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At least 1,027 records · Page 57Linked to original sources
PubMed1995

Complete 13C assignments for recombinant Cu(I) rusticyanin. Prediction of secondary structure from patterns of chemical shifts.

Complete resonance assignments for the 13C spectrum of reduced (Cu(I)) rusticyanin have been made using 13C, 15N doubly labeled recombinant material. The reported assignments include those for the carboxyl and carbonyl carbon atoms and protonated aromatic ring carbons, and were obtained using a variety of 2- and 3D inverse-detected NMR experiments, including 13C, 15N, 1H triple resonance experiments and HCCH-COSY and -TOCSY. Backbone carbonyl assignments were obtained using 3D HNCO and HCACO spectra, and modified versions of 2D H(CA)CO and HMBC spectra were used to obtain side-chain carboxyl carbon and methionine epsilon-methyl carbon assignments, respectively. A comparison of the 13C alpha, 13C beta and 13CO chemical shifts with published 'random coil' values confirms the conclusion reached from a consideration of the 3JHN alpha coupling constants and the pattern of sequential NOEs, that the protein consists largely of beta-structure.

Amino Acid Sequence↗
PubMed1993

Two-dimensional nuclear magnetic resonance study of the beta-casein peptide 1-25: resonance assignments and secondary structure.

The N-terminal 25 amino-acid residue peptide (beta-CN 1-25) obtained from a tryptic digest of bovine beta-casein was studied using two-dimensional NMR techniques. Complete sequence-specific assignment of the 1H-NMR spectrum was performed both in the presence and absence of 22 mM Ca2+. The NMR data supported earlier findings that this segment of beta-casein is highly flexible and adopts multiple conformations. The observed pattern of sequential NOEs indicated that the peptide did not contain stable folded structures. However, the structure was neither that of a fully-extended peptide nor a so-called random coil. The region Ile-12 to SerP-15 showed an enhanced population of extended structure. Furthermore, addition of Ca2+ ions induced chemical-shift changes and apparently decreased the population of conformations with extended structure in the region SerP-18 to Ile-23.

Amino Acid Sequence↗
PubMed1994

Purification and secondary structural analysis of tissue inhibitor of metalloproteinases-1.

In connective tissue diseases such as rheumatoid arthritis, the matrix metalloproteinases are the primary enzymes involved in tissue degradation. Tissue inhibitor metalloproteinases-1 (TIMP-1) is a specific inhibitor of these enzymes, which is thought to regulate their action in vivo. The structure and function of TIMP-1 may therefore be important as the basis for the rational design of therapeutic agents. This paper describes a simple and effective method for the purification of sufficient quantities of TIMP-1 for spectroscopic studies. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy have, together, showed TIMP-1 to be mostly in a beta-sheet conformation, with significant amounts of alpha-helix and beta-turn. Two-dimensional nuclear magnetic resonance spectroscopy indicated a correspondingly high proportion of beta-sheet. CD and FTIR have also shown TIMP-1 to have high thermostability.

Circular Dichroism↗
PubMed1995

The secondary structure of the insect defensin A depends on its environment. A circular dichroism study.

Defensin A is an inducible antibacterial protein isolated from the larvae of Phormia terranovae. The conformation of defensin A has been previously determined by two-dimensional 1H-NMR for concentrations in the range of 4-8 mM in water (Bonmatin JM et al (1992) J Biomol NMR 2, 235-256). CD spectroscopic data of defensin A at lower concentrations (10(-5) to 10(-3) M) are reported herein. The ellipticity in the 200-240 nm wavelength range for various solvents varies as follows: acetonitrile < water < methanol < HFIP. The magnitude of theta 222 is strongly dependent on defensin concentration in a buffer solution, suggesting an aggregation process. The helical content of defensin A is maximum at a pH value range (7.5-8) for which the optimum antibacterial activity was observed (Cociancich S et al (1993) J Biol Chem 268, 19239-19245).

Acetonitriles↗
PubMed2004

Codon optimization reveals critical factors for high level expression of two rare codon genes in Escherichia coli: RNA stability and secondary structure but not tRNA abundance.

