Biomedical subjects

C M Kay

Publications and source records attributed to C M Kay.

At least 109 records · Page 6Linked to original sources

PubMed1993

Structural prerequisites for serum amyloid A fibril formation.

Most studies of experimental amyloid A protein (AA) amyloidosis in mice have been performed in type A mice with BALB/c as the prototype. In these mice the products of two genes, SAA1 and SAA2, are the major apo-SAA isoforms on high density lipoprotein (HDL). Of these two isoforms, that differ at nine amino acids, only apo-SAA2 is rapidly cleared and deposited as amyloid fibrils. No mouse strain has ever been shown to be completely resistant to amyloid induction. We have found the CE/J mouse strain to be exceedingly resistant to amyloidogenesis. Data indicate that this resistance is not due to a lack of apo-SAA synthesis but rather resides in the unique apo-SAA isoform in this strain. CE/J mice have a single major apo-SAA isoform (pI 6.15) the product of a single gene. This is a hybrid molecule with features of both apo-SAA1 and apo-SAA2, differing from the latter at only six amino acids. When CD studies were performed to explore the structural relationship of this isoform to apo-SAA1 and apo-SAA2, we found that when bound to heparan sulfate proteoglycan the CE/J pI 6.15 isoform fails to undergo the beta-sheet folding typical for apo-SAA2. This evidence suggests that the folding effect of heparan sulfate proteoglycan on apo-SAA2 is important in amyloid formation.

Amino Acid Sequence↗

PubMed1993

Comparison of antiparallel and parallel two-stranded alpha-helical coiled-coils. Design, synthesis, and characterization.

An antiparallel coiled-coil has been designed and characterized as a model for studying protein folding and assembly. This heterostranded antiparallel coiled-coil was formed by an interchain disulfide bond between cysteine residues at position 2 of one chain and at position 33 of the other chain. Each peptide chain has 35 residues which are composed of five heptad repeats of the sequence K-L-E-A-L-E-G with a single Leu-->Ala substitution at position 16. Two homostranded parallel coiled-coils were also formed as co-products of the oxidation reaction to form the interchain disulfide bond. The CD spectra of the parallel and antiparallel peptides were very similar and their high molar ellipticities at 220 nm did not increase in the presence of 50% trifluoroethanol. These data suggest that, like the parallel peptides, the antiparallel peptide also exists in a coiled-coil structure. Urea and guanidine hydrochloride denaturation studies, in conjunction with molecular modeling studies, suggest that there are no physical restrictions to the packing of hydrophobic residues in an antiparallel coiled-coil. However, interchain electrostatic interactions can have positive or negative contributions to the overall stability of the disulfide-bridged coiled-coil. In addition, interchain electrostatic interactions appear to play a major role in protein folding by controlling the parallel or antiparallel alignment of the alpha-helical polypeptide chains. This study is also for the first time providing us with a new understanding of the information that can be obtained from urea and guanidine hydrochloride denaturation studies of proteins concerning the contributions of hydrophobic and electrostatic interactions on stability.

Amino Acid Sequence↗

PubMed1993

Physical properties of apolipoprotein A-I from the chicken, Gallus domesticus.

The amphipathic alpha-helices of exchangeable apolipoproteins (apo) function to simultaneously facilitate interaction with lipid surfaces and the aqueous environment. In contrast to mammalian apoA-I's, which self-associate in the absence of lipid, chicken apoA-I, which shares 66% sequence homology with human apoA-I, exists as a monomeric protein when dissociated from high-density lipoprotein (HDL). Sedimentation equilibrium studies conducted in the analytical ultracentrifuge yielded a weight-average molecular weight of 28,170. Corresponding sedimentation velocity and diffusion experiments gave rise to s0(20,w) = 2.23 S and D0(20,w) = 6.39 x 10(-7) cm2/s. A translational frictional ratio (f/fmin) of 1.18 and an axial ratio of 4.0 were also determined from this data. The Stokes radius (Rs,sed = 2.80 nm) and translational frictional ratio were subsequently used to calculate estimated molecular dimensions of 25.2 x 100.8 A for chicken apoA-I. Circular dichroism (CD) studies revealed a highly alpha-helical structure predicted to be 74% by Provencher-Glöckner analysis. Denaturation studies performed on lipid-free apoA-I and monitored by CD revealed a midpoint of denaturation of 0.64 M guanidine hydrochloride. From plots of delta G(app) versus guanidine hydrochloride concentration, a delta GDH2O of 1.86 kcal/mol was determined. In other studies, a midpoint of temperature-induced denaturation for apoA-I of 57 degrees C was obtained. The effect of solvent pH on the secondary structure content of apoA-I revealed a significant loss of alpha-helix below pH 4.0 and above pH 10, suggesting that lipid-free apoA-I may by partially stabilized by the formation of intra- or interhelix salt bridges between oppositely charged amino acid side chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Purification, characterization, and conformational analysis of rabbit plasma lipid transfer protein.

