The Experts below are selected from a list of 13452 Experts worldwide ranked by ideXlab platform
Robert Seckler - One of the best experts on this subject based on the ideXlab platform.
-
in vivo folding efficiencies for mutants of the p22 tailspike Beta Helix protein correlate with predicted stability changes
Biophysical Chemistry, 2009Co-Authors: Lothar Reich, Robert Seckler, Marion Becker, Thomas R WeiklAbstract:Abstract Parallel β -helices are among the simplest repetitive structural elements in proteins. The folding behavior of β -Helix proteins has been studied intensively, also to gain insight on the formation of amyloid fibrils, which share the parallel β -Helix as a central structural motif. An important system for investigating β -Helix folding is the tailspike protein from the Salmonella bacteriophage P22. The central domain of this protein is a right-handed parallel β -Helix with 13 windings. Extensive mutational analyses of the P22 tailspike protein have revealed two main phenotypes: temperature-sensitive-folding (tsf) mutations that reduce the folding efficiency at elevated temperatures, and global suppressor (su) mutations that increase the tailspike folding efficiency. A central question is whether these phenotypes can be understood from changes in the protein stability induced by the mutations. Experimental determination of the protein stability is complicated by the nearly irreversible trimerization of the folded tailspike protein. Here, we present calculations of stability changes with the program FoldX, focusing on a recently published extensive data set of 145 singe-residue alanine mutants. We find that the calculated stability changes are correlated with the experimentally measured in vivo folding efficiencies. In addition, we determine the free-energy landscape of the P22 tailspike protein in a nucleation–propagation model to explore the folding mechanism of this protein, and obtain a processive folding route on which the protein nucleates in the N-terminal region of the Helix.
-
plasticity and steric strain in a parallel Beta Helix rational mutations in the p22 tailspike protein
Proteins, 2000Co-Authors: Benjamin Schuler, Stefan Steinbacher, Robert Huber, Frank Furst, Frank Osterroth, Robert SecklerAbstract:By means of genetic screens, a great number of mutations that affect the folding and stability of the tailspike protein from Salmo- nella phage P22 have been identified. Temperature- sensitive folding (tsf) mutations decrease folding yields at high temperature, but hardly affect ther- mal stability of the native trimeric structure when assembled at low temperature. Global suppressor (su) mutations mitigate this phenotype. Virtually all of these mutations are located in the central domain of tailspike, a large parallel b-Helix. We modified tailspike by rational single amino acid replace- ments at three sites in order to investigate the influence of mutations of two types: (1) mutations expected to cause a tsf phenotype by increasing the side-chain volume of a core residue, and (2) muta- tions in a similar structural context as two of the four known su mutations, which have been sug- gested to stabilize folding intermediates and the native structure by the release of backbone strain, an effect well known for residues that are primarily evolved for function and not for stability or folding of the protein. Analysis of folding yields, refolding kinetics and thermal denaturation kinetics in vitro show that the tsf phenotype can indeed be produced rationally by increasing the volume of side chains in the b-Helix core. The high-resolution crystal struc- ture of mutant T326F proves that structural rear- rangements only take place in the remarkably plas- tic lumen of the b-Helix, leaving the arrangement of the hydrogen-bonded backbone and thus the sur- face of the protein unaffected. This supports the notion that changes in the stability of an intermedi- ate, in which the b-Helix domain is largely formed, are the essential mechanism by which tsf mutations affect tailspike folding. A rational design of su mu- tants, on the other hand, appears to be more diffi- cult. The exchange of two residues in the active site expected to lead to a drastic release of steric strain neither enhanced the folding properties nor the stability of tailspike. Apparently, side-chain interac- tions in these cases overcompensate for backbone strain, illustrating the extreme optimization of the tailspike protein for conformational stability. The result exemplifies the view arising from the statisti- cal analysis of the distribution of backbone dihedral angles in known three-dimensional protein struc- tures that the adoption of f/c angles other than the most favorable ones is often caused by side-chain interactions. Proteins 2000;39:89 -101.