Expression patterns in Escherichia coli of two small archaeal proteins with a natural content of about 30% rare codons were analyzed. The proteins, a histone-like protein from Sulfolobus shibatae (Ssh10), and a glutaredoxin-like protein from Methanobacterium thermoautotrophicum (mtGrx), were produced with expression plasmids encoding wild-type genes, codon-optimized synthetic, and GST-fusion genes. These constructs were expressed in BL21 (DE3), its LysS derivative, and modified strains carrying copies for rare codon tRNAs or deletions in the RNAseE gene. Both Ssh10 and mtGrx expression levels were constitutively high in BL21(DE3) and its derivatives, with the exception of the LysS phenotype, which prevented high level expression of the Ssh10 wild-type gene. Surprisingly, a codon-optimized mtGrx gene construct displayed undetectable levels of protein production. The translational block observed with the synthetic mtGrx gene could be circumvented by using a synthetic mtGrx-glutathione S-transferase (GST) fusion construct or by in vitro translation. Taken together, the results underscore the importance of mRNA levels and RNA stability, but not necessarily tRNA abundance for efficient heterologous protein production in E. coli.

Archaeal Proteins↗
PubMed2005

The influence of phospholipid membranes on bovine calcitonin peptide's secondary structure and induced neurotoxic effects.

The peptide hormone, calcitonin, which is associated with medullary carcinoma of the thyroid, has a marked tendency to form amyloid fibrils and may be a useful model in probing the role of peptide-membrane interactions in beta-sheet and amyloid formation and amyloid neurotoxicity. Using bovine calcitonin, we found that, like other amyloids, the peptide was toxic only when in a beta-sheet-rich, amyloid form, but was non-toxic, when it lacked an amyloid structure. We found that the peptide bound with significant affinity to membranes that contained either cholesterol and gangliosides. In addition, incubation of calcitonin with cholesterol-rich and ganglioside-containing membranes resulted in significant changes in peptide structure yielding a peptide enriched in beta-sheet and amyloid content. Because the cholesterol- and ganglioside-rich phospholipid systems enhanced the calcitonin beta-sheet and amyloid contents, and peptide amyloid content was associated with neurotoxicity, we then investigated whether depleting cellular cholesterol and gangliosides affected calcitonin neurotoxicity. We found that cholesterol and ganglioside removal significantly reduced the calcitonin-induced PC12 cell neurotoxicity. Similar results have been observed with other amyloid-forming peptides such as beta-amyloid (A beta) of Alzheimer's disease and suggest that modulation of membrane composition and peptide-membrane interactions may prove useful in the control of amyloid formation and amyloid neurotoxicity.

Animals↗
PubMed2006

Effect of RGD secondary structure and the synergy site PHSRN on cell adhesion, spreading and specific integrin engagement.

The relationship between the form of cell adhesion, ligand presentation, and cell receptor function was characterized using model Langmuir-Blodgett supported films, containing lipid-conjugated peptide ligands, in which isolated variables of the ligand presentation were systematically altered. First, the conformation of an adhesive Arginine-Glycine-Aspartic acid (RGD) peptide was varied by synthesizing linear and looped RGD peptide-containing amphiphiles and subsequently measuring the impact on the function of human umbilical vein endothelial cells. Secondly, the contribution of non-contiguous ligands to cellular engagement was assessed using multi-component biomimetic films. The peptide amphiphiles were composed of fibronectin-derived headgroups--GRGDSP, and its synergy site Pro-His-Ser-Arg-Asn (PHSRN)--attached to hydrocarbon tails. The peptide amphiphiles were diluted using polyethylene glycol (PEG) amphiphiles, where PEG inhibited non-specific cell adhesion. Cells adhered and spread on GRGDSP/PEG systems in a dose-dependent manner. The presentation of GRGDSP influenced integrin cell surface receptor specificity. Results demonstrated that beta1-containing integrins mediated adhesion to the linear GRGDSP presentation to a greater extent than did the alphavbeta3 integrin, and looped GRGDSP preferentially engaged alphavbeta3. GRGDSP/PHSRN/PEG mixtures that closely mimicked the RGD-PHSRN distance in fibronectin, enhanced cell spreading over their two-component analogues. This study demonstrated that controlling the microenvironment of the cell was essential for biomimetics to modulate specific binding and subsequent signaling events.

