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At least 883 records · Page 49Linked to original sources
PubMed2002

Synthesis, aggregation, neurotoxicity, and secondary structure of various A beta 1-42 mutants of familial Alzheimer's disease at positions 21-23.

Cerebral amyloid angiopathy (CAA) due to amyloid beta (A beta) deposition is a key pathological feature of Alzheimer's disease (AD), especially in some form of familial Alzheimer's disease (FAD) including hereditary cerebral hemorrhage with amyloidosis-Dutch type. A beta mainly consists of 40- and 42-mer peptides (Abeta 1-40 and A beta 1-42), which accumulate in senile plaques of AD brains and show neurotoxicity for cultured nerve cells. We synthesized all variant forms of A beta 1-42 associated with reported FAD, such as A21G (Flemish), E22Q (Dutch), E22K (Italian), E22G (Arctic), and D23N (Iowa) along with three potential mutants by one point missense mutation (E22A, E22D, and E22V) in a highly pure form, and examined their ability to aggregate and their neurotoxicity in PC12 cells. The mutants at positions 22 and 23 showed potent aggregative ability and neurotoxicity whereas the potential mutants did not, indicating that A beta 1-42 mutants at positions 22 and 23 play a critical role in FAD of Dutch-, Italian-, Arctic-, and Iowa-types. However, Flemish-type FAD needs alternative explanation except the aggregation and neurotoxicity of the corresponding A beta 1-42 mutant.

Alzheimer Disease↗
PubMed2002

The characterization of biomolecular secondary structures by surface plasmon resonance.

Recently there has been considerable interest in using surface plasmon resonance (SPR) for the measurement of conformational changes of immobilized biomolecules that are induced by an exogenous analyte. While a number of studies have shown the analytical utility of such measurements, there has been no report which characterizes the specific secondary structure that actuates the change in SPR signal. The use of SPR to indicate the type of secondary structure present in two immobilized polypeptides, poly-L-lysine (PL) and poly-L-glutamic acid (PGA), and a globular protein, concanavalin A (Con A) is described in this report. The PL, PGA and Con A were modified with N-succinimidyl 3-(2-pyridyldithiol) propionate (SPDP) to introduce disulfide groups to facilitate the attachment onto gold-coated surfaces via self-assembly. Ethanol and 2,2,2-trifluoroethanol (TFE) were used to induce changes in the secondary structure of the immobilized polypeptides and the protein respectively. Using both circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies, it has been demonstrated that it is possible to correlate the signal changes observed in SPR to the secondary conformation of the biomolecule. Both CD and FTIR showed that a decrease in SPR signal corresponded to a high content of beta, turn or unordered structures while an increase corresponded to a high alpha-helical content. The sensitivity of the SPR technique is comparable to that obtained in solution with CD and FTIR spectroscopies. These results are the first demonstration that SPR can be used to characterize secondary structures. There is potential, therefore, for SPR to be used as a technique to study secondary conformational changes of immobilized polypeptides and proteins.

Circular Dichroism↗
PubMed1995

1H and 15N assignment of NMR spectrum, secondary structure and global folding of the immunophilin-like domain of the 59-kDa FK506-binding protein.

FKBP59, a 59-kDa FK506 binding protein, was discovered in heterooligomeric complexes containing nontransformed, non-DNA binding, steroid receptors. Sequence similarity search and secondary structure prediction suggested that the protein has a multi-domain organization, the N-terminal domain having a great similarity to human FKBP12 (12-kDa FK506-binding protein). FKBP59 binds immunosuppressant FK506 and has peptidylprolyl cis-trans-isomerase activity, both properties being localized in the N-terminal domain (FKBP59-I). In order to characterize its conformational features and to better understand its biological significance, we overexpressed and 15N-labeled this domain (149 amino acids) in Escherichia coli and initiated an NMR structural study in solution. Almost complete sequence-specific assignment of the 1H and 15N resonances was achieved using two-dimensional and three-dimensional homonuclear and heteronuclear experiments. Localization of the secondary structure elements was derived essentially from C alpha H chemical shift distribution along the sequence, the short-range and medium-range NOE connectivities and exchange kinetics of amide protons. The domain has a structured part comprising six beta-strands and a three-turn alpha-helix between K87 and M96. The first 17 residues are highly flexible and show no regular secondary structure. The beta-sheet structure, derived from long-range connectivities between backbone protons, consists of six beta-strands defined as follows: B1, V22-I24; B2, V32-K37; B3, D50-L61; B4, T64-S68 and F76-L80; B5, E100-K107; B6, L127-F137. They are organized in an antiparallel beta-sheet with the connecting topology +1, +3, +1, -3, +1. The alpha-helix connects strand B4 to strand B5. Globally, the structure of FKBP59-I, derived from the present work, is similar to the NMR-derived structures of uncomplexed FKBP12. However, several conformational differences were noted at this level of structural analysis. The beta-sheet of the FKBP59 domain has an additional strand at the N-terminal and the alpha-helix is longer by about one helical turn. In addition, strand B4 has two components, separated by a large bulge (seven residues); the first component was observed in the X-ray or NMR structures of complexed FKBP12 but not in the NMR-derived, uncomplexed structure.

