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D R McCaslin

Publications and source records attributed to D R McCaslin.

16 recordsLinked to original sources
PubMed2001

Monomeric solution structure of the prototypical 'C' chemokine lymphotactin.

Lymphotactin, the sole identified member of the C class of chemokines, specifically attracts T lymphocytes and natural killer cells. This 93-residue protein lacks 2 of the 4 conserved cysteine residues characteristic of the other 3 classes of chemokines and possesses an extended carboxyl terminus, which is required for chemotactic activity. We have determined the three-dimensional solution structure of recombinant human lymphotactin by NMR spectroscopy. Under the conditions used for the structure determination, lymphotactin was predominantly monomeric; however, pulsed field gradient NMR self-diffusion measurements and analytical ultracentrifugation revealed evidence of dimer formation. Sequence-specific chemical shift assignments were determined through analysis of two- and three-dimensional NMR spectra of (15)N- and (13)C/(15)N-enriched protein samples. Input for the torsion angle dynamics calculations used in determining the structure included 1258 unique NOE-derived distance constraints and 60 dihedral angle constraints obtained from chemical-shift-based searching of a protein conformational database. The ensemble of 20 structures chosen to represent the structure had backbone and heavy atom rms deviations of 0.46 +/- 0.11 and 1.02 +/- 0.14 A, respectively. The results revealed that human lymphotactin adopts the conserved chemokine fold, which is characterized by a three-stranded antiparallel beta-sheet and a C-terminal alpha-helix. Two regions are dynamically disordered as evidenced by (1)H and (13)C chemical shifts and [(15)N]-(1)H NOEs: residues 1-9 of the amino terminus and residues 69-93 of the C-terminal extension. A functional role for the C-terminal extension, which is unique to lymphotactin, remains to be elucidated.

Amino Acid Sequence↗
PubMed1999

Stoichiometry of the large conductance bacterial mechanosensitive channel of E. coli. A biochemical study.

MscL, a 15 kDa transmembrane protein, is the only component involved in the formation of a 3 nS channel in the inner membrane of Escherichia coli that opens in response to mechanical or osmotic stress. While previous data had suggested that the functional MscL complex might be a hexamer, a recent crystallographic study of the MscL homologue from M. tuberculosis reveals a pentameric structure. The present work further examines the stoichiometry of the E. coli MscL using a variety of biochemical approaches. Detergent-purified 6His-MscL in solution and MscL in the membrane could be chemically crosslinked with the products displaying ladderlike patterns on SDS gels. Three crosslinking agents (EDC, DMS, and DMA) used at saturating concentrations invariably generated pentamers as the largest product. DSS produced additional bands corresponding to larger complexes although the pentamer band appeared to be the predominant product at high levels of crosslinker. It is not clear whether these extra bands reflect a difference in the crosslinking chemistry of DSS or whether its spacer arm is the longest of those used, or a combination of both facts. For the detergent-solubilized 6His-MscL both sedimentation equilibrium and gel chromatography showed the presence of multiple species. Thus the longer spacer arm could permit both intra- and intercomplex linkages. Nonetheless, the patterns obtained with all agents are consistent with and strongly suggest a pentameric organization for the MscL channel. Expression of MscL as genetically engineered double or triple subunit tandems yields low numbers of functional channels as compared to expressed monomers. The double-tandem assemblies must have an even number of subunits and crosslinking in the membrane confirmed hexamerization. Gel chromatography clearly demonstrated that the channels formed from the double tandems were larger than those formed from WT MscL, consistent with the native channel being pentameric. The observation that both double and triple tandems form channels of normal conductance implies that the pentameric assembly is to some degree independent of the number of subunit repeats in the polypeptide precursor. The channel is thus a pentameric core with the 'extra' subunits left out of the functional complex. From sedimentation equilibrium and size-exclusion chromatography, we also conclude that MscL complexes are not in a dynamic equilibrium with monomers, but are pre-assembled; and thus, their gating properties must result from changes in the conformation of the entire complex induced by the mechanical stress.