-
formation of fibrous aggregates from a non native intermediate the isolated p22 tailspike Beta Helix domain
Journal of Biological Chemistry, 1999Co-Authors: Benjamin Schuler, Reinhard Rachel, Robert SecklerAbstract:In the assembly pathway of the trimeric P22 tailspike protein, the protein conformation critical for the partitioning between productive folding and off-pathway aggregation is a monomeric folding intermediate. The central domain of tailspike, a large right-handed parallel Beta-Helix, is essentially structured in this species. We used the isolated Beta-Helix domain (Bhx), expressed with a hexahistidine tag, to investigate the mechanism of aggregation without the two terminal domains present in the complete protein. Although Bhx has been shown to fold reversibly at low ionic strength conditions, increased ionic strength induced aggregation with a maximum at urea concentrations corresponding to the midpoint of urea-induced folding transitions. According to size exclusion chromatography, aggregation appeared to proceed via a linear polymerization mechanism. Circular dichroism indicated a secondary structure content of the aggregates similar to that of the native state, but at the same time their tryptophan fluorescence was largely quenched. Microscopic analysis of the aggregates revealed a variety of morphologies; among others, fibrils with fine structure were observed that exhibited bright green birefringence if viewed under cross-polarized light after staining with Congo red. These observations, together with the effects of folding mutations on the aggregation process, indicate the involvement of a partially structured intermediate distinct from both unfolded and native Bhx.
-
p22 tailspike folding mutants revisited effects on the thermodynamic stability of the isolated Beta Helix domain
Journal of Molecular Biology, 1998Co-Authors: Benjamin Schuler, Robert SecklerAbstract:The folding of the trimeric phage P22 tailspike protein is influenced by amino acid substitutions of two types, virtually all of which affect residues in the central domain, a large parallel Beta-Helix. Temperature sensitive folding (tsf) mutations lead to drastically decreased folding yields at elevated temperature. Their phenotype can be alleviated by global suppressor (su) mutations. Both types of mutations appeared to have no influence on the stability of the native protein at the time of their first isolation and were thus suggested to carry information needed for the folding pathway exclusively. The monomeric Beta-Helix of tailspike, expressed as an isolated domain, exhibits freely reversible unfolding and refolding transitions, allowing us to analyse the effects of two well-characterised tsf and all four known su mutations on its thermodynamic stability. We find a marked decrease in stability for the tsf mutants and a striking increase in stability for all su mutants. This leads to the conception that the isolated Beta-Helix domain, although active in receptor-binding and native-like in its spectroscopic properties, is close in conformation to a crucial monomeric folding intermediate whose thermolability is responsible for the kinetic partitioning between productive folding and irreversible aggregation during the maturation process of P22 tailspike protein.
-
a reversibly unfolding fragment of p22 tailspike protein with native structure the isolated Beta Helix domain
Biochemistry, 1998Co-Authors: Stefan Miller, Benjamin Schuler, Robert SecklerAbstract:The homotrimeric tailspike endorhamnosidase of phage P22 has been used to compare in vivo and in vitro folding pathways and the influence of single amino acid substitutions thereon. Its main structural motif, which contains the known folding mutation sites, consists of three large right-handed parallel Beta-helices. A thermodynamic analysis of the stability of tailspike is prevented by the irreversibility of unfolding at high temperatures or high concentrations of denaturant, probably due to interdigitation of the domains neighboring the Beta-Helix. We therefore expressed and isolated a tailspike fragment comprising only its central Beta-Helix domain (residues 109-544). As shown by equilibrium ultracentrifugation, the isolated Beta-Helix is a monomer at concentrations below 1 microM and trimerizes reversibly at higher protein concentrations. Both the similarity of fluorescence and CD spectra, compared to the complete protein, and the specific binding and hydrolysis of substrate suggest a nativelike structure. Moreover, urea denaturation transitions of the Beta-Helix domain are freely reversible, providing the basis for a future quantitative analysis of the effects of the folding mutations on the thermodynamic stability of the domain and of structural features responsible for folding and stability of the parallel Beta-Helix motif in general.
