ATP-dependent Ca2+ transport and Ca2+-ATPase activities in an enriched plasma membrane fraction from gypsy moth larval midgut tissue

A subcellular fraction enriched in plasma membranes was obtained from gypsy moth (Lymantria dispar) larval midgut tissue. Using [⁴⁵Ca]²⁺ as a tracer, Ca²⁺ transport activity by membrane vesicles in the enriched fraction was measured and shown to be ATP-dependent, with a very high affinity for Ca²⁺ (apparent K_m for [Ca²⁺ _free]

1 Abbreviations used: [Ca²⁺free] = concentration of free (unbound) calcium ion;CaM = calmodulin; F = fraction; IOV = inside-out membrane vesicles; W-5 = N-(6-aminohexyl)-1-naphthalenesulfonamide; W-7 = N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide.

= 22 nM). Ca²⁺ transport was abolished upon addition of the calcium ionophore, A23187. Ca²⁺-stimulated, Mg²⁺-dependent ATPase activity peaked between 100 and 200 nM Ca²⁺_free. Ca²⁺-Mg²⁺-ATPase activity was inhibited by vanadate, 2 phenothiazine drugs (trifluoperazine and chlorpromazine), and the naphthalene sulfonamide, W-7; the related compound, W-5, and ouabain had a negligible effect. These results suggest the presence of a high affinity plasma membrane Ca²⁺ pump in gypsy moth larval midgut cells and are discussed in light of earlier work involving calcium transport in isolated midguts of larval Hyalophora cecropia. Ionic and other conditions that characterize the midgut physiology of larval Lepidoptera (e.g., luminal pH; electrochemical gradient for Ca²⁺; effect of certain ions and inhibitors on Ca²⁺ transport) contrast significantly with those found in adult Diptera. The implications that these differences may have for calcium regulation are discussed. © 1992 Wiley-Liss, Inc.     

Comparative studies on the mode of action of proctolin and phorbol-12,13-dibutyrate in their ability to contract the locust mandibular closer muscle.

The role of proctolin has been further investigated in the locust (Locusta migratoria) mandibular closer muscles. Radioactive calcium uptake measurements were made using protease-dissociated muscle cells. Both the phorbol ester, phorbol-12,13-dibutyrate, and proctolin produce tonic contractions which are associated with the influx of extracellular calcium. The thresholds for proctolin and the phorbol ester to contract the muscle were 1-10 nM and 10-100nM, respectively, while their respective thresholds for evoking measurable calcium influx into the muscle cells were 0.1-1 nM for proctolin, and 0.1-1 pM for phorbol-12,13-dibutyrate. The effect of phorbol-12,13-dibutyrate is blocked by a number of protein kinase inhibitors (at a concentration of 0.1 mM), suggesting that an activation of a protein kinase can lead to calcium influx. These inhibitors, however, do not block the effect of proctolin, indicating that these two compounds work through different pathways, possibly converging on the same final target. In light of this finding, a number of other compounds have been tested to try to ascertain how proctolin mediates an increased calcium influx.     

Molecular modeling of the amphipathic helices of the plasma apolipoproteins.

In this paper we propose a classification of the amphipathic helical repeats occurring in the plasma apolipoprotein sequences. It is based upon the calculation of the molecular hydrophobicity potential around the helical segments. The repeats were identified using a new autocorrelation matrix, based upon similarities of hydrophobic and hydrophilic properties of the amino acid residues within the apolipoprotein sequences. The helices were constructed by molecular modeling, the molecular hydrophobicity potential was calculated, and isopotential contour lines drawn around the helices yielded a three-dimensional visualization of the hydrophobicity potential. Two classes of apolipoproteins could be differentiated by comparing the hydrophobic angles obtained by projection of the isopotential contour lines on a plane perpendicular to the long axis of the helix. The isopotential contour lines around apo AI, AIV, and E are more hydrophilic than hydrophobic, whereas they are of similar intensity for apo AII, CI, and CIII. In both cases discoidal lipid-protein complexes are generated, with the amphipathic helices around the edge of the lipid core. The long axis of the helices is oriented parallel to the phospholipid acyl chains and the hydrophilic side of the helix toward the aqueous phase. As a result of the differences in hydrophobicity potential, the contact between the hydrophobic side of the helices and the phospholipid acyl chains is larger for apo AII, CI, and CIII than for the other apolipoproteins. This might account for the greater stability of the discoidal complexes generated between phospholipids and these apoproteins.     

