Página 1 dos resultados de 13 itens digitais encontrados em 0.002 segundos

Design and synthesis of a-helical peptides and mimetics

Garner, James; Harding, Margaret
Fonte: Royal Society of Chemistry Publicador: Royal Society of Chemistry
Tipo: Artigo de Revista Científica
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422.62152%
The α-helix is the most abundant secondary structural element in proteins and is an important structural domain for mediating protein-protein and protein-nucleic acid interactions. Strategies for the rational design and synthesis of α-helix mimetics hav

The C-type lectin-like domain superfamily

Zelensky, Alex; Gready, Jill
Fonte: Blackwell Publishing Ltd Publicador: Blackwell Publishing Ltd
Tipo: Artigo de Revista Científica
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294.60988%
The superfamily of proteins containing C-type lectin-like domains (CTLDs) is a large group of extracellular Metazoan proteins with diverse functions. The CTLD structure has a characteristic double-loop ('loop-in-a-loop') stabilized by two highly conserved disulfide bridges located at the bases of the loops, as well as a set of conserved hydrophobic and polar interactions. The second loop, called the long loop region, is structurally and evolutionarily flexible, and is involved in Ca2+-dependent carbohydrate binding and interaction with other ligands. This loop is completely absent in a subset of CTLDs, which we refer to as compact CTLDs; these include the Link/PTR domain and bacterial CTLDs. CTLD-containing proteins (CTLDcps) were originally classified into seven groups based on their overall domain structure. Analyses of the superfamily representation in several completely sequenced genomes have added 10 new groups to the classification, and shown that it is applicable only to vertebrate CTLDcps; despite the abundance of CTLDcps in the invertebrate genomes studied, the domain architectures of these proteins do not match those of the vertebrate groups. Ca2+-dependent carbohydrate binding is the most common CTLD function in vertebrates...

Valine substituted winter flounder 'antifreeze': preservation of ice growth hysteresis

Haymet, A D J; Ward, Leanne G; Harding, Margaret; Knight, Charles A
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
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513.35934%
Three mutant polypeptides of the type 1 37-residue winter flounder 'antifreeze' protein have been synthesized. All four threonine residues in the native peptide were been mutated to serine, valine and glycine respectively and two additional salt bridges were incorporated into the sequences in order to improve aqueous solubility. The peptides were analyzed by nanoliter osmometry, the 'ice hemisphere' test, the 'crystal habit' test, measurement of ice growth hysteresis and CD spectroscopy. While the valine and serine mutants retain the α-helical structure, only the valine mutant retains 'antifreeze' activity similar to that of the native protein. These data show that the threonine hydroxyl groups do not play a crucial role in the accumulation of the native 'antifreeze' protein at the ice/water interface and the inhibition of ice growth below the equilibrium melting temperature.

Type I "antifreeze" proteins: structure-activity studies and mechanisms of ice growth inhibition

Harding, Margaret; Ward, Leanne G; Haymet, A D J
Fonte: Blackwell Publishing Ltd Publicador: Blackwell Publishing Ltd
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
419.97914%
The type I 'antifreeze' proteins, found in the body fluids of fish inhabiting polar oceans, are alanine-rich α-helical proteins that are able to inhibit the growth of ice. Within this class there are two distinct subclasses of proteins: those related to

Winter Flounder "Antifreeze" Proteins: Synthesis and Ice Growth Inhibition of Analogs that Probe the Relative Importance of Hydrophobic and Hydrogen-Bonding Interactions

Haymet, A D J; Ward, Leanne G; Harding, Margaret
Fonte: American Chemical Society Publicador: American Chemical Society
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
629.67477%
Two series of mutant polypeptides of the type I, 37-residue winter flounder 'antifreeze' protein have been synthesized and analyzed by nanoliter osmometry, the 'ice hemisphere' test, measurement of ice growth hysteresis and circular dichroism (CD) spectroscopy. In series 1 peptides the central two threonines and all four threonines of the native protein were mutated to serine. In series 2 peptides two additional salt bridges (K7, E11 and K29, E33) were incorporated, and all four threonine residues in this sequence were mutated simultaneously to each of serine, valine, alanine, and glycine, respectively. The CD studies showed that all mutants are 100% helical in structure at low temperature, except for the glycine derivative which was estimated to be 70% α-helical. Dilute solutions of serine-substituted series 1 peptides showed no detectable, nonbasal faceting, or hysteresis behavior, indicating either no or extremely weak interaction with ice. The analogous serine-substituted mutant in series 2, as well as the glycine derivative, displayed unfaceted growth and showed no hysteresis. Hysteresis values, ice growth patterns, and the helicity measurements showed that the additional salt bridges present in series 2 peptides do not alter significantly the properties of the protein. The valine-substituted mutant gave a distinct etching pattern in which polypeptide accumulates on the {2 0 2 1 } plane of ice 1h...