Amino Acid Sequence↗
PubMed1987

The involvement of mRNA secondary structure in protein synthesis.

Translation initiation in eukaryotes is a complex process involving many factors. A key step in this process is the binding of mRNA to the 43S preinitiation complex. This is generally the rate-limiting step in translation initiation and consequently a major determinant of mRNA translational efficiency. The primary and secondary structure of the mRNA 5' noncoding region have been implicated in modulating translational efficiency. Translational efficiency was shown to be inversely proportional to the degree of secondary structure at the mRNA 5' noncoding region. Furthermore, it was shown that cap-binding proteins that interact with the 5' cap structure (m7GpppN) of eukaryotic mRNAs are involved in the "unwinding" of the mRNA secondary structure, in an ATP hydrolysis mediated event, to facilitate ribosome binding. Thus, cap-binding proteins can potentially regulate mRNA translation. Here, we discuss the available data supporting the notion that eukaryotic 5' mRNA secondary structure plays an important role in translation initiation and the possible regulation of this process.

Gene Expression Regulation↗
PubMed2000

Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set.

We have expanded the reference set of proteins used in SELCON3 by including 11 additional proteins (selected from the reference sets of Yang and co-workers and Keiderling and co-workers). Depending on the wavelength range and whether or not denatured proteins are included in the reference set, five reference sets were constructed with the number of reference proteins varying from 29 to 48. The performance of three popular methods for estimating protein secondary structure fractions from CD spectra (implemented in software packages CONTIN, SELCON3, and CDSSTR) and a variant of CONTIN, CONTIN/LL, that incorporates the variable selection method in the locally linearized model in CONTIN, were examined using the five reference sets described here, and a 22-protein reference set. Secondary structure assignments from DSSP were used in the analysis. The performances of all three methods were comparable, in spite of the differences in the algorithms used in the three software packages. While CDSSTR performed the best with a smaller reference set and larger wavelength range, and CONTIN/LL performed the best with a larger reference set and smaller wavelength range, the performances for individual secondary structures were mixed. Analyzing protein CD spectra using all three methods should improve the reliability of predicted secondary structural fractions. The three programs are provided in CDPro software package and have been modified for easier use with the different reference sets described in this paper. CDPro software is available at the website: http://lamar.colostate.edu/ approximately sreeram/CDPro.

Circular Dichroism↗
PubMed1987

Secondary structure of the hydrophobic myelin protein in a lipid environment as determined by Fourier-transform infrared spectrometry.

The secondary structure of a hydrophobic myelin protein (lipophilin), reconstituted with dimyristoylphosphatidylcholine or dimyristoylphosphatidylglycerol, was investigated by Fourier-transform infrared spectroscopy. Protein infrared spectra in the amide I region were analyzed quantitatively using resolution enhancement and band fitting procedures. Lipophilin in a phospholipid environment adopts a highly ordered secondary structure which at room temperature consists predominantly of alpha-helix (approximately 55%) and beta-type conformations (36%). The secondary structure of the protein is not affected by the lipid gel to liquid crystalline phase transition. Heating of the lipid-protein complex above approximately 35 degrees C results in a gradual decrease in alpha-helical content, accompanied by an increase in the amount of beta-structures. Lipophilin dissolved in 2-chloroethanol is, compared to the protein in a lipid environment, richer in the alpha-helical conformation but still contains a sizable amount of beta-structure.

Dimyristoylphosphatidylcholine↗
PubMed1993

A deductive database system PACADE for analyzing 3-D and secondary structures of protein.