Bacterial Proteins↗
PubMed1999

Bacterial expression and characterization of the ligand-binding domain of the vitamin D receptor.

The ligand-binding domain of the rat vitamin D receptor (amino acids 115-423) was expressed as an amino-terminal His-tagged protein in a bacterial expression system and purified over Ni-nitrilotriacetic acid resin and a Mono S column. The purified protein bound its ligand, 1,25-dihydroxyvitamin D3, with high affinity, similar to that of the full-length protein. Saturation of the protein with ligand quenched 90% of the tryptophan fluorescence, consistent with the purified protein being uniformly able to bind ligand. Addition of ligand produced no change in the tryptophan fluorescence lifetime, suggesting static quenching as the mechanism of fluorescence decrease. The near-UV circular dichroism spectrum showed a large increase in signal following the addition of ligand, consistent with a change in the environment of aromatic amino acid side chains. The far-UV circular dichroism spectrum was consistent with a protein of high alpha-helical content. Sedimentation equilibrium experiments demonstrated that the protein formed higher-order complexes, and the distribution of the protein among these complexes was significantly shifted by addition of ligand.

Animals↗
PubMed1998

Spontaneous inactivation of human tryptase involves conformational changes consistent with conversion of the active site to a zymogen-like structure.

The conformational changes accompanying spontaneous inactivation and dextran sulfate (DS) mediated reactivation of the serine protease human tryptase were investigated by analysis of (i) intrinsic fluorescence, (ii) inhibitor binding, and (iii) catalytic efficiency. Spontaneous inactivation produced a marked decrease in fluorescence emission intensity that was reversed by the addition of DS. Fluorescence decreases at high (4.0 microM) and low (0.1 microM) tryptase concentrations were similar at early times and coincided with loss of enzymatic activity but deviated significantly from activity loss at later times by showing a difference in the extent of change. The fluorescence losses were best described by a two-step kinetic model in which the major decrease correlated to activity loss (t1/2 of 4.3 min in 0.2 M NaCl, pH 6.8, 30 degrees C) and was followed by a further decrease (t1/2 approximately 60 min) whose extent differed with tryptase concentration. The ability to bind the competitive inhibitor p-aminobenzamidine was reversibly lost upon spontaneous inactivation, providing evidence for conformational changes affecting the major substrate binding site (S1-pocket). Estimation of catalytic efficiency using an active site titrant showed that the specific activity of tryptase remained unchanged upon inactivation and reactivation. Return of enzymatic activity, intrinsic fluorescence, and the S1 pocket appeared to occur in the same time frame (t1/2 approximately 3 min). These studies indicate that spontaneous inactivation involves reversible changes which convert the active site to a nonfunctional state. The association of activity loss with an intrinsic fluorescence decrease and loss of the S1-pocket is consistent with the disruption of a critical ionic bond at the active site. Formation of this ionic bond is the basis of zymogen activation for the chymotrypsin family of serine proteases.

Benzamidines↗
PubMed1995

Structural changes associated with the spontaneous inactivation of the serine proteinase human tryptase.