Richard W Pickersgill - One of the best experts on this subject based on the ideXlab platform.
-
the crystal structure of pectate lyase pel9a from erwinia chrysanthemi
Journal of Biological Chemistry, 2004Co-Authors: J Jenkins, Vladimir E. Shevchik, Nicole Hugouvieuxcottepattat, Richard W PickersgillAbstract:The "family 9 polysaccharide lyase" pectate lyase L (Pel9A) from Erwinia chrysanthemi comprises a 10-coil parallel Beta-Helix domain with distinct structural features including an asparagine ladder and aromatic stack at novel positions within the superhelical structure. Pel9A has a single high affinity calcium-binding site strikingly similar to the "primary" calcium-binding site described previously for the family Pel1A pectate lyases, and there is strong evidence for a common second calcium ion that binds between enzyme and substrate in the "Michaelis" complex. Although the primary calcium ion binds substrate in subsite -1, it is the second calcium ion, whose binding site is formed by the coming together of enzyme and substrate, that facilitates abstraction of the C5 proton from the sacharride in subsite +1. The role of the second calcium is to withdraw electrons from the C6 carboxylate of the substrate, thereby acidifying the C5 proton facilitating its abstraction and resulting in an E1cb-like anti-Beta-elimination mechanism. The active site geometries and mechanism of Pel1A and Pel9A are closely similar, but the catalytic base is a lysine in the Pel9A enzymes as opposed to an arginine in the Pel1A enzymes.
-
three dimensional structure of erwinia chrysanthemi pectin methylesterase reveals a novel esterase active site
Journal of Molecular Biology, 2001Co-Authors: J Jenkins, Olga Mayans, Drummond Smith, Kathryn Worboys, Richard W PickersgillAbstract:Most structures of neutral lipases and esterases have been found to adopt the common alpha/Beta hydrolase fold and contain a catalytic Ser-His-Asp triad. Some variation occurs in both the overall protein fold and in the location of the catalytic triad, and in some enzymes the role of the aspartate residue is replaced by a main-chain carbonyl oxygen atom. Here, we report the crystal structure of pectin methylesterase that has neither the common alpha/Beta hydrolase fold nor the common catalytic triad. The structure of the Erwinia chrysanthemi enzyme was solved by multiple isomorphous replacement and refined at 2.4 A to a conventional crystallographic R-factor of 17.9 % (R(free) 21.1 %). This is the first structure of a pectin methylesterase and reveals the enzyme to comprise a right-handed parallel Beta-Helix as seen in the pectinolytic enzymes pectate lyase, pectin lyase, polygalacturonase and rhamnogalacturonase, and unlike the alpha/Beta hydrolase fold of rhamnogalacturonan acetylesterase with which it shares esterase activity. Pectin methylesterase has no significant sequence similarity with any protein of known structure. Sequence conservation among the pectin methylesterases has been mapped onto the structure and reveals that the active site comprises two aspartate residues and an arginine residue. These proposed catalytic residues, located on the solvent-accessible surface of the parallel Beta-Helix and in a cleft formed by external loops, are at a location similar to that of the active site and substrate-binding cleft of pectate lyase. The structure of pectin methylesterase is an example of a new family of esterases.