Multiple protein sequence alignment from tertiary structure comparison: assignment of global and residue confidence levels.

An algorithm is presented for the accurate and rapid generation of multiple protein sequence alignments from tertiary structure comparisons. A preliminary multiple sequence alignment is performed using sequence information, which then determines an initial superposition of the structures. A structure comparison algorithm is applied to all pairs of proteins in the superimposed set and a similarity tree calculated. Multiple sequence alignments are then generated by following the tree from the branches to the root. At each branchpoint of the tree, a structure-based sequence alignment and coordinate transformations are output, with the multiple alignment of all structures output at the root. The algorithm encoded in STAMP (STructural Alignment of Multiple Proteins) is shown to give alignments in good agreement with published structural accounts within the dehydrogenase fold domains, globins, and serine proteinases. In order to reduce the need for visual verification, two similarity indices are introduced to determine the quality of each generated structural alignment. Sc quantifies the global structural similarity between pairs or groups of proteins, whereas Pij' provides a normalized measure of the confidence in the alignment of each residue. STAMP alignments have the quality of each alignment characterized by Sc and Pij' values and thus provide a reproducible resource for studies of residue conservation within structural motifs.     

Crystal structure of human immunoglobulin fragment Fab New refined at 2.0 A resolution.

The three-dimensional structure of the human immunoglobulin fragment Fab New (IgG1, lambda) has been refined to a crystallographic R-factor of 16.9% to 2 A resolution. Rms deviations of the final model from ideal geometry are 0.014 A for bond distances and 3.03 degrees for bond angles. Refinement was based on a new X-ray data set including 28,301 reflections with F > 2.5 sigma(F) from 6.0 to 2.0 A resolution. The starting model for the refinement procedure reported here is from the Brookhaven Protein Data Bank entry 3FAB (rev. 1981). Differences between the initial and final models include modified polypeptide-chain folding in the third complementarity-determining region (CDR3) and the third framework region (FR3) of VH and in some exposed loops of CL and CH1. Amino acid sequence changes were determined at a number of positions by inspection of difference electron density maps. The incorporation of amino acid sequence changes results in an improved VH framework model for the "humanization" of monoclonal antibodies.     

Recurrent alpha beta loop structures in TIM barrel motifs show a distinct pattern of conserved structural features.

A systematic survey of seven parallel alpha/beta barrel protein domains, based on exhaustive structural comparisons, reveals that a sizable proportion of the alpha beta loops in these proteins--20 out of a total of 49--belong to either one of two loop types previously described by Thornton and co-workers. Six loops are of the alpha beta 1 type, with one residue between the alpha-helix and beta-strand, and 13 are of the alpha beta 3 type, with three residues between the helix and the strand. Protein fragments embedding the identified loops, and termed alpha beta connections since they contain parts of the flanking helix and strand, have been analyzed in detail revealing that each type of connection has a distinct set of conserved structural features. The orientation of the beta-strand relative to the helix and loop portions is different owing to a very localized difference in backbone conformation. In alpha beta 1 connections, the chain enters the beta-strand via a residue adopting an extended conformation, while in alpha beta 3 it does so via a residue in a near alpha-helical conformation. Other conserved structural features include distinct patterns of side chain orientation relative to the beta-sheet surface and of main chain H-bonds in the loop and the beta-strand moieties. Significant differences also occur in packing interactions of conserved hydrophobic residues situated in the last turn of the helix. Yet the alpha-helix surface of both types of connections adopts similar orientations relative to the barrel sheet surface. Our results suggest furthermore that conserved hydrophobic residues along the sequence of the connections, may be correlated more with specific patterns of interactions made with neighboring helices and sheet strands than with helix/strand packing within the connection itself. A number of intriguing observations are also made on the distribution of the identified alpha beta 1 and alpha beta 3 loops within the alpha/beta-barrel motifs. They often occur adjacent to each other; alpha beta 3 loops invariably involve even numbered beta-strands, while alpha beta 1 loops involve preferentially odd beta-strands; all the analyzed proteins contain at least one alpha beta 3 loop in the first half of the eightfold alpha/beta barrel. Possible origins of all these observations, and their relevance to the stability and folding of parallel alpha/beta barrel motifs are discussed.     

