive, as found for the Arg171Tyr/Gln102Arg double mutant, for which the stability of the protein in 100 mM sulfate solution reaches that of L-lactate dehydrogenases from extreme thermophiles. All mutant proteins retain significant catalytic activity, both in the presence and absence of stabilizing salts, and are viable catalysts in preparative scale reactions.

Calorimetry↗

PubMed1992

Effect of particle lipid content on the structure of insect lipophorins.

Four distinct subspecies of the major insect lipoprotein, lipophorin, that range in overall lipid content from 20 to 51% of the particle mass, were isolated from the hemolymph or oocytes of the tobacco hornworm, Manduca sexta. Examination of these subspecies by electron microscopy revealed distinctive morphologies. Adult high density lipophorin (HDLp-A) was found to be an approximately spherical particle with a diameter of 15 +/- 1 nm, while HDLp-Wanderer 1 (W1), was more rectangular in shape and had a distinct cleft extending into the particle at one end. In the case of HDLp-Wanderer 2 (W2) the cleft was deeper and wider than that in HDLp-W1. In egg very high density lipophorin (VHDLp-E) the cleft was increased in size to the extent that the particle had an overall crescent-like conformation. Circular dichroism spectroscopy of the three lipophorin subspecies that contain only apolipophorin I and II revealed that only minor differences in the global protein secondary structure occur as the particle lipid content is decreased. The VHDLp-E apolipoproteins are an exception in that, while having the same alpha-helix content as HDLp-W1 and HDLp-W2, they contain less beta-structure and correspondingly more random coil. Limited digestion of the apolipoprotein components of the lipophorin subspecies with trypsin revealed that as the lipid content of the particles decreases the susceptibility of the apolipoprotein to proteolytic degradation increases. Likewise, tryptophan fluorescence quenching experiments demonstrated that the relative exposure of lipophorin apolipoprotein tryptophan residues also increases as the particle lipid content decreases.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

A comparative study of the interactions of synthetic peptides of the skeletal and cardiac troponin I inhibitory region with skeletal and cardiac troponin C.

The cardiac and skeletal TnI inhibitory regions have identical sequences except at position 110 which contains Pro in the skeletal sequence and Thr in the cardiac sequence. The effect of the synthetic TnI inhibitory peptides [skeletal TnI peptide (104-115), cardiac TnI peptide (137-148), and a single Gly-substituted analogue at position 110] on the secondary structure of skeletal and cardiac TnC was investigated. The biphasic increases in ellipticity and tyrosine fluorescence were analyzed to determine the Ca2+ binding constants for the high- and low-affinity Ca2+ binding sites of TnC. Importantly, the skeletal and cardiac TnI peptides altered Ca2+ binding at the low-affinity sites of TnC, but the magnitude and direction of the pCa shifts depended on whether the peptides were bound to skeletal or cardiac TnC. For example, binding of skeletal TnI peptide to skeletal TnC (monitored by CD) caused a pCa shift of +0.30 unit such that a lower Ca2+ concentration was required to fill sites I and II, while binding of this peptide to cardiac TnC caused a pCa shift of -0.35 unit such that a higher Ca2+ concentration was required to fill site II. This is the first report of the alteration at the low-affinity regulatory sites (located in the N-terminal domain) by the skeletal TnI inhibitory peptide, even though the primary peptide binding site is located in the C-terminal domain of TnC, a finding which strongly indicates that there is communication between the two halves of the TnC molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1991

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.

Amino Acid Sequence↗

PubMed1991

Spectral [corrected] studies on the cadmium-ion-binding properties of bovine brain S-100b protein.

