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PubMed2004

Interplay of secondary structures and side-chain contacts in the denatured state of BBA1.

The denatured state of a miniprotein BBA1 is studied under the native condition with the AMBER/Poisson-Boltzmann energy model and with the self-guided enhanced sampling technique. Forty independent trajectories are collected to sample the highly diversified denatured structures. Our simulation data show that the denatured BBA1 contains high percentage of native helix and native turn, but low percentage of native hairpin. Conditional population analysis indicates that the native helix formation and the native hairpin formation are not cooperative in the denatured state. Side-chain analysis shows that the native hydrophobic contacts are more preferred than the non-native hydrophobic contacts in the denatured BBA1. In contrast, the salt-bridge contacts are more or less nonspecific even if their populations are higher than those of hydrophobic contacts. Analysis of the trajectories shows that the native helix mostly initiates near the N terminus and propagates to the C terminus, and mostly forms from 3(10)-helix/turn to alpha helix. The same analysis shows that the native turn is important but not necessary in its formation in the denatured BBA1. In addition, the formations of the two strands in the native hairpin are rather asymmetric, demonstrating the likely influence of the protein environment. Energetic analysis shows that the native helix formation is largely driven by electrostatic interactions in denatured BBA1. Further, the native helix formation is associated with the breakup of non-native salt-bridge contacts and the accumulation of native salt-bridge contacts. However, the native hydrophobic contacts only show a small increase upon the native helix formation while the non-native hydrophobic contacts stay essentially the same, different from the evolution of hydrophobic contacts observed in an isolated helix folding.

Biophysical Phenomena↗
PubMed2001

HIV-1 nucleocapsid protein and the secondary structure of the binary complex formed between tRNA(Lys.3) and viral RNA template play different roles during initiation of (-) strand DNA reverse transcription.

In human immunodeficiency virus type 1 (HIV-1), the tRNA(Lys.3) primer and viral RNA template can form a specific complex that is characterized by extensive inter- and intramolecular interactions. Initiation of reverse transcription from this complex has been shown to be distinguished from subsequent elongation by early pausing events, such as at the +1 and +3 nucleotide positions. One major concern regarding the biological relevance of these results is that most kinetic studies of HIV-1 reverse transcription have been performed using tRNA(Lys.3)-viral (v) RNA complexes that were formed by heat annealing. In contrast, tRNA(Lys.3) in viruses is placed onto the primer binding site by nucleocapsid (NC) sequences of the Gag protein. In this study, we have further characterized the initiation features of reverse transcription in the presence of HIV-1 NC protein. In contrast to results obtained with a heat-annealed tRNA(Lys.3).vRNA complex, we found that polymerization reactions catalyzed by HIV-1 reverse transcriptase did not commonly pause at the +1 nucleotide position when a NC-annealed RNA complex was used, and that this was true regardless whether NC was actually still present during reverse transcription. This activity of NC required both zinc finger motifs, as demonstrated by experiments that employed zinc finger-mutated forms of NC protein (H23C NC and ddNC), supporting the involvement of the zinc fingers in the RNA chaperone activity of NC. However, NC was not able to help reverse transcriptase to escape the +3 pausing event. Mutagenesis of a stem structure within the tRNA(Lys.3). vRNA complex led to disappearance of the +3 pausing event as well as to significantly reduced rates of reverse transcription. Thus, this stem structure is essential for optimal reverse transcription, despite its role in promotion of the +3 pausing event.

Amino Acid Motifs↗
PubMed1997

Secondary structure and tertiary fold of the human immunodeficiency virus protein U (Vpu) cytoplasmic domain in solution.

The human immunodeficiency virus type 1 Vpu protein enhances virus particle release from infected cells, decreases the tendency of syncytia formation and induces degradation of human CD4 receptor. It is known that the cytoplasmic part of Vpu is responsible for direct interaction to and degradation of CD4. The tertiary fold of the Vpu cytoplasmic domain in aqueous solution was determined employing NMR spectroscopy and molecular-dynamics simulated-annealing protocols. We found a very well defined amphipathic alpha-helix in the membrane proximal part (40-50), a less well defined helix (60-68), and a short alpha-helix at the C-terminus (75-79). We further determined the overall tertiary structure based on long-range nuclear Overhauser enhancement effects. Correlation of results from mutation experiments of Vpu and the structure data is discussed.

Amino Acid Sequence↗
PubMed2002

Mapping the agonist binding site of the nicotinic acetylcholine receptor by cysteine scanning mutagenesis: antagonist footprint and secondary structure prediction.

