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T C Terwilliger

Publications and source records attributed to T C Terwilliger.

66 records · Page 4Linked to original sources
PubMed1986

Surface structure recognized for covalent modification of the aspartate receptor in chemotaxis.

The aspartate receptor involved in chemotaxis is modified by methyl esterification at four distinct glutamate residues during the adaptive response of this receptor. To explain the high degree of specificity of this modification, it has been proposed that the methyltransferase recognizes the sequence Glu-Glu-Xaa-Xaa-Ala-Ser/Thr in an alpha-helical conformation and methylates the second glutamate in this sequence. This hypothesis is strengthened here by localized mutagenesis studies. By reversing the alanine-threonine sequence to threonine-alanine at the principal site of methylation, Glu-309, a factor of 4 decrease in reactivity was achieved. Thus, the rate of methylation of this site is sensitive to the reversal of two residues of similar structure. These residues are somewhat distant in sequence from the glutamate that is modified but are adjacent in space if an alpha-helical structure is present. The other sites of modification, Glu-295, Glu-302, and Glu-491, are slightly increased in reactivity in the mutant. The 4-fold change in reactivity of the major site of methylation obtained with a relatively subtle change supports the recognition sequence hypothesis, including its structural implications. It is noted, in addition, that chemotaxis of bacteria expressing the mutant receptor does not seem to be greatly altered. This might be explained by the observation that the overall methylation levels of the mutant and wild-type receptors are similar.

Amino Acid Sequence↗
PubMed1986

S-methyl glutathione synthesis is catalyzed by the cheR methyltransferase in Escherichia coli.

The cheR methyltransferase, known to be necessary for the methyl esterification of receptors involved in chemotaxis, is shown to be essential to the synthesis of S-methyl glutathione from glutathione and S-adenosylmethionine in intact Escherichia coli. S-Methyl glutathione is not, however, found to be essential for chemotaxis. It is suggested that the synthesis of S-methyl glutathione may be due to a "parasitic" reaction of glutathione with S-adenosylmethionine bound to the methyltransferase.

Chemotaxis↗
PubMed1984

Sites of methyl esterification and deamination on the aspartate receptor involved in chemotaxis.

The receptors involved in bacterial chemotaxis are post-translationally modified by specific enzymes which catalyze the deamination of glutaminyl residues and the methyl esterification and demethylation of glutamyl residues. In this work we identify the sites of these covalent modifications on the aspartate receptor from Salmonella typhimurium. These were identified using the properties of the Staphylococcus aureus V8 protease which cleaves peptide bonds following glutamyl but not glutaminyl residues. We show here that bonds following methyl-esterified glutamyl residues are also resistant to the protease. A comparison of the fragments obtained after V8 protease cleavage of methyl-esterified (or deaminated) peptides with the fragments from the corresponding unmodified peptides immediately yields the sites of modification. Three of the four methyl-esterified glutamyl residues are located near the middle of the receptor amino acid sequence; one of these is synthesized as a glutaminyl residue and is deaminated by the esterase to form a glutamyl residue. The fourth site of methyl esterification is located near the carboxyl terminus. All four sites occupy analogous positions in a well-conserved arrangement of residues which may form a binding site for the esterase and the methyltransferase.

Amino Acid Sequence↗
PubMed1984

The hydrophobic moment detects periodicity in protein hydrophobicity.

Periodicities in the polar/apolar character of the amino acid sequence of a protein can be examined by assigning to each residue a numerical hydrophobicity and searching for periodicity in the resulting one-dimensional function. The strength of each periodic component is the quantity that has been termed the hydrophobic moment. When proteins of known three-dimensional structure are examined, it is found that sequences that form alpha helices tend to have, on average, a strong periodicity in the hydrophobicity of 3.6 residues, the period of the alpha helix. Similarly, many sequences that form strands of beta sheets tend to have a periodicity in their hydrophobicity of about 2.3 residues, the period typical of beta structure. Also, the few sequences known to form 3(10) helices display a periodicity of about 2.5 residues, not far from the period of 3 for an ideal 3(10) helix. This means that many protein sequences tend to form the periodic structure that maximizes their amphiphilicity. This observation suggests that the periodicity of the hydrophobicity of the protein primary structure is a factor in the formation of secondary structures. Moreover, the observation that many protein sequences tend to form segments of maximum amphiphilicity suggests that segments of secondary structure fold at a hydrophobic surface, probably formed from other parts of the folding protein.

