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Combinatorial synthesis and directed evolution applied to the production of alpha-helix forming antimicrobial peptides analogues

Castro, Mariana S.; Cilli, Eduardo Maffud; Fontes, Wagner
Fonte: Bentham Science Publ Ltd Publicador: Bentham Science Publ Ltd
Tipo: Revisão Formato: 473-478
ENG
Relevância na Pesquisa
678.06484%
Antimicrobial peptides (AMPs) are effector molecules of innate immune systems found in different groups of organisms, including microorganisms, plants, insects, amphibians and humans. These peptides exhibit several structural motifs but the most abundant AMPs assume an amphipathic alpha-helical structure. The alpha-helix forming antimicrobial peptides are excellent candidates for protein engineering leading to an optimization of their biological activity and target specificity. Nowadays several approaches are available and this review deals with the use of combinatorial synthesis and directed evolution in order to provide a high-throughput source of antimicrobial peptides analogues with enhanced lytic activity and specificity.

Charged histidine affects alpha-helix stability at all positions in the helix by interacting with the backbone charges.

Armstrong, K M; Baldwin, R L
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/12/1993 EN
Relevância na Pesquisa
483.7822%
To determine whether a charged histidine side chain affects alpha-helix stability only when histidine is close to one end of the helix or also when it is in the central region, we substitute a single histidine residue at many positions in two reference peptides and measure helix stability and histidine pKa. The position of a charged histidine residue has a major effect on helix stability in 0.01 M NaCl: the helix content of a 17-residue peptide is 24% when histidine is at position 3 compared to 76% when it is at position 17. This dependence of helix content on histidine position decreases sharply in 1 M NaCl, as expected for counterion screening of the charge-helix dipole interaction. Results at interior positions indicate that the position of a charged histidine residue affects helix stability at these positions. Unexpectedly high values of the helix content are found when either neutral or charged histidine is at one of the last three C-terminal positions, suggesting that either form can stabilize an isolated helix by hydrogen bonding to a main-chain CO group.

Alpha-helix stabilization by natural and unnatural amino acids with alkyl side chains.

Lyu, P C; Sherman, J C; Chen, A; Kallenbach, N R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/06/1991 EN
Relevância na Pesquisa
488.4712%
Knowledge of the role of individual side chains in forming different secondary structures such as the alpha-helix would be useful for prediction of protein structure from sequence or de novo protein design. Experimental and theoretical studies on natural and synthetic peptides and proteins indicate that individual side chains differ in their helix-forming potential. Four aliphatic side chains occur in the standard complement of amino acids: alanine and leucine are helix stabilizing, whereas isoleucine and valine are weakly destabilizing. We have synthesized a series of helical peptides containing unnatural aliphatic side chains having two to four carbons to explore some of the factors involved in alpha-helix stabilization and the basis for selection of the natural set. We find that linear side chains with two, three, or four carbons are as strongly helix stabilizing as the single methyl in alanine and that all linear side chains are stronger helix promoters than leucine. In addition, a t-butyl side chain is significantly more helix destabilizing than the sec-butyl side chain of isoleucine, the isopropyl side chain of valine, or even the unrestricted side chain of glycine. These results provide experimental evidence that restriction in conformational freedom of a side chain imposed by alpha-helix formation is a major component of the role of a side chain in stabilizing helical structure.

Energetics of the structure of the four-alpha-helix bundle in proteins.

Chou, K C; Maggiora, G M; Némethy, G; Scheraga, H A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1988 EN
Relevância na Pesquisa
483.1042%
The main features of the four-alpha-helix bundle, one of the characteristic structural elements of many proteins, can be explained in terms of noncovalent interactions between the constituent helices. Conformational energy computations have been carried out on four types of four-alpha-helix bundles, each consisting of four CH3CO-(L-Ala)10-NHCH3 polypeptide chains, with various combinations of parallel and antiparallel orientations of the helices. In the bundle with the most favorable energy, all pairs of neighboring helices are oriented antiparallel--i.e., in the orientation that is favored by electrostatic interactions between the helices. In this structure, the orientation angle between neighboring helix axes is -168 degrees, within +/- 7 degrees, in close agreement with the orientation angles observed in proteins and with the value that we computed earlier for the most favorable packing of pairs of interacting alpha-helices. This orientation corresponds to a left-handed twisting of the helical bundle. The preferred handedness of this twisting arises as a result of favorable nonbonded interactions between the alpha-helices.