Amino Acid Sequence↗
PubMed2006

PSSARD (2.0): a database server for making flexible queries relating amino acid sequences to main-chain secondary structure conformations for proteins of known three-dimensional structure and certain useful applications.

We have updated the Protein Sequence-Structure Analysis Relational Database (PSSARD) first published in the Int. J. Biol. Macromol. 36 (2005) 259-262 corresponding to 1573 representative protein chains selected from the Protein Data Bank (PDB). In this, the updated and revised PSSARD (Version 2.0), we have included all proteins in the Protein Data Bank available at the time of developing this database including the NMR PDB entries. The current database corresponds to 22,752 XRAY PDB entries and 3977 NMR PDB entries and is separated accordingly in order to facilitate the appropriate database search. The representative protein chains can also be separately accessed within the current database. We have made a provision to combine more than one field to query the database and the results of any search can be used to carry out further nested searches using a combination of queries. We have provided hyperlinks to the individual PDB entries obtained as the result of any search in PSSARD in order to obtain additional details relevant to the protein structure. Certain applications useful to identify domains and structural motifs are discussed.

Amino Acid Sequence↗
PubMed2004

Mapping abeta amyloid fibril secondary structure using scanning proline mutagenesis.

Although the amyloid fibrils formed from the Alzheimer's disease amyloid peptide Abeta are rich in cross-beta sheet, the peptide likely also exhibits turn and unstructured regions when it becomes incorporated into amyloid. We generated a series of single-proline replacement mutants of Abeta(1-40) and determined the thermodynamic stabilities of amyloid fibrils formed from these mutants to characterize the susceptibility of different residue positions of the Abeta sequence to proline substitution. The results suggest that the Abeta peptide, when engaged in the amyloid fibril, folds into a conformation containing three highly structured segments, consisting of contiguous sequence elements 15-21, 24-28, and 31-36, that are sensitive to proline replacement and likely to include the beta-sheet portions of the fibrils. Residues relatively insensitive to proline replacement fall into two groups: (a) residues 1-14 and 37-40 are likely to exist in relatively unstructured, flexible elements extruded from the beta-sheet-rich amyloid core; (b) residues 22, 23, 29 and 30 are likely to occupy turn positions between these three structured elements. Although destabilized, fibrils formed from Abeta(1-40) proline mutants are very similar in structure to wild-type fibrils, as indicated by hydrogen-deuterium exchange and other analysis. Interestingly, however, some proline mutations destabilize fibrils while at the same time increasing the number of amide protons protected from hydrogen exchange. This suggests that the stability of amyloid fibrils, rather than being driven exclusively by the formation of H-bonded beta-sheet, is achieved, as in globular proteins, through a balance of stabilizing and destabilizing forces. The proline scanning data are most compatible with a model for amyloid protofilament structure loosely resembling the parallel beta-helix folding motif, such that each Abeta(15-36) core region occupies a single layer of a prismatic, H-bonded stack of peptides.

Amino Acid Substitution↗
PubMed2002

Folding, stability, and secondary structure of a new dimeric cysteine proteinase inhibitor.

Clitocypin, a new type of cysteine proteinase inhibitor from the mushroom Clitocybe nebularis, is a 34-kDa homodimer lacking disulphide bonds, reported to have unusual stability properties. Sequence similarity is limited solely to certain proteins from mushrooms. Infrared spectroscopy shows that clitocypin is a high beta-structure protein which was lost at high temperatures. The far UV circular dichroism spectrum is not that of classical beta-structure, but similar to those of a group of small beta-strand proteins, with a peak at 189nm and a trough at 202nm. An aromatic peak at 232nm and infrared bands at 1633 and 1515cm(-1) associated with the peptide backbone and the tyrosine microenvironment, respectively, were used to characterize the thermal unfolding. The reversible transition has a midpoint at 67 degrees C, with DeltaG=34kJ/mol and DeltaH=300kJ/mol, and is, unusually, independent of protein concentration. The kinetics of thermal unfolding and refolding are slow, with activation energies of 167 and 44kJ/mol, respectively. A model for folding and assembly is discussed.