Amino Acid Sequence↗
PubMed1983

Sites of methyl esterification on the aspartate receptor involved in bacterial chemotaxis.

The methyl esterification of the aspartate receptor involved in chemotaxis has been studied in order to clarify the role of receptor modification. Receptors were methyl esterified in an in vitro system using S-adenosyl-L-[methyl-3H]methionine as a methyl donor. Methyl esterified receptors were digested with trypsin and radioactive tryptic peptides were purified using high performance liquid chromatography. Comparing the amino acid composition of the modified peptides with the DNA sequence of the receptor gene, two regions of the polypeptide chain which contain methyl esterified residues were identified. The regions are homologous and contain a strongly conserved 13 amino acid sequence. One region, containing up to three modified residues, is near the middle of the protein; the other, containing one modified residue, is near the carboxyl terminus.

Chemotaxis↗
PubMed1982

The helical hydrophobic moment: a measure of the amphiphilicity of a helix.

The spatial distribution of the hydrophobic side chains in globular proteins is of considerable interest. It was recognized previously that most of the alpha-helices of myoglobin and haemoglobin are amphiphilic; that is, one surface of each helix projects mainly hydrophilic side chains, while the opposite surface projects mainly hydrophobic side chains. To quantify the amphiphilicity of a helix, here we define the mean helical hydrophobic moment, (mu H) = [sigma Ni = 1Hi]/N, to be the mean vector sum of the hydrophobicities Hi of the side chains of a helix of N residues. The length of a vector Hi is the signed numerical hydrophobicity associated with the type of side chain, and its direction is determined by the orientation of the side chain about the helix axis. A large value of (mu H) means that the helix is amphiphilic perpendicular to its axis. We have classified alpha-helices by plotting their mean helical moment versus the mean hydrophobicity of their residues, and report that transmembrane helices, helices from globular proteins and helices which are believed to seek surfaces between aqueous and nonpolar phases, cluster in different regions of such a plot. We suggest that this classification may be useful in identifying helical regions of proteins which bind to the surface of biological membranes. The concept of the hydrophobic moment can be generalized also to non-helical protein structures.

Membrane Proteins↗
PubMed1982

The structure of melittin. I. Structure determination and partial refinement.

Melittin is the principal protein component of bee venom and is thought to function as a lytic agent. Despite its predominantly hydrophobic character, melittin is soluble as a tetramer in aqueous salt solutions. We report here on the determination of the crystal structure of tetrameric melittin at 2.8-A resolution by the method of multiple isomorphous replacement, followed by partial atomic refinement at 2.0-A resolution. The melittin tetramer contains a noncrystallographic 2-fold axis of symmetry in addition to a crystallographic 2-fold axis, so that the four polypeptide chains have nearly identical structures. The noncrystallographic 2-fold axis was utilized twice during the determination of the structure. The multiple isomorphous replacement electron density map was averaged over this 2-fold axis before model building and strict noncrystallographic symmetry was assumed during the initial stages of atomic refinement. The 2.8-A resolution electron density map suggests that the melittin monomer contains two alpha-helical regions separated by a non-alpha-helical segment at residues 11 and 12. Difference maps at 2.0-A resolution tend to confirm this structure and reveal that at least six solvent molecules are bound to the melittin tetramer in the crystal. The relatively high occupancies of four of these suggest that they are ions of crystallization rather than water molecules.

Bee Venoms↗
PubMed1982

The structure of melittin in the form I crystals and its implication for melittin's lytic and surface activities.