Alpha-helix and mixed 3(10)/alpha-helix in cocrystallized conformers of Boc-Aib-Val-Aib-Aib-Val-Val-Val-Aib-Val-Aib-OMe.

Karle, I L; Flippen-Anderson, J L; Uma, K; Balaram, H; Balaram, P
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1989 EN
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484.2867%
Two molecules of Boc-Aib-Val-Aib-Aib-Val-Val-Val-Aib-Val-Aib-OMe (where Boc is t-butoxycarbonyl and Aib is alpha-aminoisobutyryl) cocrystallize in a triclinic cell with different helical conformations. One molecule is completely alpha-helical with seven 5----1 intramolecular hydrogen bonds. It forms three head-to-tail NH...O = C hydrogen bonds to other molecules of the same conformation. The second molecule has a mixed 3(10)/alpha-helix conformation with three 4----1 hydrogen bonds and four 5----1 hydrogen bonds; furthermore, there is a helix reversal at both termini. The second molecule forms only two head-to-tail hydrogen bonds with molecules of the same type, and the N(3)H group does not participate in any hydrogen bonding. The two different types of helices occur in alternate sheets in the crystal, where each sheet is composed of adjacent rods of helices formed by head-to-tail hydrogen bonding. Within each sheet, containing helices of only one type of conformation, the helices aggregate in a parallel mode. Between the sheets of different helices, the aggregation is antiparallel. The peptide, with formula C51H92N10O13, crystallizes in space group P1 with Z = 2 and cell parameters a = 10.047 +/- 0.002 A, b = 16.684 +/- 0.003 A, c = 19.198 +/- 0.004 A...

Conformation of a 16-residue zervamicin IIA analog peptide containing three different structural features: 3(10)-helix, alpha-helix, and beta-bend ribbon.

Karle, I L; Flippen-Anderson, J; Sukumar, M; Balaram, P
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1987 EN
Relevância na Pesquisa
485.436%
Boc-Trp-Ile-Ala-Aib-Ile-Val-Aib-Leu-Aib-Pro-Ala-Aib-Pro-Aib-Pro-Phe-OMe (where Boc is t-butoxycarbonyl and Aib is alpha-aminoisobutyric acid), a synthetic apolar analog of the membrane-active fungal peptide antibiotic zervamycin IIA, crystallizes in space group P1 with Z = 1 and cell parameters a = 9.086 +/- 0.002 A, b = 10.410 +/- 0.002 A, c = 28.188 +/- 0.004 A, alpha = 86.13 +/- 0.01 degrees, beta = 87.90 +/- 0.01 degrees, and gamma = 89.27 +/- 0.01 degrees; overall agreement factor R = 7.3% for 7180 data (F0 greater than 3 sigma) and 0.91-A resolution. The peptide backbone makes a continuous spiral that begins as a 3(10)-helix at the N-terminus, changes to an alpha-helix for two turns, and ends in a spiral of three beta-bends in a ribbon. Each of the beta-bends contains a proline residue at one of the corners. The torsion angles phi i range from -51 degrees to -91 degrees (average value -64 degrees), and the torsion angles psi i range from -1 degree to -46 degrees (average value -31 degrees). There are 10 intramolecular NH...OC hydrogen bonds in the helix and two direct head-to-tail hydrogen bonds between successive molecules. Two H2O and two CH3OH solvent molecules fill additional space with appropriate hydrogen bonding in the head-to-tail region...

Mouse interleukin-2 structure-function studies: substitutions in the first alpha-helix can specifically inactivate p70 receptor binding and mutations in the fifth alpha-helix can specifically inactivate p55 receptor binding.