The effect of Cd2+ binding on bovine brain S-100b protein was studied using c.d. u.v. difference spectroscopy and fluorescence measurements. At pH 7.5, S-100b protein binds two Cd2+ ions per monomer with a Kd value of 3 x 10(-5) M. Addition of Cd2+ resulted in perturbing the single tyrosine residue (Tyr17) in the protein as indicated by u.v. difference spectroscopy and aromatic c.d. measurements. In the presence of Cd2+, the tyrosine residue moves to a more non-polar environment, since a red shift was observed in the u.v. difference spectrum. When the protein was excited at 278 nm, the tyrosine fluorescence emission maximum was centred at 306 nm. Cd2+ addition resulted in an increase in intrinsic fluorescence intensity. Fluorescence titration with Cd2+ indicated the protein binds Cd2+ with a Kd value of 3 x 10(-5) M. 2-p-Toluidinylnaphthalene-6-sulphonate-labelled protein, when excited at 345 nm, had a fluorescence emission maximum at 440 nm. Addition of Cd2+ to labelled protein resulted in a 5-fold increase in fluorescence intensity accompanied by a 5 nm blue shift in the emission maximum, suggesting that the probe, in the presence of Cd2+, moves to a hydrophobic domain. U.v. difference spectroscopic studies indicated a unique Cd2(+)-binding site on the protein, since Cd2+ addition yielded a large positive absorption band in the 240 nm region that is not found with either Ca2+ or Zn2- ions. Similar absorption bands have been observed in Cd-protein complexes such as Cd-metallothionein [Vasak, Kagi & Hill (1981) Biochemistry 20, 2852-2856] and also in model complexes of Cd2+ with 2-mercaptoethanol. This absorption band is believed to arise as a result of charge-transfer transitions between the thiolate and Cd2+. Of the two Cd2- -binding sites on the beta-chain, one must be located at the N-terminal end near the single tyrosine residue, since Cd2- and Zn2+ produced similar effects on the intrinsic protein fluorescence. The other Cd2+ site which is unique to Cd2+ must be Cys84, located at the C-terminal end.

Amino Acid Sequence↗

PubMed1991

Solution structure of a polypeptide dimer comprising the fourth Ca(2+)-binding site of troponin C by nuclear magnetic resonance spectroscopy.

The structure of a 39 amino acid proteolytic fragment of rabbit skeletal troponin C containing the fourth Ca(2+)-binding site has been determined by an approach involving nuclear magnetic resonance (NMR) spectroscopy combined with hybrid distance geometry-dynamical simulated annealing calculations. Hydrodynamic and NMR evidence establishes unambiguously that the fragment forms a stable dimer in solution in the presence of excess Ca2+. The calculation of the dimeric structure is based on a total of 1056 experimental restraints comprising 422 interproton distances, 35 phi, 28 psi, and 28 chi 1 torsion angle restraints within each subunit, 30 intermonomer distance restraints, and 6 Ca2+ restraints per subunit. A total of 48 final structures were calculated having an rms deviation about the mean atomic backbone coordinate positions of 1.0 A for residues Asp128-Glu156. The solution structure consists of a dimer of helix-loop-helix motifs related by a 2-fold axis of symmetry. The overall architecture of the dimer is very similar to the C-terminal domain in the crystal structure of chicken skeletal troponin C.

Amino Acid Sequence↗

PubMed1990

Electrochemical, kinetic, and circular dichroic consequences of mutations at position 82 of yeast iso-1-cytochrome c.

Replacement of Phe-82 in yeast iso-1-cytochrome c with Tyr, Leu, Ile, Ser, Ala, and Gly produces a gradation of effects on (1) the reduction potential of the protein, (2) the rate of reaction with Fe(EDTA)2-, and (3) the CD spectra of the ferricytochromes in the Soret region under conditions where contributions from the alkaline forms of these proteins are absent. The reduction potential of cytochrome c is lowered by as little as 10 mV (Tyr-82) or by as much as 43 mV (Gly-82; pH 6.0) as the result of these substitutions. The second-order rate constants for reduction of these cytochromes range from a low of 6.20 (2) x 10(4) for the Tyr-82 variant to a high of 14.8 x 10(4) M-1 s-1 for the Ser-82 variant [pH 6.0, 25 degrees C, mu = 0.1 M (sodium phosphate)]. Analysis of these rates by use of relative Marcus theory produces values of k11corr that range from 10.9 M-1 s-1 for the wild-type protein to 190 M-1 s-1 for the Gly-82 mutant [25 degrees C, mu = 0.1 M, pH 6.0 (sodium phosphate)]. Reinvestigation of the effect of substituting Phe-82 by a Tyr residue on the CD spectrum of the protein now reveals little alteration of the intense, negative Cotton effect in the Soret CD spectrum of ferricytochrome c. On the other hand, substitution of nonaromatic residues of various sizes at this position results in loss of this spectroscopic feature, consistent with previous findings.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1990