-
structure and evolution of parallel Beta Helix proteins
Journal of Structural Biology, 1998Co-Authors: John R Jenkins, Olga Mayans, Richard W PickersgillAbstract:Three bacterial pectate lyases, a pectin lyase from Aspergillus niger, the structures of rhamnogalacturonase A from Aspergillus aculeatus, RGase A, and the P22-phage tailspike protein, TSP, display the right-handed parallel Beta-Helix architecture first seen in pectate lyase. The lyases have 7 complete coils while RGase A and TSP have 11 and 12, respectively. Each coil contains three Beta-strands and three turn regions named PB1, T1, PB2, T2, PB3, and T3 in their order of occurrence. The lyases have homologous sequences but RGase A and TSP do not show obvious sequence homology either to the lyases or to each other. However, the structural similarities between all these molecules are so extensive that divergence from a common ancestor is much more probable than convergence to the same fold. The region PB2-T2-PB3 is the best conserved region in the lyases and shows the clearest structural similarity. Not only is the pleating and the direction of the hydrogen bonding in the sheets conserved, but so is the unusual alphaL-conformation turn between the two sheets. However, the overall shape, the position of long loops, a conserved alpha-Helix that covers the amino-terminal end of the parallel Beta-Helix and stacks of residues in alphaR-conformation at the start of PB1 all suggest a common ancestor. The functional similarity, that the enzymes all bind alpha-galactose containing polymers at an equivalent site involving PB1 and its two flanking turn regions, further supports divergent evolution. We suggest that the stacking of the coils and the unusual near perpendicular junction of PB2 and PB3 make the parallel Beta-Helix fold especially likely to maintain similar main chain conformations during divergent evolution even after all vestige of similarity in primary structure has vanished.
-
the structure of bacillus subtilis pectate lyase in complex with calcium
Nature Structural & Molecular Biology, 1994Co-Authors: Richard W Pickersgill, William Nasser, John A Jenkins, G W Harris, Janine RobertbaudouyAbstract:We have solved the structure of the Bacillus subtilis pectate lyase (BsPel) in complex with calcium. The structure consists of a parallel Beta-Helix domain and a loop region. The alpha L-bounded Beta-strand seen in BsPel is a new element of protein structure and its frequent occurrence suggests it is an important characteristic of the parallel Beta-Helix. A pronounced cleft is formed between the loops and the parallel Beta-Helix domain and we propose that this is the active site cleft. Calcium, essential for the activity of the enzyme, binds at the bottom of this cleft and an arginine residue close to the calcium, which is conserved across all pectin and pectate lyases, may be involved in catalysis.
Frances Jurnak - One of the best experts on this subject based on the ideXlab platform.
-
Functional Implications of Structure-Based Sequence Alignment of Proteins in the Extracellular Pectate Lyase Superfamily
Plant Physiology, 1995Co-Authors: Bernard Henrissat, Marilyn D Yoder, Susan E. Lietzke, Susan E. Heffron, Frances JurnakAbstract:Pectate lyases are plant virulence factors that degrade the pectate component of the plant cell wall. The enzymes share considerable sequence homology with plant pollen and style proteins, suggesting a shared structural topology and possibly functional relationships as well. The three-dimensional structures of two Erwinia chrysanthemi pectate lyases, C and E, have been superimposed and the structurally conserved amino acids have been identified. There are 232 amino acids that superimpose with a root-mean-square deviation of 3 A or less. These amino acids have been used to correct the primary sequence alignment derived from evolution-based techniques. Subsequently, multiple alignment techniques have allowed the realignment of other extracellular pectate lyases as well as all sequence homologs, including pectin lyases and the plant pollen and style proteins. The new multiple sequence alignment reveals amino acids likely to participate in the parallel Beta Helix motif, those involved in binding Ca2+, and those invariant amino acids with potential catalytic properties. The latter amino acids cluster in two well-separated regions on the pectate lyase structures, suggesting two distinct enzymatic functions for extracellular pectate lyases and their sequence homologs.