Human skin tryptase, a serine proteinase stored within mast cell secretory granules, rapidly loses enzymatic activity in solutions of physiological salt concentration, pH, and temperature. The inactivation of tryptase can be slowed and even reversed by addition of heparin, a highly sulfated glycosaminoglycan also found in the secretory granules. These properties may be relevant to tryptase regulation after secretion from mast cells. To further characterize the molecular changes underlying the functional instability of tryptase, circular dichroism (CD) and analytical ultracentrifugation were used to investigate structural changes during spontaneous inactivation. The CD spectra of active and spontaneously inactivated tryptase are different, particularly in the region around 230 nm where active tryptase displays a distinct negative peak. This peak is also observed in the CD spectrum of bovine chymotrypsin but not in trypsin, elastase, or chymotrypsinogen. Loss of activity resulting from spontaneous inactivation was accompanied by a diminution of the 230-nm signal. The kinetics for the signal loss appeared to be first-order and closely paralleled the rate of enzymatic activity loss. Dextran sulfate, a highly sulfated polysaccharide, was capable of reactivating tryptase and restoring the CD signal. After 2 h of decay (> 90% loss of activity), addition of dextran sulfate resulted in an almost immediate return of the CD signal to that of active tryptase. The return of the CD signal appeared to be more rapid than the return of enzymatic activity, thereby suggesting the presence of an unidentified step which is rate-limiting for activity return (and loss) and subsequent (prior) to the CD change accompanying activity loss. Ultracentrifugation analysis of tryptase showed a marked change in its association state upon inactivation. Sedimentation equilibrium under stabilizing conditions demonstrated the presence of a single species with the molecular weight of a tetramer. After spontaneous inactivation, a mixture of species was evident, which was characterized as monomers and tetramers in equilibrium. These results demonstrate that spontaneous inactivation of tryptase is associated with reversible conformational changes and that a consequence of inactivation is the formation of a destabilized tetrameric form. Although the molecular mechanism initiating these changes remains unclear, possible insights into the process are discussed on the basis of the similarity between the CD spectra of tryptase and chymotrypsin.

Chymases↗
PubMed1993

Human skin tryptase: kinetic characterization of its spontaneous inactivation.

The spontaneous loss of human tryptase hydrolytic activity was investigated. Time course studies monitoring the loss in catalytic activity were biphasic and correlated with a reduction in the concentration of catalytic sites. There was an initial rapid phase leading to greater than 85% loss in activity. The remaining activity gradually decayed toward completion over a 40-h period. The initial phase could be described as a first-order process with a t1/2 of approximately 6.0 min in 0.2 M NaCl (pH 6.8, 30 degrees C). The rate constant for this phase showed little, if any, sensitivity to changes in enzyme concentration, consistent with a first-order process, and analysis of the reaction as a function of temperature was consistent with a single rate-determining step. The rate of this process, however, showed marked sensitivity to changes in NaCl concentration and pH. Increasing the NaCl concentration as well as decreasing the pH below the pI (pH 6.3) reduced the rate of activity loss, whereas increasing the pH above pH 8.0 markedly increased the rate of activity loss. The effect of NaCl concentration and pH on the rate of activity loss suggests that the rate-limiting step governing the fast phase of the reaction involves electrostatic interactions. The presence of a fast and a slow phase in the decay process may suggest heterogeneity in the sample or the rapid formation of an inactive, but reversible, intermediate. A reversible intermediate was demonstrated when "inactivated tryptase" was incubated in the presence of heparin, and an increase in tryptase catalytic activity was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Substructure of human von Willebrand factor. Proteolysis by V8 and characterization of two functional domains.

The effects of Staphylococcus aureus V8 protease (V8) on the multimeric structure of human von Willebrand factor (vWF) were studied to test and expand our model for the substructure of vWF. Electron microscopy of V8 digests of vWF revealed that the multimers were cleaved where the flexible rod (R) domains join the large elongated globular (G) domains. The resulting two major fragments, which were purified by affinity and hydrophobic interaction chromatography and by glycerol-gradient ultracentrifugation, are disulfide-linked homodimers of these domains (i.e. RR and GG) and are morphologically identical to the alternating RR and GG domains of intact vWF. The glycoprotein fragment GG (6.5 X 35 nm) has mass 343 kDa by sedimentation equilibrium and the amino-terminal sequence of intact plasma vWF. It contains the binding site for heparin within 300 residues of its amino terminus and a separate site for the platelet GPIb receptor responsible for platelet agglutination in the presence of ristocetin. With approximately 18% alpha-helix and approximately 15% beta-pleated sheet, fragment GG accounts for most of the ordered secondary structure present in whole vWF. The two thin flexible rod domains (1.8-2.0 X 30-34 nm) of fragment RR are joined at a small central nodule (approximately 5 nm diameter) and also have a small nodule at each free end. Fragment RR contains an extraordinarily high cystine content, lower than average amounts of other hydrophobic residues, and essentially no alpha-helix, as judged by circular dichroism. The amino-terminal sequence and amino acid composition of fragment RR corresponded to that of the COOH-terminal 685 residues of the intact vWF subunit (Titani, K., Kumar, S., Takio, K., Ericsson, L. H., Wade, R. D., Ashida, K., Walsh, K. A., Chopek, M. W., Sadler, J. E., and Fujikawa, K. (1986) Biochemistry 25, 3171-3184). This sequence analysis gives a mass of 180 kDa for glycosylated fragment RR, somewhat higher than the 130 kDa we obtained by sedimentation equilibrium. Our sequence analysis of a 110-kDa plasmic vWF peptide also permitted identification of a major plasmin cleavage site 705 residues from the COOH terminus and a half-cystine residue (1360) involved in maintaining the multimeric structure of plasmin-degraded vWF.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1985