Purification and spectral study of a microbial fatty acyltransferase: activation by limited proteolysis.

A fatty acyltransferase with a reaction mechanism similar to that of mammalian lecithin: cholesterol acyltransferase has been purified from culture supernatants of a mutant Aeromonas salmonicida containing the cloned Aeromonas hydrophila structural gene. Typically, more than 35 mg of protein were isolated from 2 L of culture supernatant. The amino-terminal sequence, amino acid composition, and molecular weight of the purified protein corresponded to predictions based on the sequence of the gene, indicating that the signal sequence had been correctly removed during export but that no further processing had occurred. Analysis of the far-UV circular dichroic (CD) spectrum of the enzyme showed that it consists of 31% alpha-helix, 21% beta-sheet, and 16% beta-turn, with 12% of aperiodic form. Treatment of the purified protein with a variety of proteases resulted in nicking near the C-terminus. This led to an increase in enzyme activity against lipids in erythrocyte membranes and increased rate of hydrolysis of p-nitrophenyl butyrate. Activation was accompanied by a change in the CD spectrum and a change in its aggregation state. The trypsin cut site was located between the two cysteines in the enzyme. Evidence is presented that the cysteines are joined by a disulfide bond and therefore cannot participate in acyl transfer. This may distinguish the microbial enzyme from lecithin:cholesterol acyltransferase. This is the second extracellular A. hydrophila protein that we have shown can be activated by proteolysis after it is released.

Acyltransferases↗

PubMed1990

Biophysical studies on the lipid transfer particle from the hemolymph of the tobacco hornworm, Manduca sexta.

Hydrodynamic studies conducted in the analytical ultracentrifuge provided evidence for two populations of lipid transfer particle (LTP) when centrifuged in a buffer solution containing 10 mM Tris, pH 8.0/100 mM KCl. The apparent sedimentation coefficients of the two species was 23.3 S and 15.3 S. Upon changing the buffer pH to 7.0 or 5.7, two species of LTP were still present but the ratio of their relative abundance was altered. When the KCl concentration in the buffer was lowered to 50 mM the sample sedimented as a single species with an apparent S20,w of 22.9 S. In higher ionic strength buffers (10 mM succinate, pH 5.7/500 mM KCl) LTP sedimented with an apparent S20,w of 14.8 S. Further experiments revealed that these two forms are interconvertable as a function of buffer ionic strength. Given previous estimates of the molecular size of LTP we concluded that the slower sedimenting peak observed at high ionic strength represents monomeric LTP while the faster sedimenting material observed at low ionic strength is likely to be an aggregated state of LTP. This interpretation is supported by molecular weight determinations made by sedimentation equilibrium experiments conducted in 10 mM succinate, pH 5.7/500 mM KCl which yielded a particle Mr = 887,000. Circular dichroism spectra of monomeric LTP sample revealed 6% alpha-helix, 49% beta-sheet, 7% beta-turn and 35% random coil while aggregated LTP contained 13% alpha-helix, 66% beta-sheet and 21% random coil. The transfer activity of the two LTP forms was assayed and found to be the same indicating that either the state of LTP aggregation did not affect transfer activity or that upon exposure to a large excess of lipoprotein substrate disaggregation, without loss of activity, occurs.

Animals↗

PubMed1990

Isolation and characterization of a novel molecular weight 11,000 Ca2(+)-binding protein from smooth muscle.