Melittin from bee venom is water-soluble, yet integrates into membranes and lyses cells. Each melittin chain consists of 26 amino acid residues and in aqueous salt solutions it exists as a tetramer. We have determined the molecular structure of the tetramer in two crystal forms grown from concentrated salt solutions. In both crystal forms the melittin polypeptide is a bent alpha-helical rod, with the "inner" surface largely consisting of hydrophobic sidechains and the "outer" surface consisting of hydrophilic side chains. Thus, the helix is strongly amphiphilic. In the tetramer, four such helices contribute their hydrophobic side chains to the center of the molecule. The packing of melittin tetramers is also very similar in the two crystal forms: they are packed in planar layers with the outsides forming hydrophilic surfaces and the insides (the centers of melittin tetramers) forming a hydrophobic surface. We suggest that the surface activity of melittin can be rationalized in terms of these surfaces. The lytic activity of melittin can also be interpreted in terms of the molecular structure observed in the crystals: the hydrophobic inner surface of a melittin helix may integrate into the apolar region of a bilayer with the helix axis approximately parallel to the plane of the bilayer, and with the hydrophilic surface exposed to the aqueous phase. This integration would be expected to disrupt the bilayer because of melittin helix would penetrate only a short distance into it. Additionally, the integration of melittin from one side of a bilayer would produce a surface area difference across the bilayer, perhaps leading to lysis. In this view, melittin is distinct from membrane proteins that penetrate evenly into both leaflets of a bilayer or exactly halfway through a bilayer, and hence we refer to melittin as a surface-active protein.

Animals↗
PubMed1981

Methylation of membrane proteins in human erythrocytes. Identification and characterization of polypeptides methylated in lysed cells.

An in vitro system was developed for studying protein methylation reactions in human red blood cells. Packed erythrocytes were lysed by freeze-thawing in the presence of S-adenosyl[methyl-3H]methionine. Specific incorporation of base-labile methyl groups into the band 3 anion transport protein and the major sialoglycoprotein (glycophorin, periodic acid-Schiff reagent-1) was demonstrated by dodecyl sulfate gel electrophoresis at pH 2.4, selective extractions with Triton X-100 and lithium diiodosalicylate, and protease sensitivity. Two other unidentified intrinsic membrane proteins with Mr = 96,000 and 23,500 were also methylated. Little radioactivity was incorporated into membrane proteins when membranes were incubated with S-adenosyl-L-[methyl-3H]methionine in the absence of cytosol. No evidence was obtained for incorporation of methyl label into extrinsic proteins including bands 1, 2, 2.1, 4, 5, 6, or in zone 4.5. Proteolytic digestion of intact cells and isolated membranes revealed that one site of methylation on the band 3 polypeptide may be at the inner surface of the membrane near the junction of the cytoplasmic domain and the membrane domain. The rates of hydrolysis of the incorporated methyl groups were characterized over a range of pH values. These rates were compared to those of methyl esterified amino acids and peptides, including aspartic acid beta-methyl ester which has been isolated from proteolytic digests of methylated erythrocyte membranes (Janson, C. A., and Clarke, S. (1980) J. Biol. Chem. 225, 11640-11643). We find that the rates of base-catalyzed hydrolysis of beta-methyl esters of aspartic acid and gamma-methyl esters of glutamic acid are highly sensitive to the presence of substituents on the alpha-carboxyl and alpha-amino groups. The rate of hydrolysis of the membrane-incorporated methyl groups are consistent with those of internal aspartic acid and glutamic acid methyl ester residues.

Chymotrypsin↗
PubMed1981

Osmotic water permeability of human red cells.

The osmotic water permeability of human red cells has been reexamined with a stopped-flow device and a new perturbation technique. Small osmotic gradients are used to minimize the systematic error caused by nonlinearities in the relationship between cell volume and light scattering. Corrections are then made for residual systematic error. Our results show that the hydraulic conductivity, Lp, is essentially independent of the direction of water flow and of osmolality in the range 184-365 mosM. the mean value of Lp obtained obtained was 1.8 +/- 0.1 (SEM) X 10-11 cm3 dyne -1 s-1.

Cell Membrane Permeability↗
PubMed1980

Melittin forms crystals which are suitable for high resolution X-ray structural analysis and which reveal a molecular 2-fold axis of symmetry.

Melittin is the principal protein component of bee venom and is believed to function as a lytic agent. In aqueous salt solution, it is a tetramer of identical peptides, each with 26 amino acid residues. Although its amino acid composition is unusually nonpolar, and although it is believed to integrate into membranes while lysing cells, melittin is water-soluble at neutral pH. Two crystal forms have been grown from solutions containing ammonium sulfate and sodium formate, and their x-ray diffraction patterns indicate that the melittin tetramer contains at least one 2-fold axis of rotation. Both crystal forms are suitable for high resolution x-ray structural studies. Moreover, both crystals bind several heavy atoms as judged by changes in buoyancy, so that phase determination by the method of isomorphous replacement is possible. Crystallized melittin retains its lytic activity even under the conditions of crystallization (about 70% saturated ammonium sulfate).

Animals↗