Zurawski, S M; Zurawski, G
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1989 EN
Relevância na Pesquisa
493.1176%
The function of two alpha-helical regions of mouse interleukin-2 were analyzed by saturation substitution analysis. The functional parts of the first alpha-helix (A) was defined as residues 31-39 by the observation that proline substitutions within this region inactivate the protein. Four residues within alpha-helix A, Leu31, Asp34, Leu35 and Leu38, were found to be crucial for biological activity. Structural modeling suggested that these four residues are clustered on one face of alpha-helix A. Residues 31 and 35 had to remain hydrophobic for the molecule to be functional. At residue 38 there was a preference for hydrophobic side chain residues, while at residue 34 some small side chain residues as well as acidic or amide side chain residues were functionally acceptable. Inactivating changes at residue 34 had no effect upon the ability of the protein to interact with the p55 receptor. Disruption of the fifth alpha-helix (E), which had little effect upon biological activity, resulted in an inability of the protein to interact with the p55 receptor. Mutagenesis of the alpha-helix E region demonstrated that alpha-helicity and the nature of the side chain residues in this region were unimportant for biological activity. The region immediately proximal to alpha-helix E was important only for the single intramolecular disulfide linkage.

Predicted alpha-helix/beta-sheet secondary structures for the zinc-binding motifs of human papillomavirus E7 and E6 proteins by consensus prediction averaging and spectroscopic studies of E7.

Ullman, C G; Haris, P I; Galloway, D A; Emery, V C; Perkins, S J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/10/1996 EN
Relevância na Pesquisa
488.74766%
The E7 and E6 proteins are the main oncoproteins of human papillomavirus types 16 and 18 (HPV-16 and HPV-18), and possess unknown protein structures. E7 interacts with the cellular tumour-suppressor protein pRB and contains a zinc-binding site with two Cys-Xaa2-Cys motifs spaced 29 or 30 residues apart. E6 interacts with another cellular tumour-suppressor protein p53 and contains two zinc-binding sites, each with two Cys-Xaa2-Cys motifs at a similar spacing of 29 or 30 residues. By using the GOR I/III, Chou-Fasman, SAPIENS and PHD methods, the effectiveness of consensus secondary structure predictions on zinc-finger proteins was first tested with sequences for 160 transcription factors and 72 nuclear hormone receptors. These contain Cys2His2 and Cys2Cys2 zinc-binding regions respectively, and possess known atomic structures. Despite the zinc- and DNA-binding properties of these protein folds, the major alpha-helix structures in both zinc-binding regions were correctly identified. Thus validated, the use of these prediction methods with 47 E7 sequences indicated four well-defined alpha-helix (alpha) and beta-sheet (beta) secondary structure elements in the order beta beta alpha beta in the zinc-binding region of E7 at its C-terminus. The prediction was tested by Fourier transform infrared spectroscopy of recombinant HPV-16 E7 in H2O and 2H2O buffers. Quantitative integration showed that E7 contained similar amounts of alpha-helix and beta-sheet structures...

Free-energy determinants of alpha-helix insertion into lipid bilayers.

Ben-Tal, N; Ben-Shaul, A; Nicholls, A; Honig, B
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1996 EN
Relevância na Pesquisa
482.77703%
A detailed treatment is provided of the various free-energy terms that contribute to the transfer of a polyalanine alpha-helix from the aqueous phase into lipid bilayers. In agreement with previous work, the hydrophobic effect is found to provide the major driving force for helix insertion. However, an opposing effect of comparable magnitude is also identified and is attributed to the large free-energy penalty associated with the desolvation of peptide hydrogen bonds on transfer to the low dielectric environment of the bilayer. Lipid perturbation effects as well as the entropy loss associated with helix immobilization in the bilayer are also evaluated. Two configurations of a membrane-bound 25mer polyalanine helix were found to be lower in free energy than the isolated helix in the aqueous phase. The first corresponds to the case of vertical insertion, in which a helix terminus protrudes from each side of the bilayer. The second minimum is for the case of horizontal insertion, for which the helix is adsorbed upon the surface of the bilayer. The calculated free-energy minima are found to be in good agreement with recent measurements of related systems. Large free-energy barriers resulting from desolvation of unsatisfied hydrogen-bonding groups at the helix termini are obtained for both insertion processes. The barriers for insertion are significantly reduced if the helix termini are assumed to be "capped" through the formation of hydrogen bonds with polar sidechains. For uncapped helices...