-
protein motifs 3 the parallel Beta Helix and other coiled folds
The FASEB Journal, 1995Co-Authors: Marilyn D Yoder, Frances JurnakAbstract:A new type of structural domain, composed of all parallel Beta strands, has been observed within the last year. An analysis of the basic types suggests that there are two distinct classes: the parallel Beta helices, which belong to a tri Beta-strand category, and the Beta roll, which belongs to a di Beta-strand category. The novel structural features of each class are described and the proteins belonging to each category are summarized. Proteins with the parallel Beta Helix fold include three pectate lyases and the tailspike protein from P22 phage. Proteins with the Beta roll fold include two alkaline proteases. Although the parallel Beta composition is emphasized, the same set of proteins share another common structural feature with several other proteins containing alpha helices: the polypeptide backbone is folded into a coiled structure in which each coil has the same 3-dimensional arrangement of a group of secondary structural elements. In addition to parallel Beta domains, the other groups include th...
-
the three dimensional structure of pectate lyase e a plant virulence factor from erwinia chrysanthemi
Plant Physiology, 1994Co-Authors: Susan E. Lietzke, Marilyn D Yoder, Noel T. Keen, Frances JurnakAbstract:The three-dimensional structure of pectate lyase E (PelE) has been determined by crystallographic techniques at a resolution of 2.2 A. The model includes all 355 amino acids but no solvent, and refines to a crystallographic refinement factor of 20.6%. The polypeptide backbone folds into a large right-handed cylinder, termed a parallel [Beta] Helix. Loops of various sizes and conformations protrude from the central Helix and probably confer function. A putative Ca2+-binding site as well as two cationic sites have been deduced from the location of heavy atom derivatives. Comparison of the PelE and recently determined pectate lyase C (PelC) structures has led to identification of a putative polygalacturonate-binding region in PelE. Structural differences relevant to differences in the enzymatic mechanism and maceration properties of PelE and PelC have been identified. The comparative analysis also reveals a large degree of structural conservation of surface loops in one region as well as an apparent aromatic specificity pocket in the amino-terminal branch. Also discussed is the sequence and possible functional relationship of the pectate lyases with pollen and style plant proteins.
-
unusual structural features in the parallel Beta Helix in pectate lyases
Structure, 1993Co-Authors: Marilyn D Yoder, Susan E. Lietzke, Frances JurnakAbstract:Abstract Background: A new type of domain structure, an all parallel β class, has recently been observed in two pectate lyases, PelC and PelE. The atomic models have been analyzed to determine whether the new tertiary fold exhibits unusual structural features. Results: The polypeptide backbone exhibits no new types of secondary structural elements. However, novel features occur in the amino acid side chain interactions. The side chain atoms form linear stacks that include asparagine ladders, serine stacks, aliphatic stacks, and ringed-residue stacks. A new type of β-sandwich between parallel β-sheets is observed with properties that are more characteristic of antiparallel β-sheets. Conclusion: An analysis of the PelC and PelE structures, belonging to an all parallel β structural class, reveals novel amino acid side chain interactions, a new type of β-sandwich and an atypical amino acid composition of parallel β-sheets. The findings are relevant to three-dimensional structural predictions.
-
New domain motif: The structure of pectate lyase C, a secreted plant virulence factor
Science, 1993Co-Authors: Marilyn D Yoder, Noel T. Keen, Frances JurnakAbstract:Pectate lyases are secreted by pathogens and initiate soft-rot diseases in plants by cleaving polygalacturonate, a major component of the plant cell wall. The three-dimensional structure of pectate lyase C from Erwinia chrysanthemi has been solved and refined to a resolution of 2.2 angstroms. The enzyme folds into a unique motif of parallel Beta strands coiled into a large Helix. Within the core, the amino acids form linear stacks and include a novel asparagine ladder. The sequence similarities that pectate lyases share with pectin lyases, pollen and style proteins, and tubulins suggest that the parallel Beta Helix motif may occur in a broad spectrum of proteins.
Benjamin Schuler - One of the best experts on this subject based on the ideXlab platform.