Functional roles of amino acid residues involved in forming the alpha-helix-turn-alpha-helix operator DNA binding motif of Tet repressor from Tn10.

The Tn10 derived Tet repressor contains an amino acid segment with high homology to the alpha-helix-turn-alpha-helix motif (HTH) of other DNA binding proteins. The five most conserved amino acids in HTH are probably involved in structural formation of the motif. Their functional role was probed by saturation mutagenesis yielding 95 single amino acid replacement mutants of Tet repressor. Their binding efficiencies to tet operator were quantitatively determined in vivo. All functional mutants contain amino acid substitutions consistent with their proposed role in a HTH. In particular, only the two smallest amino acids (serine, glycine) can substitute a conserved alanine in the proposed first alpha-helix without loss of activity. The last position of the first alpha-helix, the second position in the turn, and the fourth position in the second alpha-helix require mostly hydrophobic residues. The proposed C-terminus of the first alpha-helix is supported by a more active asparagine compared to glutamine replacement mutant of the wt leucine residue. The turn is located close to the protein surface as indicated by functional lysine and arginine replacements for valine. A glycine residue at the first position in the turn can be replaced by any amino acid yielding mutants with at least residual tet operator affinity. A structural model of the HTH of Tet repressor is presented.     

Amino acid substitution analysis of E. coli thymidylate synthase: The study of a highly conserved region at the N-terminus

Amino acid substitution analysis within a highly conserved region of Escherichia coli thymidylate synthase (TS), using suppression of amber mutations by tRNA suppressors, has yielded a bank of 124 new mutationally altered TS proteins. These mutant proteins have been used to study the structure-function relationship of the Escherichia coli TS protein at the N-terminus corresponding to residues 20 through 35. This region contains a block of amino acids whose sequence has been well conserved among other known TS proteins from various organisms. Positions 20 through 25 contain a surface loop structure and positions 26 through 35 encompass a β-strand. We find that residues surrounding a β-bulge structure within the β-strand are particularly sensitive to amino acid substitution, suggesting that this structure is maintained by a highly ordered packing arrangement. Three residues in the surface loop that are present at the base of the substrate binding pocket are also sensitive to amino acid substitution. The remainder of the conserved sites, including those at the dimer interface, are tolerant to most, if not all, of the substitutions tested. © 1992 Wiley-Liss, Inc.     

Detection of native-like models for amino acid sequences of unknown three-dimensional structure in a data base of known protein conformations.

We present an approach which can be used to identify native-like folds in a data base of protein conformations in the absence of any sequence homology to proteins in the data base. The method is based on a knowledge-based force field derived from a set of known protein conformations. A given sequence is mounted on all conformations in the data base and the associated energies are calculated. Using several conformations and sequences from the globin family we show that the native conformation is identified correctly. In fact the resolution of the force field is high enough to discriminate between a native fold and several closely related conformations. We then apply the procedure to several globins of known sequence but unknown three dimensional structure. The homology of these sequences to globins of known structures in the data base ranges from 49 to 17%. With one exception we find that for all globin sequences one of the known globin folds is identified as the most favorable conformation. These results are obtained using a force field derived from a data base devoid of globins of known structure. We briefly discuss useful applications in protein structural research and future development of our approach.