Type I shorthorn sculpin antifreeze protein. Recombinant synthesis, solution conformation, and ice growth inhibition studies

Fairley, Kayesh; Westman, Belinda J; Pham, Linda H; Haymet, A D J; Harding, Margaret; Mackay, Joel Peter
Fonte: American Society for Biochemistry and Molecular Biology Inc Publicador: American Society for Biochemistry and Molecular Biology Inc
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
627.2542%
A number of structurally diverse classes of "antifreeze" proteins that allow fish to survive in sub-zero ice-laden waters have been isolated from the blood plasma of cold water teleosts. However, despite receiving a great deal of attention, the one or more mechanisms through which these proteins act are not fully understood. In this report we have synthesized a type I antifreeze polypeptide (AFP) from the shorthorn sculpin Myoxocephalus scorpius using recombinant methods. Construction of a synthetic gene with optimized codon usage and expression as a glutathione S-transferase fusion protein followed by purification yielded milligram amounts of polypeptide with two extra residues appended to the N terminus. Circular dichroism and NMR experiments, including residual dipolar coupling measurements on a 15N-labeled recombinant polypeptide, show that the polypeptides are α-helical with the first four residues being more flexible than the remainder of the sequence. Both the recombinant and synthetic polypeptides modify ice growth, forming facetted crystals just below the freezing point, but display negligible thermal hysteresis. Acetylation of Lys-10, Lys-20, and Lys-21 as well as the N terminus of the recombinant polypeptide gave a derivative that displays both thermal hysteresis (0.4°C at 15 mg/ml) and ice crystal faceting. These results confirm that the N terminus of wild-type polypeptide is functionally important and support our previously proposed mechanism for all type I proteins...

Solution structure of a hydrophobic analogue of the winter flounder antifreeze protein

Liepinsh, Edvards; Otting, Gottfried; Harding, Margaret; Ward, Leanne G; Mackay, Joel Peter; Haymet, A D J
Fonte: Blackwell Publishing Ltd Publicador: Blackwell Publishing Ltd
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
832.9532%
The solution structure of a synthetic mutant type I antifreeze protein (AFP I) was determined in aqueous solution at pH 7.0 using nuclear magnetic resonance (NMR) spectroscopy. The mutations comprised the replacement of the four Thr residues by Val and the introduction of two additional Lys-Glu salt bridges. The antifreeze activity of this mutant peptide, VVVV2KE, has been previously shown to be similar to that of the wild type protein, HPLC6 (defined here as TTTT). The solution structure reveals an ahelix bent in the same direction as the more bent conformer of the published crystalstructure of TTTT, while the side chain χ1 rotamers of VVVV2KE are similar to those of the straighter conformer in the crystal of TTTT. The Val side chains of VVVV2KE assume the same orientations as the Thr side chains of TTTT, confirming the conservative nature of this mutation. The combined data suggest that AFP I undergoes an equilibrium between straight and bent helices in solution, combined with independent equilibria between different side chain rotamers for some of the amino acid residues. The present study presents the first complete sequence-specific resonance assignments and the first complete solution structure determination by NMR of any AFP I protein.

"Antifreeze" glycoproteins from polar fish

Harding, Margaret; Anderberg, Pia I; Haymet, A D J
Fonte: Blackwell Publishing Ltd Publicador: Blackwell Publishing Ltd
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
529.7689%
Antifreeze glycoproteins (AFGPs) constitute the major fraction of protein in the blood serum of Antarctic notothenioids and Arctic cod. Each AFGP consists of a varying number of repeating units of (Ala-Ala-Thr)n, with minor sequence variations, and the disaccharide β-D-galactosyl-(1→3)-α-N-acetyl-D-galactosamine joined as a glycoside to the hydroxyl oxygen of the Thr residues. These compounds allow the fish to survive in subzero ice-laden polar oceans by kinetically depressing the temperature at which ice grows in a noncolligative manner. In contrast to the more widely studied antifreeze proteins, little is known about the mechanism of ice growth inhibition by AFGPs, and there is no definitive model that explains their properties. This review summarizes the structural and physical properties of AFGPs and advances in the last decade that now provide opportunities for further research in this field. High field NMR spectroscopy and molecular dynamics studies have shown that AFGPs are largely unstructured in aqueous solution. While standard carbohydrate degradation studies confirm the requirement of some of the sugar hydroxyls for antifreeze activity, the importance of following structural elements has not been established: (a) the number of hydroxyls required...

The effect of hydrophobic analogues of the type I winter flounder antifreeze protein on lipid bilayers

Tomczak, Melanie M; Hincha, Dirk K; Crowe, John H; Harding, Margaret; Haymet, A D J
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
833.99195%
The effect of four synthetic analogues of the 37-residue winter flounder type I antifreeze protein (AFP), which contain four Val, Ala or Ile residues in place of Thr residues at positions 2, 13, 24 and 37 and two additional salt bridges, on the binary lipid system prepared from a 1:1 mixture of the highly unsaturated DGDG and saturated DMPC has been determined using FTIR spectroscopy. In contrast to the natural protein, which increases the thermotropic phase transition, the Thr, Val and Ala analogues decreased the thermotropic phase transitions of the liposomes by 2.2°C, 3.4°C and 2.4°C, while the Ile analogue had no effect on the transition. Experiments performed using perdeuterated DMPC showed that the Ala and Thr peptides interacted preferentially with the DGDG in the lipid mixture, while the Val peptide showed no preference for either lipid. The results are consistent with interactions involving the hydrophobic face of type I AFPs and model bilayers, i.e. the same face of the protein that is responsible for antifreeze properties. The different effects correlate with the helicity of the peptides and suggest that the solution conformation of the peptides has a significant role in determining the effects of the peptides on thermotropic membrane phase transitions.