Calculation of electrostatic effects at the amino terminus of an alpha helix.

Sitkoff, D; Lockhart, D J; Sharp, K A; Honig, B
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1994 EN
Relevância na Pesquisa
485.436%
It is generally believed that the electrostatic field arising from the dipolar charge distribution in alpha helices is important for protein structure and function. We report a calculation of the electrostatic potential and field at the amino terminus of an alpha helix in water, obtained from a finite difference solution to the Poisson-Boltzmann equation. This method takes into account the detailed helix shape and charge distribution, as well as solvent, and generalized ionic strength effects. The calculated potential and field are found to be in good agreement with the experimentally observed helix-induced Stark effect and pKa shifts of a probe at the N-terminus of a stable, monomeric alpha-helical peptide (Lockhart and Kim, 1992, 1993). Ionic screening effects are reproduced at low salt concentrations. Deviations at higher salt concentrations may result from specific ion effects (specific ion-solute and/or ion-solvent interactions). The FDPB method was used to analyze the contributions from each residue, charged side chains, and solvent to the helix potential and field. Backbone contributions come primarily from the first one to two helical turns. Charged side chains contribute to helix-induced pKa shifts for certain probe-peptide combinations...

Analysis of the three-alpha-helix motif in the spectrin superfamily of proteins.

Parry, D A; Dixon, T W; Cohen, C
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1992 EN
Relevância na Pesquisa
485.1808%
Members of the spectrin superfamily of proteins contain different numbers of homologous repeats arranged in tandem. Each of these consists of a three-alpha-helix motif, comprising two similarly and one oppositely directed alpha-helical segment joined by nonhelical linkers of characteristic length. The right-handed alpha-helices each display a heptad repeat in their amino acid sequences indicative of left-handed coiled-coil-like packing. We have calculated the potential number of inter-helix ionic interactions that specify the spatial arrangement of the helices in the motif in terms of both the handedness of helix connectivity (left or right) and the relative axial stagger between the three alpha-helices. All of the models examined were constrained to have optimal coiled-coil packing. For alpha-spectrin and alpha-actinin the results provide strong support for a left-handed connectivity of the three helices and axial repeat lengths of 5.05 and 6.24 nm, respectively. Furthermore, the axial staggers between homologous segments in the preferred models are identical. The insights provided into the topography of this widespread tertiary fold may prove of value to those concerned with the problem of de novo protein design.

Multiple alanine replacements within alpha-helix 126-134 of T4 lysozyme have independent, additive effects on both structure and stability.

Zhang, X. J.; Baase, W. A.; Matthews, B. W.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /06/1992 EN
Relevância na Pesquisa
485.436%
In a systematic attempt to identify residues important in the folding and stability of T4 lysozyme, five amino acids within alpha-helix 126-134 were substituted by alanine, either singly or in selected combinations. Together with three alanines already present in the wild-type structure this provided a set of mutant proteins with up to eight alanines in sequence. All the variants behaved normally, suggesting that the majority of residues in the alpha-helix are nonessential for the folding of T4 lysozyme. Of the five individual alanine substitutions it is inferred that four result in slightly increased protein stability and one, the replacement of a buried leucine with alanine, substantially decreased stability. The results support the idea that alanine is a residue of high helix propensity. The change in protein stability observed for each of the multiple mutants is approximately equal to the sum of the energies associated with each of the constituent substitutions. All of the variants could be crystallized isomorphously with wild-type lysozyme, and, with one trivial exception, their structures were determined at high resolution. Substitution of the largely solvent-exposed residues Asp 127, Glu 128, and Val 131 with alanine caused essentially no change in structure except at the immediate site of replacement. Substitutions of the partially buried Asn 132 and the buried Leu 133 with alanine were associated with modest (< or = 0.4 A) structural adjustments. The structural changes seen in the multiple mutants were essentially a combination of those seen in the constituent single replacements. The different replacements therefore act essentially independently not only so far as changes in energy are concerned but also in their effect on structure. The destabilizing replacement Leu 133-->Ala made alpha-helix 126-134 somewhat less regular. Incorporation of additional alanine replacements tended to make the helix more uniform. For the penta-alanine variant a distinct change occurred in a crystal-packing contact...