-
plasticity and steric strain in a parallel Beta Helix rational mutations in the p22 tailspike protein
Proteins, 2000Co-Authors: Benjamin Schuler, Stefan Steinbacher, Robert Huber, Frank Furst, Frank Osterroth, Robert SecklerAbstract:By means of genetic screens, a great number of mutations that affect the folding and stability of the tailspike protein from Salmo- nella phage P22 have been identified. Temperature- sensitive folding (tsf) mutations decrease folding yields at high temperature, but hardly affect ther- mal stability of the native trimeric structure when assembled at low temperature. Global suppressor (su) mutations mitigate this phenotype. Virtually all of these mutations are located in the central domain of tailspike, a large parallel b-Helix. We modified tailspike by rational single amino acid replace- ments at three sites in order to investigate the influence of mutations of two types: (1) mutations expected to cause a tsf phenotype by increasing the side-chain volume of a core residue, and (2) muta- tions in a similar structural context as two of the four known su mutations, which have been sug- gested to stabilize folding intermediates and the native structure by the release of backbone strain, an effect well known for residues that are primarily evolved for function and not for stability or folding of the protein. Analysis of folding yields, refolding kinetics and thermal denaturation kinetics in vitro show that the tsf phenotype can indeed be produced rationally by increasing the volume of side chains in the b-Helix core. The high-resolution crystal struc- ture of mutant T326F proves that structural rear- rangements only take place in the remarkably plas- tic lumen of the b-Helix, leaving the arrangement of the hydrogen-bonded backbone and thus the sur- face of the protein unaffected. This supports the notion that changes in the stability of an intermedi- ate, in which the b-Helix domain is largely formed, are the essential mechanism by which tsf mutations affect tailspike folding. A rational design of su mu- tants, on the other hand, appears to be more diffi- cult. The exchange of two residues in the active site expected to lead to a drastic release of steric strain neither enhanced the folding properties nor the stability of tailspike. Apparently, side-chain interac- tions in these cases overcompensate for backbone strain, illustrating the extreme optimization of the tailspike protein for conformational stability. The result exemplifies the view arising from the statisti- cal analysis of the distribution of backbone dihedral angles in known three-dimensional protein struc- tures that the adoption of f/c angles other than the most favorable ones is often caused by side-chain interactions. Proteins 2000;39:89 -101.
-
formation of fibrous aggregates from a non native intermediate the isolated p22 tailspike Beta Helix domain
Journal of Biological Chemistry, 1999Co-Authors: Benjamin Schuler, Reinhard Rachel, Robert SecklerAbstract:In the assembly pathway of the trimeric P22 tailspike protein, the protein conformation critical for the partitioning between productive folding and off-pathway aggregation is a monomeric folding intermediate. The central domain of tailspike, a large right-handed parallel Beta-Helix, is essentially structured in this species. We used the isolated Beta-Helix domain (Bhx), expressed with a hexahistidine tag, to investigate the mechanism of aggregation without the two terminal domains present in the complete protein. Although Bhx has been shown to fold reversibly at low ionic strength conditions, increased ionic strength induced aggregation with a maximum at urea concentrations corresponding to the midpoint of urea-induced folding transitions. According to size exclusion chromatography, aggregation appeared to proceed via a linear polymerization mechanism. Circular dichroism indicated a secondary structure content of the aggregates similar to that of the native state, but at the same time their tryptophan fluorescence was largely quenched. Microscopic analysis of the aggregates revealed a variety of morphologies; among others, fibrils with fine structure were observed that exhibited bright green birefringence if viewed under cross-polarized light after staining with Congo red. These observations, together with the effects of folding mutations on the aggregation process, indicate the involvement of a partially structured intermediate distinct from both unfolded and native Bhx.