Solution Structure of a Recombinant Type I Sculpin Antifreeze Protein

Kwan, Ann H-Y; Fairley, Kayesh; Anderberg, Pia I; Liew, Chu W; Harding, Margaret; Mackay, Joel Peter
Fonte: American Chemical Society Publicador: American Chemical Society
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
737.8502%
We have determined the solution structure of rSS3, a recombinant form of the type I shorthorn sculpin antifreeze protein (AFP), at 278 and 268 K. This AFP contains an unusual sequence of N-terminal residues, together with two of the 11-residue repeats that are characteristic of the type I winter flounder AFP. The solution conformation of the N-terminal region of the sculpin AFP has been assumed to be the critical factor that results in recognition of different ice planes by the sculpin and flounder AFPs. At 278 K, the two repeats units (residues 11-20 and 21-32) in rSS3 form a continuous α-helix, with the residues 30-33 in the second repeat somewhat less well defined. Within the N-terminal region, residues 2-6 are well defined and helical and linked to the main helix by a more flexible region comprising residues A7-T11. At 268 K the AFP is overall more helical but retains the apparent hinge region. The helical conformation of the two repeats units is almost identical to the corresponding repeats in the type I winter flounder AFP. We also show that while tetracetylated rSS3 has antifreeze activity comparable to the natural AFP, its overall structure is the same as that of the unacetylated peptide. These data provide some insight into the structural determinants of antifreeze activity and should assist in the development of models that explain the recognition of different ice interfaces by the sculpin and flounder type I AFPs.

Diffusion NMR studies on fish antifreeze proteins and synthetic analogues

Inglis, Steven R; McGann, Matthew J; Price, William Sydney; Harding, Margaret
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
625.67895%
Pulsed field gradient spin echo NMR spectroscopy was used to measure diffusion coefficients of the α-helical type I antifreeze protein from the winter flounder, two synthetic derivatives in which the four Thr residues were replaced with Val and Ala, resp

Applications of type I antifreeze proteins: Studies with model membranes & cryoprotectant properties

Inglis, Steven R; Turner, Jennifer J; Harding, Margaret
Fonte: Bentham Science Publishers Ltd Publicador: Bentham Science Publishers Ltd
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
631.87734%
Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs), found in the body fluids of many species of polar fish allow them to survive in waters colder than the equilibrium freezing point of their blood and other internal fluids. Despite their structural diversity, all AF(G)Ps kinetically depress the temperature at which ice grows in a non-colligative manner and hence exhibit thermal hysteresis. AF(G)Ps also share the ability to interact with and protect mammalian cells and tissues from hypothermic damage (e.g., improved storage of human blood platelets at low temperatures), and are able to stabilize or disrupt membrane composition during low temperature and freezing stress (e.g., cryoprotectant properties in stabilization of sperm and oocytes). This review will summarize studies of AFPs with phospholipids and plant lipids, proposed mechanisms for inhibition of leakage from membranes, and cryoprotectant studies with biological samples. The major focus will be on the α-helical type I antifreeze proteins, and synthetic mutants, that have been most widely studied. For completeness, data on glycoproteins will also be presented. While a number of models to explain stabilization and destabilization of different lipid systems have been proposed...

A solid-state NMR study of the interaction of fish antifreeze proteins with phospholipid membranes

Garner, James; Inglis, Steven R; Hook, J; Separovic, Frances; Harding, Margaret
Fonte: Springer Publicador: Springer
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
525.67895%
Fish antifreeze proteins and glycoproteins (AF(G)Ps) prevent ice crystal growth and are able to protect mammalian cells and tissues from hypothermic damage in the sub-zero Polar oceans. This protective mechanism is not fully understood, and further data is required to explain how AF(G)Ps are able to stabilize lipid membranes as they pass through their phase transition temperatures. Solid-state NMR spectroscopy was used as a direct method to study the interaction of the 37-residue α-helical type I AFP, TTTT, and the low molecular weight fraction glycoprotein, AFGP8, with dimyristoylphosphatidylcholine membranes above and below the gel-fluid phase transition temperature. In contrast to previous studies in fluid phase bilayers these experiments have provided direct information regarding both the mobility of the phosphate headgroups and perturbation of the acyl chains at a range of temperatures under identical conditions on the same sample. At 5°C changes in the 2H and 31P spectra and a dramatic increase in the 31P T1 relaxation times were consistent with a significant disruption of the membrane by TTTT. Heating to 30°C appeared to expel the peptide from the lipid and re-cooling showed that the interaction of TTTT was not reversible. By contrast...