Models for the 3(10)-helix/coil, pi-helix/coil, and alpha-helix/3(10)-helix/coil transitions in isolated peptides.

Rohl, C. A.; Doig, A. J.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /08/1996 EN
Relevância na Pesquisa
490.1085%
Models for the 3(10)-helix/coil and pi-helix/coil equilibria have been derived. The theory is based on classifying residues into helical or nonhelical (coil) conformations. Statistical weights are assigned to residues in a helical conformation with an associated helical hydrogen bond, a helical conformation with no hydrogen bond, an N-cap position, a C-cap position, or the reference coil conformation. The models for alpha-helix formation and 3(10)-helix formation have also been combined to describe a three-state equilibrium in which alpha-helical, 3(10)-helical, and coil conformations are populated. The results are compared with the modified Lifson-Roig theory for the alpha-helix/coil equilibrium. The comparison accounts for the experimental observations that 3(10)-helices tend to be short and pi-helices are not favored for any length. This work may provide a framework for quantitatively rationalizing experimental work on isolated 3(10)-helices and mixed 3(10)-/alpha-helices.

Residues in the alpha helix 7 of the bacterial maltose binding protein which are important in interactions with the Mal FGK2 complex.

Szmelcman, S.; Sassoon, N.; Hofnung, M.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /03/1997 EN
Relevância na Pesquisa
485.40883%
The periplasmic maltose binding protein, MalE, is a major element in maltose transport and in chemotaxis towards this sugar. Previous genetic analysis of the MalE protein revealed functional domains involved in transport and chemotactic functions. Among them the surface located alpha helix 7, which is part of the C-lobe, one of the two lobes forming the three dimensional structure of MalE. Small deletions in this region abolished maltose transport, although maintaining wild-type affinity and specificity as well as a normal chemoreceptor function. It was suggested that alpha helix 7 may be implicated in interactions between the maltose binding protein and the membrane-bound protein complex (Duplay P, Szmelcman S. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. II. Chemotaxis towards maltose. J Mol Biol 194:675-678: Duplay P, Szmelcman S, Bedouelle H, Hofnung M. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. I: Transport of maltose. J Mol Biol 194:663-673). In this study, we submitted a region of 14 residues--Asp 207 to Gly 220--encompassing alpha helix 7, to genetic analysis by oligonucleotide mediated random mutagenesis. Out of 127 identified mutations...

Addition of side-chain interactions to 3(10)-helix/coil and alpha-helix/3(10)-helix/coil theory.

Sun, J. K.; Doig, A. J.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /11/1998 EN
Relevância na Pesquisa
496.60734%
An increasing number of experimental and theoretical studies have demonstrated the importance of the 3(10)-helix/ alpha-helix/coil equilibrium for the structure and folding of peptides and proteins. One way to perturb this equilibrium is to introduce side-chain interactions that stabilize or destabilize one helix. For example, an attractive i, i + 4 interaction, present only in the alpha-helix, will favor the alpha-helix over 3(10), while an i, i + 4 repulsion will favor the 3(10)-helix over alpha. To quantify the 3(10)/alpha/coil equilibrium, it is essential to use a helix/coil theory that considers the stability of every possible conformation of a peptide. We have previously developed models for the 3(10)-helix/coil and 3(10)-helix/alpha-helix/ coil equilibria. Here we extend this work by adding i, i + 3 and i, i + 4 side-chain interaction energies to the models. The theory is based on classifying residues into alpha-helical, 3(10)-helical, or nonhelical (coil) conformations. Statistical weights are assigned to residues in a helical conformation with an associated helical hydrogen bond, a helical conformation with no hydrogen bond, an N-cap position, a C-cap position, or the reference coil conformation plus i, i + 3 and i, i + 4 side-chain interactions. This work may provide a framework for quantitatively rationalizing experimental work on isolated 3(10)-helices and mixed 3(10)-/alpha-helices and for predicting the locations and stabilities of these structures in peptides and proteins. We conclude that strong i...