We have developed a deductive database system PACADE for analyzing 3-D and secondary structures of protein. The PACADE system consists of a relational database created from Protein Data Bank and a deductive engine DEE based on logic programming. It has the following features: (1) The system has an inference mechanism. This means by which users can easily write and check biological hypotheses using logical and declarative rules instead of procedural programs. (2) The relational database of the PACADE system stores data on both 3-D and secondary structures of protein. The integration of this two level structure makes feasible an abstract representation of the protein structure. We describe herein the design, functions, and implementation of this PACADE system.

Computer Graphics↗
PubMed1975

Method for predicting RNA secondary structure.

We report a method for predicting the most stable secondary structure of RNA from its primary sequence of nucleotides. The technique consists of a series of three computer programs interfaced to take the nucleotide sequence of any RNA and (a) list all possible helical regions, using modified Watson-Crick base-pairing rules; (b) create all possible secondary structures by forming permutations of compatible helical regions; and (c)evaluate each structure for total free energy of formation from a completely extended chain. A free energy distribution and the base-by-base bonding interactions of each possible structure are catalogued by the system and are readily available for examination. The method has been applied to 62 tRNA sequences. The total free-energy of the predicted most stable structures ranged from -19 to -41 kcal/mole (-22 to -49 kJ/mole). The number of structures created was also highly sequence-dependent and ranged from 200 to 13,000. In nearly all cases the cloverleaf is predicted to be the structure with the lowest free energy of formation.

Base Sequence↗
PubMed1992

A native RNA secondary structure controls alternative splice-site selection and generates two human growth hormone isoforms.

Consensus sequences at the splice donor, splice acceptor, and lariat branch point regions are necessary but insufficient determinants of splice-site selection in nuclear precursor mRNAs. Sequences outside of these regions can have a significant effect on the utilization of splice sites. Although the mode of action of such sequences is undefined in most cases, higher order RNA structures have been suggested as a potential contributor to splice-site selection. During a detailed analysis of the splicing patterns of the human growth hormone transcript, we located 2 bases in the vicinity of the exon 3 major splice-acceptor site (B) which facilitate the utilization of a competing downstream acceptor (B'). The effects of a series of site-specific mutations on the splicing pattern demonstrate that these 2 bases function by stabilizing a specific stem-loop structure in the native transcript. This defined secondary structure selectively encompasses the upstream B splice-acceptor site together with its lariat branch point region. Increasing the predicted stability of this stem by point mutations results in a corresponding shift in splicing towards the alternative B' splice-acceptor site. These results indicate that a specific secondary structure within the native human growth hormone transcript controls the relative utilization of two competing splice-acceptor sites with the consequent generation of two functionally distinct hormone isoforms.

Animals↗
PubMed2006

Efficient characterization of protein secondary structure in terms of screw motions.

A simple and efficient method is presented to describe the secondary structure of proteins in terms of orientational distances between consecutive peptide planes and local helix parameters. The method uses quaternion-based superposition fits of the protein peptide planes in conjunction with Chasles' theorem, which states that any rigid-body displacement can be described by a screw motion. The helix parameters are derived from the best superposition of consecutive peptide planes and the ;worst' fit is used to define the orientational distance. Applications are shown for standard secondary-structure motifs of peptide chains for several proteins belonging to different fold classes and for a description of structural changes in lysozyme under hydrostatic pressure. In the latter case, published reference data obtained by X-ray crystallography and by structural NMR measurements are used.

Algorithms↗
PubMed1988

Secondary structure prediction and determination of proteins--a review.

The rapid increase in sequence data in combination with a greater understanding of the forces regulating protein structure has been the impetus for an upsurge in the development of theoretical prediction methods. These methods have afforded protein chemists the ability to identify and quantify the various secondary structures along the protein chain. Concurrently, various physico-chemical techniques have been developed such as nuclear Overhauser enhancement n.m.r. and laser Raman spectroscopy. In addition, traditional methods such as infrared and circular dichroism spectroscopy have been refined. Although both predictive and physico-chemical techniques are limited in the types of secondary structure they are capable of determining, they have provided valuable information with regards to protein folding and topology in the absence of X-ray data, and have formed the basis for the development of improved methods for secondary structure determination. This paper reviews some of the predictive and physico-chemical methods presently used to determine protein secondary structure.