A procedure for rapid isolation of lipid transfer protein (LTP) from commercially available rabbit plasma is described. Use of protease inhibitors was important for obtaining intact, stable LTP. After lipoproteins were precipitated from the plasma by dextran sulfate, column chromatographies through Butyl-Toyopearl 650M, CM-Toyopearl 650M, and Butyl-Toyopearl 650M were employed. Overall purification from plasma was (3830 +/- 710)-fold with a yield of 3-5%. The isolated LTP migrated as a single band during sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M(r) = 74K and had an NH2-terminal amino acid sequence and amino acid composition closely matching those predicted by its cDNA. This band was recognized by immunoblotting with an anti-human LTP monoclonal antibody, TP2. Gel permeation chromatography revealed that LTP behaved as a globular protein of M(r) = 83K. Isoelectric focusing of the isolated LTP demonstrated a ladder of bands with pI's of 5.7-5.9. The specific activity of rabbit LTP was similar to that of human LTP. Monoclonal antibody TP2, that blocked human plasma LTP activity almost completely, only partially inhibited purified rabbit LTP, and rabbit plasma LTP activity to a similar extent. By a centrifugation binding assay, rabbit LTP was shown to predominantly associate with lipid microemulsion in its presence. Circular dichroism spectroscopy indicated a high content of beta structure, and Provencher and Glöckner analysis gave estimated fractional values of 0.30, 0.39, 0.12, and 0.19 for alpha-helix, beta-sheet, beta-turn, and remainder content, respectively. Upon lipid binding, the helical content did not change drastically, although there was some disordering of beta structure.

Amino Acid Sequence↗

PubMed1993

A single-stranded amphipathic alpha-helix in aqueous solution: design, structural characterization, and its application for determining alpha-helical propensities of amino acids.

In order to investigate the positional effect of alpha-helical propensities of amino acids in an amphipathic alpha-helix, an amphipathic alpha-helical model peptide (Ac-Glu-Ala-Glu-Lys-Ala-Ala-Lys-Glu-Ala-Glu-Lys-Ala-Ala-Lys-Glu-Ala-Glu-Lys- amide) was designed and characterized by circular dichroism and 2D-NMR spectroscopies. This peptide contains 65% alpha-helical structure in solution, and its monomeric molecular weight in aqueous solution was determined by size-exclusion chromatography. The independence of alpha-helical structure and stability on peptide concentration demonstrates that helix formation of this peptide is a monomolecular process. To compare the effect of substitutions in the hydrophobic and hydrophilic face of the helix on structure and stability, a leucine, alanine, or glycine was individually substituted in the hydrophobic face (position 9) or hydrophilic face (position 7) of the model peptide. The change in helix content and stability upon substitution was measured by circular dichroism studies in the absence and presence of TFE or urea. The results indicate that each amino acid has a different helix propensity when it is located in the hydrophobic face versus hydrophilic face and the effect of substitution is more significant in the hydrophobic face. This single-stranded amphipathic alpha-helical peptide provides an appropriate model system to determine helix propensities of amino acids on both hydrophobic and hydrophilic faces.

Amino Acid Sequence↗

PubMed1993

Disulfide bond contribution to protein stability: positional effects of substitution in the hydrophobic core of the two-stranded alpha-helical coiled-coil.