Localization of basic residues required for receptor binding to the single alpha-helix of the receptor binding domain of human alpha2-macroglobulin.

Huang, W.; Dolmer, K.; Liao, X.; Gettins, P. G.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /12/1998 EN
Relevância na Pesquisa
485.40883%
To better understand the structural basis for the binding of proteinase-transformed human alpha2-macroglobulin (alpha2M) to its receptor, we have used three-dimensional multinuclear NMR spectroscopy to determine the secondary structure of the receptor binding domain (RBD) of human alpha2M. Assignment of the backbone NMR resonances of RBD was made using 13C/15-N and 15N-enriched RBD expressed in Escherichia coli. The secondary structure of RBD was determined using 1H and 13C chemical shift indices and inter- and intrachain nuclear Overhauser enhancements. The secondary structure consists of eight strands in beta-conformation and one alpha-helix, which together comprise 44% of the protein. The beta-strands form three regions of antiparallel beta-sheet. The two lysines previously identified as being critical for receptor binding are located in (Lys1374), and immediately adjacent to (Lys1370) the alpha-helix, which also contains an (Arg1378). Secondary structure predictions of other alpha-macroglobulins show the conservation of this alpha-helix and suggest an important role for this helix and for basic residues within it for receptor binding.

Determination of alpha-helix N1 energies after addition of N1, N2, and N3 preferences to helix/coil theory.

Sun, J. K.; Penel, S.; Doig, A. J.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /04/2000 EN
Relevância na Pesquisa
482.12664%
Surveys of protein crystal structures have revealed that amino acids show unique structural preferences for the N1, N2, and N3 positions in the first turn of the alpha-helix. We have therefore extended helix-coil theory to include statistical weights for these locations. The helix content of a peptide in this model is a function of N-cap, C-cap, N1, N2, N3, C1, and helix interior (N4 to C2) preferences. The partition function for the system is calculated using a matrix incorporating the weights of the fourth residue in a hexamer of amino acids and is implemented using a FORTRAN program. We have applied the model to calculate the N1 preferences of Gln, Val, Ile, Ala, Met, Pro, Leu, Thr, Gly, Ser, and Asn, using our previous data on helix contents of peptides Ac-XAKAAAAKAAGY-CONH2. We find that Ala has the highest preference for the N1 position. Asn is the most unfavorable, destabilizing a helix at N1 by at least 1.4 kcal mol(-1) compared to Ala. The remaining amino acids all have similar preferences, 0.5 kcal mol(-1) less than Ala. Gln, Asn, and Ser, therefore, do not stabilize the helix when at N1.

An Amphipathic Alpha-Helix in the Prodomain of Cocaine and Amphetamine Regulated Transcript Peptide Precursor Serves as Its Sorting Signal to the Regulated Secretory Pathway

Blanco, Elías H.; Lagos, Carlos F.; Andrés, María Estela; Gysling, Katia
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 19/03/2013 EN
Relevância na Pesquisa
488.25582%
Cocaine and Amphetamine Regulated Transcript (CART) peptides are anorexigenic neuropeptides. The L34F mutation in human CART peptide precursor (proCART) has been linked to obesity (Yanik et al. Endocrinology 147: 39, 2006). Decrease in CART peptide levels in individuals carrying the L34F mutation was attributed to proCART subcellular missorting. We studied proCART features required to enter the regulated secretory pathway. The subcellular localization and the secretion mode of monomeric EGFP fused to the full-length or truncated forms of human proCART transiently transfected in PC12 cells were analyzed. Our results showed that the N-terminal 1–41 fragment of proCART was necessary and sufficient to sort proCART to the regulated secretory pathway. In silico modeling predicted an alpha-helix structure located between residues 24–37 of proCART. Helical wheel projection of proCART alpha-helix showed an amphipathic configuration. The L34F mutation does not modify the amphipathicity of proCART alpha-helix and consistently proCARTL34F was efficiently sorted to the regulated secretory pathway. However, four additional mutations to proCARTL34F that reduced its alpha-helix amphipathicity resulted in the missorting of the mutated proCART toward the constitutive secretory pathway. These findings show that an amphipathic alpha-helix is a key cis-structure for the proCART sorting mechanism. In addition...