-
p22 tailspike folding mutants revisited effects on the thermodynamic stability of the isolated Beta Helix domain
Journal of Molecular Biology, 1998Co-Authors: Benjamin Schuler, Robert SecklerAbstract:The folding of the trimeric phage P22 tailspike protein is influenced by amino acid substitutions of two types, virtually all of which affect residues in the central domain, a large parallel Beta-Helix. Temperature sensitive folding (tsf) mutations lead to drastically decreased folding yields at elevated temperature. Their phenotype can be alleviated by global suppressor (su) mutations. Both types of mutations appeared to have no influence on the stability of the native protein at the time of their first isolation and were thus suggested to carry information needed for the folding pathway exclusively. The monomeric Beta-Helix of tailspike, expressed as an isolated domain, exhibits freely reversible unfolding and refolding transitions, allowing us to analyse the effects of two well-characterised tsf and all four known su mutations on its thermodynamic stability. We find a marked decrease in stability for the tsf mutants and a striking increase in stability for all su mutants. This leads to the conception that the isolated Beta-Helix domain, although active in receptor-binding and native-like in its spectroscopic properties, is close in conformation to a crucial monomeric folding intermediate whose thermolability is responsible for the kinetic partitioning between productive folding and irreversible aggregation during the maturation process of P22 tailspike protein.
-
a reversibly unfolding fragment of p22 tailspike protein with native structure the isolated Beta Helix domain
Biochemistry, 1998Co-Authors: Stefan Miller, Benjamin Schuler, Robert SecklerAbstract:The homotrimeric tailspike endorhamnosidase of phage P22 has been used to compare in vivo and in vitro folding pathways and the influence of single amino acid substitutions thereon. Its main structural motif, which contains the known folding mutation sites, consists of three large right-handed parallel Beta-helices. A thermodynamic analysis of the stability of tailspike is prevented by the irreversibility of unfolding at high temperatures or high concentrations of denaturant, probably due to interdigitation of the domains neighboring the Beta-Helix. We therefore expressed and isolated a tailspike fragment comprising only its central Beta-Helix domain (residues 109-544). As shown by equilibrium ultracentrifugation, the isolated Beta-Helix is a monomer at concentrations below 1 microM and trimerizes reversibly at higher protein concentrations. Both the similarity of fluorescence and CD spectra, compared to the complete protein, and the specific binding and hydrolysis of substrate suggest a nativelike structure. Moreover, urea denaturation transitions of the Beta-Helix domain are freely reversible, providing the basis for a future quantitative analysis of the effects of the folding mutations on the thermodynamic stability of the domain and of structural features responsible for folding and stability of the parallel Beta-Helix motif in general.
-
a reversibly unfolding fragment of p22 tailspike protein with native structure the isolated Beta Helix domain
Biochemistry, 1998Co-Authors: Stefan Miller, Benjamin Schuler, Robert SecklerAbstract:The homotrimeric tailspike endorhamnosidase of phage P22 has been used to compare in vivo and in vitro folding pathways and the influence of single amino acid substitutions thereon. Its main structural motif, which contains the known folding mutation sites, consists of three large right-handed parallel ‚-helices. A thermodynamic analysis of the stability of tailspike is prevented by the irreversibility of unfolding at high temperatures or high concentrations of denaturant, probably due to interdigitation of the domains neighboring the ‚-Helix. We therefore expressed and isolated a tailspike fragment comprising only its central ‚-Helix domain (residues 109-544). As shown by equilibrium ultracentrifugation, the isolated ‚-Helix is a monomer at concentrations below 1 IM and trimerizes reversibly at higher protein concentrations. Both the similarity of fluorescence and CD spectra, compared to the complete protein, and the specific binding and hydrolysis of substrate suggest a nativelike structure. Moreover, urea denaturation transitions of the ‚-Helix domain are freely reversible, providing the basis for a future quantitative analysis of the effects of the folding mutations on the thermodynamic stability of the domain and of structural features responsible for folding and stability of the parallel ‚-Helix motif in general.
Bernardo Celda - One of the best experts on this subject based on the ideXlab platform.