Molecular structure of the beta-adrenergic receptor.

The beta-adrenergic receptor from several tissues has been purified to homogeneity or photoaffinity radiolabeled and its subunit molecular weight determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. In this study we have examined the oligomeric structure of nondenatured beta 1- and beta 2-adrenergic receptor proteins, as solubilized with the detergent digitonin. Model systems used were frog and turkey red blood cell as well as rat, rabbit, and bovine lung plasma membrane preparations. To correct for the effects of detergent binding, sedimentation equilibrium analysis in various solvents, as adapted for the air-driven ultracentrifuge, was used. With this approach an estimate of 6 g of digitonin/g of protein binding was determined, corresponding to a ratio of 180 mol of digitonin/mol of protein. Protein molecular weights estimated by this method were 43 500 for the turkey red blood cell beta 1 receptor and 54 000 for the frog red blood cell beta 2 receptor. Molecular weights of 60 000-65 000 were estimated for beta 1 and beta 2 receptors present in mammalian lungs. These values agree with estimates of subunit molecular weight obtained by SDS gel electrophoresis of purified or photoradiolabeled preparations and suggest beta-adrenergic receptors to be digitonin solubilized from the membrane as single polypeptide chains.

Amino Acids↗
PubMed1982

Phosphorylation of calcium adenosinetriphosphatase by inorganic phosphate: reversible inhibition at high magnesium ion concentrations.

Magnesium stimulates phosphorylation of the calcium pump protein of the sarcoplasmic reticulum by inorganic phosphate, but the effect is reversed by high [Mg2+]. This reversal is readily explained in terms of the generally accepted existence of two conformational states of the enzyme, E1 and E2. E2 is the form of the enzyme that can be phosphorylated by Pi, and it has one binding site for Mg2+. E1 is the form of the enzyme that has two high-affinity Ca2+ binding sites, and it is phosphorylated by ATP when Ca2+ is bound. Mg2+ can bind weakly to the two Ca2+ sites and to a third site known to be present on E1; this stabilizes E1 at the expense of E2 when [Mg2+] is large. Stabilization of E1 at pH 6.2 and 25 degrees C was found to be a highly cooperative function of [Mg2+] and was not prevented by increasing [Pi]. The latter result requires the existence of a binding site for Pi on E1, with an affinity for Pi comparable to that of E2. Cooperativity with respect to [Mg2+] requires that E2 is the stable state of the enzyme in the absence of ligands, with an equilibrium constant [E2]/[E1] on the order of 10(3) or higher at pH 6.2 and 25 degrees C.

Binding Sites↗
PubMed1982

Two-dimensional rhodopsin crystals from disk membranes of frog retinal rod outer segments.

Two-dimensional crystals of rhodopsin have been prepared from purified frog disk membranes by using the detergent Tween 80. The space group of the orthorhombic crystals is p22121; the unit cell dimensions are 47 X 151 A. Projection maps of negatively stained preparations have been calculated to a resolution of approximately 22 A. The rhodopsin molecules are associated as dimers that appear to be slightly sigmoidal and are 20-25 A in width and 70-80 A in length.