A new low molecular weight calcium-binding protein, designated as SMCaBP-11, has been isolated from chicken gizzard using a phenyl-Sepharose affinity column followed by ion-exchange and gel filtration chromatographies. The isolated protein was homogeneous by the criteria of gel electrophoresis in the absence and presence of sodium dodecyl sulfate (NaDodSO4). Molecular weight studies by both sedimentation equilibrium in 6 M guanidine hydrochloride and 15% polyacrylamide-SDS gels indicated the subunit molecular weight to be 11,000, and since a molecular weight of 21,000 was obtained in native solvents, the protein exists as a dimer in benign medium. The amino acid composition of this protein is similar but distinct from other known low molecular weight Ca2(+)-binding proteins. Ca2(+)-binding assays using Arsenazo III (Sigma) indicated the protein to bind 2 mol of Ca2+/subunit. In non-SDS gels, the protein moved faster in the presence of EDTA, suggesting that Ca2+ binding affects its mobility in a manner similar to other smooth muscle calcium-binding proteins such as calmodulin and 67-kDa calcimedin. Upon binding calcium, the protein underwent a conformational change as revealed by UV difference spectroscopy and circular dichroism studies in the aromatic and far-ultraviolet range. When the protein was excited at 280 nm, the tyrosine fluorescence emission maximum was centered at 306 nm. Ca2+ addition resulted in a nearly 15% decrease in intrinsic fluorescence intensity. Fluorescence titration with Ca2+ exhibited two classes of calcium-binding sites with Kd values of 0.2 and 80 microM, in agreement with UV difference spectral data.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1990

Functional degeneracy of residues in a T cell peptide epitope contributes to its recognition by different T cell hybridomas.

Synthetic antigen Poly EYK(EYA)5 induces T cells of narrowly defined fine specificity as represented by the two I-Ad-restricted T cell hybridomas, A.1.1 and B.1.1. Both these hybridomas recognize the minimum 15-amino-acid peptide sequence EYK(EYA)4. We have characterized the residues involved in the recognition of EYK(EYA)4 peptide by these hybridomas with synthetic peptides and discovered a distinct functional hierarchy for the residues in the sequence. Even with the repeating tripeptide (EYA)5, which is recognized by B.1.1 cells, the residues that are essential cluster near the middle of the sequence but not near the N- or C-terminal region. Different MHC binding and TCR contacting residues were found for each of the hybridomas. The results suggest that different T cells either recognize different parts of the peptide MHC complex or that the peptide binds to MHC in multiple conformations. This was supported by the fact that Poly EYK(EYA)5 is alpha-helical but the peptides used here showed only a slight propensity to adopt this structure and it did not correlate with their functional activity. We also found that (EYA)5 does not compete with EYK(EYA)4 in the stimulation of A.1.1 cells despite its obvious capacity to interact with I-Ad when it stimulates B.1.1 cells. This may be because these peptides have a low affinity for Ia and therefore only appropriate TCR interactions would stabilize the antigen-Ia complex. In conclusion, antigen-MHC-TCR interaction appears to be a dynamic process which allows recognition of different residues of a T cell determinant by different T cells.

Amino Acid Sequence↗

PubMed1989

Secondary structural features of the bacteriophage Mu-encoded A and B transposition proteins.

The role of the bacteriophage Mu-encoded A and B proteins is to direct the transposition of Mu DNA. These are the first active DNA transposition proteins to have been purified and their mechanism of action at the biochemical level is under intensive study. Structural studies on these proteins, however, have lagged behind their biochemical characterization. We report here near- and far-u.v. c.d. spectra for these proteins and their secondary structural features derived from these data. The Mu A protein appears to be composed of primarily beta-sheet (40%) with 24% alpha-helix, 9% beta-turn and 27% random coil. In contrast, the Mu B protein contains 55% alpha-helix with only 13% beta-sheet and 3+ beta-turn and 29% random coil. The near-u.v. c.d. spectrum of the A protein was not unusual; however, the profile of the B protein suggested either buried or restricted chromophores within the protein or short-range interactions between aromatic residues.

Circular Dichroism↗