-
conformational and structural analysis of the equilibrium between single and double strand Beta Helix of a d l alternating oligonorleucine
Biopolymers, 2004Co-Authors: E Navarro, E Fenude, Bernardo CeldaAbstract:Alternating sequences of D and L residues in peptides are directly related to the formation of several kinds of regular helical conformations usually called Beta-helices. The major feature of these structures is that they can be associated with the transmembrane ion-conducting channel activity in some natural antibacterial peptides. The study of alternating D,L synthetic peptides is critical to understand how factors such as surrounding media, main chain length, type of side chain and terminal groups, among others, can determine the adoption of a specific kind of Beta-Helix. Early studies pointed out that the peptides Boc-(D-NLeu-L-NLeu)(6)-D-MeNLe-L-Nl-D-Nl-L-Nl-OMe (Boc: tert-butyloxycarbonyl) and Boc-L-Nle-(D-Nle-L-Nle)(5)-D-MeNle-L-Nle-D-Nle-L-Nle-OMe adopt in chloroform a unique detectable conformation single Beta(4.4)- and double Beta(5.6) upward arrow downward arrow -Helix, respectively. The influence of terminal groups on the final stable conformation of N-formylated peptides has been studied in this work. The initial basic NMR data analysis of a synthetic alternating D,L-oligopeptide with ten norleucines, N-methylated on the residue 7 and having HCO- and -OMe as terminal groups clearly indicates the coexistence of two different conformations in equilibrium. NMR data and molecular dynamics calculations point to a dimeric antiparallel Beta-Helix structure Beta(5.6) upward arrow downward arrow for the main conformation. On the other hand, NMR data suggest a single Beta-Helix structure Beta(4.4) for the second conformation. Finally, a thermodynamic analysis of the equilibrium between both conformations has been carried out by one-dimensional NMR measurements at ten different temperatures. The temperature at which 50% of dimer conformation is dissociated is 319 K. In addition, the dimer-monomer equilibrium curve obtained shows a DeltaG>0 for the whole range of studied temperatures, and its behavior can be considered similar to the thermodynamic denaturation protein processes.
-
solution structure of a d l alternating oligonorleucine as a model of double stranded antiparallel Beta Helix
Biopolymers, 2002Co-Authors: E Navarro, E Fenude, Bernardo CeldaAbstract:Conformational characteristics of alternating D,L linear peptides are of particular interest because of their capacity to form transmembrane channels with different transport properties, as some natural antibiotics do. Single- and double-stranded Beta-helical structures are common for alternating D,L peptides. The stability of the Beta-Helix depends on several structural factors, such as the backbone peptide length, type and position of side chains, and nature of terminal groups. The NMR and molecular dynamics solution conformation of a synthetic alternating D,L-oligopeptide with 15 norleucines (XVMe) has been used as a model to get insight in to the conformational features of double-stranded Beta-Helix structures. The NH chemical shift values (delta(NH)) and long-range nuclear Overhauser effects (NOE) cross peaks, in particular interstrand connectivities, clearly point to an antiparallel double-stranded Beta-Helix for the XVMe major conformation in solution. An extensive set of distances (from NOE cross peaks) and H-bonds (from delta(NH)) has been included in the molecular dynamics calculations. The experimental NMR data and theoretical calculations clearly indicate that the most probable conformation of XVMe in solution is a double-strand antiparallel Beta(5.6) increasing decreasing-Helix structure.
-
solution nmr structure of a d l alternating oligonorleucine as a model of Beta Helix
Biopolymers, 2001Co-Authors: E Navarro, R Tejero, E Fenude, Bernardo CeldaAbstract:Beta-Helix structures are of particular interest due to their capacity to form transmembrane channels with different transport properties. However, the relatively large number of Beta-helices configurations does not allow a direct conformational analysis of Beta-helical oligopeptides. A synthetic alternating D,L-oligopeptide with twelve norleucines (XIIMe) has been used as a model to get insight in the conformational features of Beta-Helix structures. The spatial configuration of XIIMe in solution has been determined by NMR. An extensive set of distances (nuclear Overhauser effect) and dihedral (J coupling constants) constraints have been included in molecular dynamics calculations. The NMR experimental data and theoretical calculations clearly indicate that the XIIMe adopts a single Beta(4.4)-Helix-type conformation in nonpolar solvents.