To investigate the positional effect of the disulfide bond on the structure and stability of a two-stranded alpha-helical coiled-coil, an interchain disulfide bond was systematically introduced into the hydrophobic core of a de novo designed model coiled-coil at the N-terminus (position 2), C-terminus (position 33), and nonterminal positions a (positions 9, 16, 23, and 30) and d (positions 5, 12, 19, and 26). The rate of formation of a disulfide bond is faster at position d compared to at the corresponding position a under nondenaturing conditions, suggesting that position d is more suitable for engineering a disulfide bond. The structure and stability of the reduced and oxidized coiled-coils were determined by circular dichroism studies in the absence and presence of guanidine hydrochloride. Our results demonstrate that the improvement of protein stability by introduction of a disulfide bond is very relevant to its location and the most effective disulfide bonds are those that can be introduced in the hydrophobic core without any disruption of the protein structure. The disulfide bond at position d with near-optimal geometry does not perturb the coiled-coil structure and makes the largest contribution to coiled-coil stability. In contrast, the inappropriate geometry of the disulfide bond at nonterminal position a introduces a high strain energy on the disulfide bond which disrupts the coiled-coil structure. At positions a, the closer the disulfide bridge is to the center of the coiled-coil, the larger the disruption on the coiled-coil structure and the smaller the contribution the disulfide bond makes to coiled-coil stability. The computer modeling results also suggest that an insertion of an interchain disulfide bond at position a in the GCN4 leucine zipper X-ray structure has a higher potential energy than insertion at position d. The energy-minimized coiled-coil structure with an interchain disulfide bond at position a has a larger root mean square difference from the X-ray structure of GCN4 than the coiled-coil with a disulfide bond at position d. Because interhelical interactions are common in globular proteins as well as coiled-coils, the results obtained in this study will have general utility for selecting the sites for engineering disulfide bonds between alpha-helices.

Amino Acid Sequence↗

PubMed1993

Characterization of the smooth muscle calponin and calmodulin complex.

Calponin interacts with several Ca2+ binding proteins in a Ca(2+)-dependent manner. In order to determine the possible biological relevance of these interactions in smooth muscle function, it is necessary to characterize the strength and stoichiometry of the complexes formed. The interaction between calponin and calmodulin can be monitored through an acrylodan label on a cysteine of calponin. The fluorescently labeled calponin possesses the same biological function and physical behavior in binding to calmodulin as the native calponin. This probe is very environment-sensitive and responds to the calponin-calmodulin interaction by the emission peak blue-shifting 20 nm and by the fluorescent quantum yield increasing 3.5 times at 460 nm. The stoichiometric nature of this complex has been determined using analytical ultracentrifugation and is two calmodulins to one calponin, and the interaction is Ca(2+)-sensitive with a Kd1 of < or = 0.22 microM and a Kd2 of 2.5-3.4 microM. Calmodulin is not the only protein which interacts with calponin in this manner, but rather this interaction seems to be a general feature attributable to all hydrophobic patch exposing proteins, suggesting that it may be nonspecific, occurring because of a generalized mode of interaction. Two other proteins, S-100b from bovine brain and SMCaBP-11 from smooth muscle, had stronger affinities for calponin, and in particular interaction of SMCaBP-11 with calponin may be biologically relevant. In determining the nature of calponin's interaction with these Ca2+ binding proteins, it was apparent there was no effect of Ca2+ upon calponin itself and physical studies could find no evidence that calponin interacts with calcium.

2-Naphthylamine↗

PubMed1993

Conformational, thermodynamic, and stability properties of Manduca sexta apolipophorin III.

Apolipophorin III (apoLp-III) is a major protein in hemolymph of adult Manduca sexta. Although it normally exists in a lipid-free state, during sustained flight, apoLp-III functions as an apolipoprotein, reversibly associating with the surface of lipoprotein particles. In an effort to gain a fuller understanding of this dual existence, we have investigated its solution properties using spectroscopic methods. The UV absorption spectrum of apoLp-III is distinctive owing to the absence of tryptophan and the presence of a single tyrosine residue. Circular dichroism experiments revealed an enhancement of apoLp-III alpha-helical content when spectra were obtained in 50% trifluoroethanol versus aqueous buffer. The helical content in buffer was unaffected by protein concentration, suggesting that apoLp-III exists in solution as a monomeric species. At pH values > 10 and < 4, there was a marked loss of helical content. Increasing the temperature of apoLp-III solutions also caused a loss of secondary structure, with a temperature-induced denaturation midpoint of 52 degrees C. Upon recooling of heat-denatured apoLp-III, approximately 95% of the secondary structure was restored. In guanidine HCl denaturation studies monitored by CD, a 50% transition midpoint of 0.355 M was determined, corresponding to a delta GDH2O of 1.29 kcal/mol. Fluorescence studies indicated that guanidine HCl induced an enhancement of tyrosine fluorescence emission at 300 nm when excited at 277 nm. In native apoLp-III, we propose that tyrosine fluorescence is quenched to a large extent due to a hydrophobic stacking interaction of its side chain with that of a neighboring phenylalanine residue. delta GDH2O was determined from the fluorescence data to be 2.1 kcal/mol, with a transition midpoint occurring at 0.25 M guanidine HCl. The lower concentration of guanidine HCl required to induce half-maximal tyrosine fluorescence enhancement versus the transition midpoint detected by CD may be a reflection of the fact that this residue is located near the COOH-terminal end of the protein and as such may be more susceptible to denaturation. The results presented indicate that apoLp-III assumes a relatively labile conformation in solution that appears to be partially stabilized by side chain charge-charge interactions within predicted alpha-helical segments.