Agaricales↗
PubMed1999

Effects of the binding of a dextran derivative on fibroblast growth factor 2: secondary structure and receptor-binding studies.

CMDB (carboxymethyldextran-benzylamide) are dextrans statistically substituted with carboxymethyl and benzylamide groups which can mimick some of the biological properties of heparin. It has previously been shown that CMDB inhibit autocrine growth of breast tumor cells (Bagheri-Yarmand et al., Biochem. Biophys. Res. Commun. 239: 424-428, 1997) and selectively displace fibroblast growth factor 2 (FGF-2) from its receptor. Here, we used circular dichroism and fluorescence anisotropy measurements to show that the conformation of FGF-2 was significantly altered upon its binding to CMDB and to short CMDB fragments prepared within this study. CMDB and fragments formed a stable 1:1 complex with FGF-2, with affinities being estimated as 20+/-10 nM from fluorescence anisotropy analysis. No such a complex was formed with insulin-like growth factor (IGF-1) or epidermal growth factor (EGF). CMDB competed with the FGF-2 receptor for binding to FGF-2 but did not disturb the binding of IGF-1 and EGF to their receptors. Thus, our results highlight the selectivity of CMDB and their fragments towards FGF-2. Heparin, however, competes with CMDB and their fragments for binding to FGF-2. The carboxymethyl and benzylamide groups of these molecules likely interact directly with a heparin-binding region of FGF-2. The resulting change in conformation disturbs the binding of FGF-2 to its receptor and consecutively its mitogenic activity.

Binding, Competitive↗
PubMed1999

1H-NMR and circular dichroism spectroscopic studies on changes in secondary structures of the sodium channel inactivation gate peptides as caused by the pentapeptide KIFMK.

The pentapeptide KIFMK, which contains three clustered hydrophobic amino acid residues of isoleucine, phenylalanine, and methionine (IFM) in the sodium channel inactivation gate on the cytoplasmic linker between domains III and IV (III-IV linker), is known to restore fast inactivation to the mutant sodium channels having a defective inactivation gate or to accelerate the inactivation of the wild-type sodium channels. To investigate the docking site of KIFMK and to clarify the mechanisms for restoring the fast inactivation, we have studied the interactions between KIFMK and the fragment peptide in the III-IV linker GGQDIFMTEEQK (MP-1A; G1484-K1495 in rat brain IIA) by one- and two-dimensional (1)H-NMR and circular dichroism (CD) spectroscopies. KIFMK was found to increase the helical content of MP-1A in 80% trifluoroethanol (TFE) solution by approximately 11%. A pentapeptide, KIFMT, which can restore inactivation but less effectively than KIFMK, also increased the helical content of MP-1A, but to a lesser extent ( approximately 6%) than did KIFMK. In contrast, KDIFMTK, which is ineffective in restoring inactivation, decreased the helical content ( approximately -4%). Furthermore, we studied the interactions between KIFMK and modified peptides from MP-1A, that is, MP-1NA (D1487N), MP-1QEA (E1492Q), or MP-1EQA (E1493Q). The KIFMK was found to increase the helical content of MP-1EQA to an extent nearly identical to that of MP-1A, whereas it was found to decrease those of MP-1NA and MP-1QEA. These findings mean that KIFMK, by allowing each of the Lys residues to interact with D1487 and E1492, respectively, stabilized the helical structure of the III-IV linker around the IFM residues. This helix-stabilizing effect of KIFMK on the III-IV linker may restore and/or accelerate fast inactivation to the sodium channels having a defective inactivation gate or to wild-type sodium channels.