Chemical Phenomena↗
PubMed1995

Solution secondary structure of calcium-saturated troponin C monomer determined by multidimensional heteronuclear NMR spectroscopy.

The solution secondary structure of calcium-saturated skeletal troponin C (TnC) in the presence of 15% (v/v) trifluoroethanol (TFE), which has been shown to exist predominantly as a monomer (Slupsky CM, Kay CM, Reinach FC, Smillie LB, Sykes BD, 1995, Biochemistry 34, forthcoming), has been investigated using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. The 1H, 15N, and 13C NMR chemical shift values for TnC in the presence of TFE are very similar to values obtained for calcium-saturated NTnC (residues 1-90 of skeletal TnC), calmodulin, and synthetic peptide homodimers. Moreover, the secondary structure elements of TnC are virtually identical to those obtained for calcium-saturated NTnC, calmodulin, and the synthetic peptide homodimers, suggesting that 15% (v/v) TFE minimally perturbs the secondary and tertiary structure of this stably folded protein. Comparison of the solution structure of calcium-saturated TnC with the X-ray crystal structure of half-saturated TnC reveals differences in the phi/psi angles of residue Glu 41 and in the linker between the two domains. Glu 41 has irregular phi/psi angles in the crystal structure, producing a kink in the B helix, whereas in calcium-saturated TnC, Glu 41 has helical phi/psi angles, resulting in a straight B helix. The linker between the N and C domains of calcium-saturated TnC is flexible in the solution structure.

Amino Acid Sequence↗
PubMed2005

Resonance assignments and secondary structure analysis of E. coli thioredoxin by magic angle spinning solid-state NMR spectroscopy.

De novo site-specific 13C and 15N backbone and sidechain resonance assignments are presented for uniformly enriched E. coli thioredoxin, established using two-dimensional homo- and heteronuclear solid-state magic angle spinning NMR correlation spectroscopy. Backbone dihedral angles and secondary structure were derived from the statistical analysis of the secondary chemical shifts, and are in good agreement with solution values for the intact full-length thioredoxin, with the exception of a small number of residues located at the termini of the individual secondary structure elements. A large number of cross-peaks observed in the DARR spectra with long mixing times correspond to the pairs of carbon atoms separated by 4-6 angstroms, suggesting that DARR could be efficiently employed for observation of medium- and long-range correlations. The 108 amino acid residue E. coli thioredoxin is the largest uniformly enriched protein assigned to this degree of completeness by solid-state NMR spectroscopy to date. It is anticipated that with a combination of two-dimensional correlation experiments and high magnetic fields, resonance assignments and secondary structure can be generally derived for other noncrystalline proteins.

Carbon Isotopes↗
PubMed2002

Single-pass attenuated total reflection Fourier transform infrared spectroscopy for the prediction of protein secondary structure.

Principal component regression (PCR) was applied to a spectral library of proteins in H2O solution acquired by single-pass attenuated total reflectance (ATR) Fourier transform infrared (FT-IR) spectroscopy. PCR was used to predict the secondary structure content, principally alpha-helical and the beta-sheet content, of proteins within a spectral library. Quantitation of protein secondary structure content was performed as a proof of principle that use of single-pass ATR-FT-IR is an appropriate method for protein secondary structure analysis. The ATR-FT-IR method permits acquisition of the entire spectral range from 700 to 3900 cm(-1) without significant interference from water bands. An "inside model space" bootstrap and a genetic algorithm (GA) were used to improve prediction results. Specifically, the bootstrap was utilized to increase the number of replicates for adequate training and validation of the PCR model. The GA was used to optimize PCR parameters, particularly wavenumber selection. The use of the bootstrap allowed for adequate representation of variability in the amide A, amide B, and C-H stretching regions due to differing levels of sample hydration. Implementation of the bootstrap improved the robustness of the PCR models significantly; however, the use of a GA only slightly improved prediction results. Two spectral libraries are presented where one was better suited for beta-sheet content prediction and the other for alpha-helix content prediction. The GA-optimized PCR method for alpha-helix content prediction utilized 120 wavenumbers within the amide I, II, A, B, and IV and the C-H stretching regions and 18 factors. For beta-sheet content predictions, 580 wavenumbers within the amide I, II, A, and B and the C-H stretching regions and 18 factors were used. The validation results using these two methods yielded an average absolute error of 1.7% for alpha-helix content prediction and an average absolute error of 2.3% for beta-sheet content prediction. After the PCR models were developed and validated, they were used to predict the alpha-helix and beta-sheet content of two unknowns, casein and immunoglobulin G.