Animals↗

PubMed1993

Packing and hydrophobicity effects on protein folding and stability: effects of beta-branched amino acids, valine and isoleucine, on the formation and stability of two-stranded alpha-helical coiled coils/leucine zippers.

The aim of this study was to examine the differences between hydrophobicity and packing effects in specifying the three-dimensional structure and stability of proteins when mutating hydrophobes in the hydrophobic core. In DNA-binding proteins (leucine zippers), Leu residues are conserved at positions "d," and beta-branched amino acids, Ile and Val, often occur at positions "a" in the hydrophobic core. In order to discern what effect this selective distribution of hydrophobes has on the formation and stability of two-stranded alpha-helical coiled coils/leucine zippers, three Val or three Ile residues were simultaneously substituted for Leu at either positions "a" (9, 16, and 23) or "d" (12, 19, and 26) in both chains of a model coiled coil. The stability of the resulting coiled coils was monitored by CD in the presence of Gdn.HCl. The results of the mutations of Ile to Val at either positions "a" or "d" in the reduced or oxidized coiled coils showed a significant hydrophobic effect with the additional methylene group in Ile stabilizing the coiled coil (delta delta G values range from 0.45 to 0.88 kcal/mol/mutation). The results of mutations of Leu to Ile or Val at positions "a" in the reduced or oxidized coiled coils showed a significant packing effect in stabilizing the coiled coil (delta delta G values range from 0.59 to 1.03 kcal/mol/mutation). Our results also indicate the subtle control hydrophobic packing can have not only on protein stability but on the conformation adopted by the amphipathic alpha-helices. These structural findings correlate with the observation that in DNA-binding proteins, the conserved Leu residues at positions "d" are generally less tolerant of amino acid substitutions than the hydrophobic residues at positions "a."

Amino Acid Sequence↗

PubMed1993

Structural properties of the myelin-associated glycoprotein ectodomain.

Myelin-associated glycoprotein (MAG) has been proposed to mediate adhesive interactions during myelin development. We have used the baculovirus expression system to produce a truncated form of this molecule [soluble extracellular domain of MAG (sMAG)] consisting of the complete extracellular ectodomain. Spectroscopic studies indicate a high beta-sheet content, consistent with the prediction of Ig-like structure. Hydrodynamic studies indicate an asymmetric monomer, with a Stokes radius of 4.1-4.6 nm, a sedimentation coefficient of 3.6S, and a frictional ratio of approximately 1.6. We postulate that the outer two Ig-like domains form a unit that folds back over the rest of the molecule. Fluorescence quenching studies indicate that sMAG interacts with divalent cations and may have a functional lectin domain.

Amino Acids↗

PubMed1993

DNA-based diagnostic tests for Salmonella species targeting agfA, the structural gene for thin, aggregative fimbriae.