Amino Acid Sequence↗
PubMed2001

Secondary structure of human apolipoprotein A-I(1-186) in lipid-mimetic solution.

The solution structure of an apoA-I deletion mutant, apoA-I(1-186) was determined by the chemical shift index (CSI) method and the torsion angle likelihood obtained from shift and sequence similarity (TALOS) method, using heteronuclear multidimensional NMR spectra of [u-(13)C, u-(15)N, u-50% (2)H]apoA-I(1-186) in the presence of sodium dodecyl sulfate (SDS). The backbone resonances were assigned from a combination of triple-resonance data (HNCO, HNCA, HN(CO)CA, HN(CA)CO and HN(COCA)HA), and intraresidue and sequential NOEs (three-dimensional (3D) and four-dimensional (4D) 13C- and 15N-edited NOESY). Analysis of the NOEs, H(alpha), C(alpha) and C' chemical shifts shows that apoA-I(1-186) in lipid-mimetic solution is composed of alpha-helices (which include the residues 8-32, 45-64, 67-77, 83-87, 90-97, 100-140, 146-162, and 166-181), interrupted by short irregular segments. There is one relatively long, irregular and mostly flexible region (residues 33-44), that separates the N-terminal domain (residues 1-32) from the main body of protein. In addition, we report, for the first time, the structure of the N-terminal domain of apoA-I in a lipid-mimetic environment. Its structure (alpha-helix 8-32 and flexible linker 33-44) would suggest that this domain is structurally, and possibly functionally, separated from the other part of the molecule.

Apolipoprotein A-I↗
PubMed2002

Use of molecular mechanics for secondary structure prediction. Is it possible to reveal alpha-helix?

A new approach to predicting protein standard conformations is suggested. The idea consists in modeling by molecular mechanics tools a continuous alpha-helical conformation for the whole protein. The profile of energy along the model alpha-helix reveals minima corresponding to real alpha-helical segments in the native protein. The 3/10-helices and beta-turns including a local alpha-helical conformation may be detected as well. All alpha-helical segments in the test sample are delineated; mean residue by residue accuracy Q(3alpha) is 79%. This non-statistical approach can shed light on the physical grounds of alpha-helix formation.

Carrier Proteins↗
PubMed1997

Sequential assignments and secondary structure of the RNA-binding transcriptional regulator NusB.

The NusB protein is involved in transcriptional regulation in bacteriophage lambda. NusB binds to the RNA form of the nut site and along with N, NusA, NusE and NusG, stabilizes the RNA polymerase transcription complex and allows stable, persistent antitermination. NusB contains a 10 residue Arg-rich RNA-binding motif (ARM) at the N-terminus but is not sequentially homologous to any other proteins. In contrast to other known ARM-containing proteins, NusB forms a stable structure in solution in the absence of RNA. NMR spectroscopy was used to determine that NusB contains six alpha-helices: R10-Q21, 127-F34, V45-L65, Q79-S93, Y100-F114 and D118-L127. The structure of NusB makes it a member of a newly emerging class of alpha-helical RNA-binding proteins.

Amino Acid Sequence↗
PubMed1998

Secondary structure and limited proteolysis give experimental evidence that the precursor of pulmonary surfactant protein B contains three saposin-like domains.

The 42 kDa precursor of surfactant protein B generates the 79 residue mature SP-B polypeptide, which belongs to the family of saposin-like proteins and has unique functional roles in pulmonary surfactant. From sequence comparisons it has been suggested that proSP-B, in addition to SP-B, contains two saposin-like domains, but their existence has until now not been experimentally verified. The 381 residue human proSP-B was now fused to an N-terminal poly-His tag, expressed in Escherichia coli, and purified from inclusion bodies by resolubilisation with 2.5% (w/v) SDS and, after removal of SDS, subsequent metal affinity chromatography. Recombinant proSP-B thus obtained exhibits about 35% alpha-helical structure in sodium phosphate buffer and is proteolytically cleaved preferentially between the three saposin-like domains. These results experimentally support that proSP contains, in addition to SP-B, two further saposin-like domains.

Animals↗