Conversion of a beta-strand to an alpha-helix induced by a single-site mutation observed in the crystal structure of Fis mutant Pro26Ala.

Yang, W. Z.; Ko, T. P.; Corselli, L.; Johnson, R. C.; Yuan, H. S.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /09/1998 EN
Relevância na Pesquisa
490.4084%
The conversion from an alpha-helix to a beta-strand has received extensive attention since this structural change may induce many amyloidogenic proteins to self-assemble into fibrils and cause fatal diseases. Here we report the conversion of a peptide segment from a beta-strand to an alpha-helix by a single-site mutation as observed in the crystal structure of Fis mutant Pro26Ala determined at 2.0 A resolution. Pro26 in Fis occurs at the point where a flexible extended beta-hairpin arm leaves the core structure. Thus it can be classified as a "hinge proline" located at the C-terminal end of the beta2-strand and the N-terminal cap of the A alpha-helix. The replacement of Pro26 to alanine extends the A alpha-helix for two additional turns in one of the dimeric subunits; therefore, the structure of the peptide from residues 22 to 26 is converted from a beta-strand to an alpha-helix. This result confirms the structural importance of the proline residue located at the hinge region and may explain the mutant's reduced ability to activate Hin-catalyzed DNA inversion. The peptide (residues 20 to 26) in the second monomer subunit presumably retains its beta-strand conformation in the crystal; therefore, this peptide shows a "chameleon-like" character since it can adopt either an alpha-helix or a beta-strand structure in different environments. The structure of Pro26Ala provides an additional example where not only the protein sequence...

Facile transition between 3(10)- and alpha-helix: structures of 8-, 9-, and 10-residue peptides containing the -(Leu-Aib-Ala)2-Phe-Aib- fragment.

Karle, I. L.; Flippen-Anderson, J. L.; Gurunath, R.; Balaram, P.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /09/1994 EN
Relevância na Pesquisa
489.5143%
A structural transition from a 3(10)-helix to an alpha-helix has been characterized at high resolution for an octapeptide segment located in 3 different sequences. Three synthetic peptides, decapeptide (A) Boc-Aib-Trp-(Leu-Aib-Ala)2-Phe-Aib-OMe, nonapeptide (B) Boc-Trp-(Leu-Aib-Ala)2-Phe-Aib-OMe, and octapeptide (C) Boc-(Leu-Aib-Ala)2-Phe-Aib-OMe, are completely helical in their respective crystals. At 0.9 A resolution, R factors for A, B, and C are 8.3%, 5.4%, and 7.3%, respectively. The octapeptide and nonapeptide form ideal 3(10)-helices with average torsional angles phi(N-C alpha) and psi(C alpha-C') of -57 degrees, -26 degrees C and -60 degrees, -27 degrees for B. The 10-residue peptide (A) begins as a 3(10)-helix and abruptly changes to an alpha-helix at carbonyl O(3), which is the acceptor for both a 4-->1 hydrogen bond with N(6)H and a 5-->1 hydrogen with N(7)H, even though the last 8 residues have the same sequence in all 3 peptides. The average phi, psi angles in the decapeptide are -58 degrees, -28 degrees for residues 1-3 and -63 degrees, -41 degrees for residues 4-10. The packing of helices in the crystals does not provide any obvious reason for the transition in helix type. Fourier transform infrared studies in the solid state also provide evidence for a 3(10)- to alpha-helix transition with the amide I band appearing at 1...