Salmonella enteritidis 27655-3b and a few diarrheagenic Escherichia coli strains produce morphologically and antigenically related, thin, aggregative fimbriae, collectively named GVVPQ fimbriae (S. K. Collinson, L. Emödy, T. J. Trust, and W. W. Kay, J. Bacteriol. 174:4490-4495, 1992). To determine whether GVVPQ fimbriae are common to Salmonella spp. and other enteropathogenic members of the family Enterobacteriaceae, 113 isolates were phenotypically screened for Congo red binding and aggregative colony morphology. Presumptive positive and representative negative strains were examined by Western blotting (immunoblotting) by using antiserum to SEF 17, the native GVVPQ fimbria of S. enteritidis. Only four S. enteritidis strains and six E. coli isolates possessed substantial amounts of GVVPQ fimbriae after 24 h of incubation on T medium. Following 5 days of incubation, 56 of 93 Salmonella isolates (60%) and 1 of 7 additional E. coli clinical isolates possessed detectable levels of GVVPQ fimbriae. Since variable expression of GVVPQ fimbriae was observed among Salmonella isolates and some E. coli strains produced scant amounts, as revealed by immunoelectron microscopy, the ability to produce these fimbriae was evaluated by genotypic screening. The structural gene for the SEF 17 fimbrin, agfA, was amplified by the polymerase chain reaction, cloned, and sequenced to provide a characterized DNA probe. An agfA DNA fragment hybridized strongly to 603 of 604 (99.8%) Salmonella isolates but very weakly to 31 of 266 other members of the family Enterobacteriaceae including 26 of 137 E. coli strains, 3 of 14 Citrobacter spp., and single isolates of Shigella sonnei and Enterobacter cloacae. The agfA DNA probe proved to be a valuable diagnostic tool for Salmonella isolates arrayed on hydrophobic grid membrane filters. Unique agfA sequences were targeted in the development of a polymerase chain reaction assay specific for Salmonella spp.

Amino Acid Sequence↗

PubMed1992

Calcium-dependent regulation of caldesmon by an 11-kDa smooth muscle calcium-binding protein, caltropin.

Caldesmon from chicken gizzard muscle has been examined for its ability to interact with caltropin using affinity chromatography and the fluorescent probe acrylodan. The action of caltropin on the inhibitory effect of caldesmon on actomyosin ATPase was also studied. Like calmodulin, caltropin could release the inhibitory effect of caldesmon in the presence of Ca2+. Complete reversal was obtained when 1 mol of caltropin was added per mol of caldesmon. When caldesmon was applied to caltropin-Sepharose in the presence of Ca2+, most of the caldesmon was bound to the column and could be eluted with EGTA, indicating that there is a direct interaction between caldesmon and caltropin. Acrylodan-labeled caldesmon, when excited at 375 nm, had an emission maximum at 504 nm. Addition of caltropin in the presence of Ca2+ resulted in a nearly 50% increase in fluorescence intensity, and this was accompanied by a blue shift in the emission maximum (i.e., lambda em,max 492 nm), suggesting that the probe now occupies a more nonpolar environment. Titration of caltropin with labeled caldesmon indicated a strong affinity for this protein (Kd was in the order of 8 x 10(-8)-2 x 10(-7) M). However, when caltropin was added to labeled caldesmon in the presence of EGTA, there was no indication of any interaction. Caltropin was at least as potent as calmodulin, if not better, in reversing the inhibitory effect of caldesmon in the presence of calcium, making it a potential Ca2+ factor in regulating caldesmon in smooth muscle.

2-Naphthylamine↗

PubMed1992

Roles of structural domains in the morphology and surface anchoring of the tetragonal paracrystalline array of Aeromonas hydrophila. Biochemical characterization of the major structural domain.

The tetragonally arranged S-layer of Aeromonas hydrophila contains two morphological domains. The mature S-layer protein of A. hydrophila has a subunit molecular weight of 52,000, and has been reported to contain two structural domains. Here a mutant has been isolated which produces an S-layer of subunit molecular weight 38,650 as determined by sedimentation analysis. This truncated S-protein was exported via the periplasm to the cell surface, but could not self-assemble into a tetragonal array or be anchored to the cell surface. Instead the truncated protein formed cup-like structures which were purified and characterized biochemically. Automated Edman degradation showed that the truncated protein comprised the amino-terminal structural domain of the S-protein. This domain had an increased hydrophobic amino acid content relative to the wild-type protein, and contained approximately 42% beta-sheet, 10% alpha-helix, and 19% beta-turn. Differences in alpha-helix and beta-turn contents between the wild-type and truncated proteins were observed when the effects of pH and SDS were examined, indicating that the carboxy terminus influences the effects of environmental change on the conformation of the S-protein. This lesser carboxy-terminal array also appears to be required for both correct array morphology, and array anchoring, while the greater amino-terminal domain appears to comprise the major morphological core of the surface array.