Animals↗
PubMed1981

Effects of detergent micelles on the recombination reaction of opsin and 11-cis-retinal.

When detergent-solubilized proteins interact with hydrophobic or amphiphilic molecules in the presence of detergent micelles, the solubility of the latter species in the micelles must be included in both thermodynamic and kinetic treatments. In this paper, we derive equations which describe the distribution of species present at equilibrium for a system in which a detergent-solubilized protein binds a hydrophobic (or amphiphilic) ligand. We have applied the formalism developed in this paper to the reaction describing the formation of rhodopsin from its apoprotein and 11-cis-retinal. Qualitatively, the results demonstrate that a significant portion of the observed decrease in the extent of recombination for rhodopsin solubilized in either sodium cholate or Tween 80 may be attributed to the partition of retinal into detergent micelles and that a detergent-induced protein denaturation need not be invoked to explain the data. We also discuss results for rhodopsin solubilized in a nonionic detergent (octaethylene glycol n-dodecyl ether) in which the detergent is clearly causing irreversible loss of the capability to recombine with 11-cis-retinal.

Detergents↗
PubMed1981

Different states of aggregation for unbleached and bleached rhodopsin after isolation in two different detergents.

Phospholipid-free rhodopsin has been purified in the detergents sodium cholate and octaethylene glycol n-dodecyl ether (C12E8). In both detergents, the native absorption spectrum of the unbleached protein is maintained; however, upon photolysis, the preparation in C12E8 loses its ability to recombine with 11-cis-retinal, whereas the preparation in cholate does not. The circular dichroic spectra of the protein in the two detergents are nearly identical, indicating that the secondary structure of the protein is the same in the two detergents. The state of association of the protein in the two detergents is different. In sodium cholate, the smallest species present was found to be a trimer of the rhodopsin polypeptide chain, and this association was unaffected by exposure to light. On the other hand, in C12E8, the protein is monomeric and undergoes a nonspecific aggregation process on exposure to light. These results suggest that protein--protein interactions may play an important role in the stabilization of the native structure of rhodopsin.

Animals↗
PubMed1976

Adrenodoxin reductase-adrenodexin complex.

Adrenodoxin reductase and adrenodoxin have been shown (Chu, J.-W., and Kimura, T. (1973) J. Biol. Chem. 248, 5183-5187) to form a low dissociation constant, 1:1 complex when both proteins are in the oxidized form. We have found that when adrenodoxin: adrenodoxin reductase ratios are varied by increasing the adrenodoxin concentration, with adrenodoxin reductase held constant, an increasing rate of cytochrome c reduction, with NADPH as reductant, is seen up to a ratio of 1:1, indicating that cytochrome c reduction occurs via the protein-protein complex. Spectra observed during titration of this protein-protein complex with NADH were resolved into components by the linear programming method, using a computer program written in Fortran IV. Analysis of the data has shown that the flavoprotein is reduced prior to the iron sulfur protein, and that the midpoint oxidation-reduction potentials (pH 7.5) of the two proteins are -295 and -331 mV, respectively, when both are present in the complex. Complex formation does not alter the potential of adrenodoxin reductase, but changes that of adrenodoxin by -40 mV. Equilibrium constants derived from potential measurements show that the strength of the protein-protein interaction in the complex is unaltered by reduction of adrenodoxin reductase, but is decreased by about 1 kcal due to reduction of adrenodoxin. The low dissociation constants for both oxidized reduced forms of the adrenodoxin reductase-adrenodoxin complex indicate that the complex must remain associated throughout its catalytic cycle. Titration of the adrenodoxin reductase-adrenodoxin complex with the physiologic reductant, NADPH, was followed by EPR and visible spectra, and yielded an order of reduction of the components identical with that seen when NADH was used as reductant. Reduction of the protein-protein complex with NADPH yielded a ternary complex between NADP+, flavoprotein, and iron sulfur protein, with the two electrons located in a "charge transfer" complex between flavoprotein and pyridine nucleotide.

Adrenal Cortex↗