Multivariate Analysis↗
PubMed1998

Accuracy of secondary structure and solvent accessibility predictions for a clostridial neurotoxin C-fragment.

Earlier studies used Rost and Sander's artificial neural network [(1993a), J. Mol. Biol. 232, 584-599] to predict the secondary structures [Lebeda and Olson (1994), Proteins 20, 293-300] and residue solvent accessibilities [Lebeda and Olson (1997), J. Protein Chem. 16, 607-618] of the clostridial neurotoxins. Because the X-ray crystal structure of the 50-kDa C-terminal half of the heavy chain of tetanus toxin was recently determined, this report evaluates the accuracy of these network-derived predictions. For this predominantly beta-strand-containing fragment, predictions, on a per-residue basis, for both secondary structure and solvent accessibility were about 70% accurate. A more flexible and realistic analysis based on overlapping segments yielded accuracies of over 80% for the three-state secondary structure and for the two-state accessibility predictions. Because the accuracies of these predictions are comparable to those made by Rost and Sander using a dataset of 126 nonhomologous globular proteins, our predictions provide a quantitative foundation for gauging the results when building by homology the structures of related proteins.

Botulinum Toxins↗
PubMed1993

Sequence-specific 1H and 15N resonance assignments and secondary structure of GDP-bound human c-Ha-Ras protein in solution.

All the backbone 1H and 15N magnetic resonances (except for Pro residues) of the GDP-bound form of a truncated human c-Ha-ras proto-oncogene product (171 amino acid residues, the Ras protein) were assigned by 15N-edited two-dimensional NMR experiments on selectively 15N-labeled Ras proteins in combination with three-dimensional NMR experiments on the uniformly 15N-labeled protein. The sequence-specific assignments were made on the basis of the nuclear Overhauser effect (NOE) connectivities of amide protons with preceding amide and/or C alpha protons. In addition to sequential NOEs, vicinal spin coupling constants for amide protons and C alpha protons and deuterium exchange rates of amide protons were used to characterize the secondary structure of the GDP-bound Ras protein; six beta stands and five helices were identified and the topology of these elements was determined. The secondary structure of the Ras protein in solution was mainly consistent with that in crystal as determined by X-ray analyses. The deuterium exchange rates of amide protons were examined to elucidate the dynamic properties of the secondary structure elements of the Ras protein in solution. In solution, the beta-sheet structure in the Ras protein is rigid, while the second helix (A66-R73) is much more flexible, and the first and fifth helices (S17-124 and V152-L171) are more rigid than other helices. Secondary structure elements at or near the ends of the effector-region loop were found to be much more flexible in solution than in the crystalline state.

Amino Acid Sequence↗
PubMed1999

Insight into the secondary structure of non-native proteins bound to a molecular chaperone alpha-crystallin. An isotope-edited infrared spectroscopic study.

alpha-Crystallin, the major lens protein, acts as a molecular chaperone by preventing the aggregation of proteins damaged by heat and other stress conditions. To characterize the backbone conformation of protein folding intermediates that are recognized by the chaperone, we prepared the uniformly (13)C-labeled alphaA-crystallin. The labeling greatly reduced the overlapping between the conformation-sensitive amide I bands of alpha-crystallin and unlabeled substrate proteins. This procedure has allowed us to gain insight into the secondary structure of alpha-crystallin-bound species, an understanding which has previously been unattainable. Analysis of the infrared spectra of two substrate proteins (gamma- and beta(L)-crystallins) indicates that heat-destabilized conformers captured by alpha-crystallin are characterized by a high proportion of native-like secondary structure. In contrast to the chaperone-bound species, the same proteins subjected to heat treatment in the absence of alpha-crystallin preserve very little native secondary structure. These data show that alpha-crystallin specifically recognizes very early intermediates on the denaturation pathway of proteins. These aggregation-prone species are characterized by native-like secondary structure but compromised tertiary interactions. The experimental approach described in this study can be further applied to probe the backbone conformation of proteins bound to chaperones other than alpha-crystallin.

Crystallins↗