Aeromonas hydrophila↗

PubMed1992

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.

Animals↗

PubMed1992

Spectroscopic study of the activation and oligomerization of the channel-forming toxin aerolysin: identification of the site of proteolytic activation.

The channel-forming protein aerolysin is secreted as a protoxin which can be activated by proteolytic removal of a C-terminal peptide. The activation and subsequent oligomerization of aerolysin were studied using a variety of spectroscopic techniques. Mass spectrometric determination of the molecular weights of proaerolysin and aerolysin permitted identification of the sites at which the protoxin is processed by trypsin and chymotrypsin. The results of far- and near-UV circular dichroism measurements indicated that processing with trypsin does not lead to major changes in secondary or tertiary structure of the protein. An increase in tryptophan fluorescence intensity and a small red shift in the maximum emission wavelength of tryptophans could be observed, suggesting that there is a change in the environment of some of the tryptophans. There was also a dramatic increase in the binding of the hydrophobic fluorescent probe 1-anilino-8-naphthalenesulfonate during activation, leading us to conclude that a hydrophobic region in the protein is exposed by trypsin treatment. Using measurements of light scattering, various parameters influencing oligomerisation of trypsin-activated aerolysin were determined. Oligomerization rates were found to increase with the concentration of aerolysin, whereas they decreased with increasing ionic strength.

Aeromonas hydrophila↗

PubMed1992

Purification and characterization of a novel 12,000-Da calcium binding protein from smooth muscle.

A new low molecular weight calcium binding protein, designated 12-kDa CaBP, 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 and has a molecular weight of 12,000 based on sodium dodecyl sulfate-gel electrophoresis. The amino acid composition of this protein is similar to but distinct from other known low molecular weight Ca2+ binding proteins. Ca2+ binding assays using Arsenazo III (Sigma) indicated that the protein binds 1 mol of Ca2+/mol of protein. The 12-kDa CaBP underwent a conformational change upon binding Ca2+, as revealed by uv difference spectroscopy and circular dichroism studies in the aromatic and far-ultraviolet range. Addition of Ca2+ to the 12-kDa CaBP labeled with 2-p-toluidinylnaphthalene-6-sulfonate (TNS) resulted in a sevenfold increase in fluorescence intensity, accompanied by a blue shift of the emission maximum from 463 to 445 nm. Hence, the probe in the presence of Ca2+ moves to a more nonpolar microenvironment. Like calmodulin and other related Ca2+ binding proteins, this protein also exposes a hydrophobic site upon binding calcium. Fluorescence titration with Ca2+ using TNS-labeled protein revealed the presence of a single high affinity calcium binding site (kd approximately 1 x 10(-6) M).

Amino Acids↗

PubMed1992

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)

Animals↗

PubMed1992

Synthetic model proteins: the relative contribution of leucine residues at the nonequivalent positions of the 3-4 hydrophobic repeat to the stability of the two-stranded alpha-helical coiled-coil.

Our de novo designed coiled-coil model protein consists of two identical 35-residue polypeptide chains arranged in a parallel and in-register alignment via interchain hydrophobic interactions and a disulfide bridge at the position 2 between two helices. To quantitate the relative contribution of leucine residues at the nonequivalent position of the 3-4 hydrophobic repeat to the stability of the two-stranded alpha-helical coiled-coil, a single alanine was systematically substituted for a leucine in each chain at position "a" (9, 16, 23, or 30) or "d" (5, 12, 19, 26, or 33). The formation and stability of the coiled-coils were determined by circular dichroism studies in the absence and presence of guanidine hydrochloride. All the proteins with an alanine substituted at position a have a similar stability ([Gdn.HCl]1/2 ranges from 2.6 to 2.9 M), while all the proteins with an alanine substituted at position d have similar stability ([Gdn.HCl]1/2 ranges from 3.6 to 4.2 M), except for the proteins with an alanine substituted in the C-terminal heptad. The greater decrease in stability observed for a Leu----Ala mutation at position a (the average delta delta Gu value is 3.3 kcal/mol) compared to those where the substitution was effected at position d (the average delta delta Gu value is 2.0 kcal/mol) indicates that an Ala mutation at position a has a greater effect on the side-chain packing and hydrophobic interactions in the coiled-coil than an Ala mutation at position d.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