To further define the surface of the Torpedo californica nicotinic acetylcholine receptor (nAChR) contributing to the agonist binding site structure, we used the substituted Cys accessibility method to identify novel residues and determined the "footprint" of residues protected from modification by the reversible competitive antagonist d-tubocurarine (dTC). nAChRs containing single Cys substitutions within regions of the alpha- or gamma-subunit primary structure known to contribute to the agonist binding site were expressed in Xenopus laevis oocytes. Cys substitutions in binding site segments A (alphaTyr-93 and alphaAsn-94), C (alphaTyr-198), and D (gammaGlu-57) had been shown previously to be accessible for modification. We now introduced cysteines from alphaAsp-195 to alphaIle-201 and from gammaAla-106 to gammaAsp-113 and identified positions accessible for modification in segments C (alphaAsp-195, alphaThr-196, alphaPro-197, alphaAsp-200, and alphaIle-201) and E (gammaAsn-107 and gammaLeu-109). dTC protected against alkylation in segments D (gammaGlu-57) and E (gammaLeu-109) but not in segment A (alphaTyr-93 and alphaAsn-94). In segment C, dTC protection experiments revealed a pattern in which every other residue (alpha196, alpha198, and alpha200, but not alpha197 or alpha201) was protected from alkylation. This pattern of protection provides evidence that bound dTC is near amino acids in segments C, D, and E but not in segment A, and identifies a beta-strand surface within segment C contributing to the binding site. These results are discussed in terms of a homology model, based on the molluscan acetylcholine binding protein crystal structure, of the T. californica nAChR agonist binding site.

Amino Acid Substitution↗
PubMed2005

Secondary structure, 1H, 13C and 15N resonance assignments and molecular interactions of the dishevelled DIX domain.

Dishevelled (Dvl) is a positive regulator of the canonical Wnt signaling pathway, which regulates the levels of beta-catenin. The beta-catenin oncoprotein depends upon the association of Dvl and Axin proteins through their DIX domains, and its accumulation directs the expression of specific developmental-related genes at the nucleus. Here, the (1)H, (13)C and (15)N resonances of the human Dishevelled 2 DIX domain are assigned using heteronuclear nuclear magnetic resonance (NMR) spectroscopy. In addition, helical and extended elements are identified based on the NMR data. The results establish a structural context for characterizing the actin and phospholipid interactions and binding sites of this novel domain, and provide insights into its role in protein localization to stress fibers and cytoplasmic vesicles during Wnt signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Hydrophobicity and prediction of the secondary structure of membrane proteins and peptides.

Reliability of the hydropathy method to predict the formation of membrane-spanning alpha-helices by integral membrane proteins and peptides whose structure is known from X-ray crystallography is analysed. It is shown that Kyte-Doolittle hydropathy plots do not predict accurately 22 transmembrane alpha-helices in the reaction centres (RC) of the photosynthetic bacteria Rhodopseudomonas viridis and Rhodobacter sphaeroides (R-26). The accuracy of prediction for these proteins was improved using an optimised Kyte-Doolittle hydrophobicity scale. However, this hydrophobicity scale did not improve the predictions for the alphabeta-peptides of the B800-850 (LH2) complexes of the photosynthetic bacteria Rhodopseudomonas acidophila and Rhodospirillum molischianum, which were excluded from the optimisation procedure. The best and worst predictions of membrane-spanning alpha-helices for the RC proteins and LH2 peptides, respectively, were obtained with a propensity scale (PRC) calculated from the amino acid sequences and X-ray data for the RC proteins. A propensity scale (PLH) obtained using the amino acid sequences and X-ray data for the alphabeta-peptides of the LH2 complexes did not give an acceptable prediction of the transmembrane segments in the LH2 peptides; moreover, it markedly contradicted the PRC scale. Amino acids have been concluded to have no significant preference to localisation in transmembrane segments. Therefore, the predictive ability of the hydropathy methodology appears to be limited: the number of transmembrane segments can be correctly calculated for the best case only, and the lengths and positions of membrane-spanning alpha-helices in a protein amino acid sequence can not be predicted exactly.

Algorithms↗
PubMed1995

The peptide-binding domain of the chaperone protein Hsc70 has an unusual secondary structure topology.

Modern NMR methods were used to determine the secondary structure topology of the 18 kDa peptide binding domain of the chaperone protein Hsc70 in solution. This report constitutes the first experimental conformational information on this important domain of the class of Hsp70 proteins. The domain consists of two four-stranded antiparallel beta-sheets and a single alpha-helix. The topology does not resemble at all the topology observed in the human leukocyte antigen (HLA) proteins of the major histocompatibility complex. This is significant because such resemblance was predicted on the basis of limited amino acid homology, secondary structure prediction, and related function. Moreover, the exact meander-type beta-sheet topology identified in Hsc70 has to our best knowledge not been observed in any other known protein structure.

Amino Acid Sequence↗
PubMed1994

Combining evolutionary information and neural networks to predict protein secondary structure.

Using evolutionary information contained in multiple sequence alignments as input to neural networks, secondary structure can be predicted at significantly increased accuracy. Here, we extend our previous three-level system of neural networks by using additional input information derived from multiple alignments. Using a position-specific conservation weight as part of the input increases performance. Using the number of insertions and deletions reduces the tendency for overprediction and increases overall accuracy. Addition of the global amino acid content yields a further improvement, mainly in predicting structural class. The final network system has sustained overall accuracy of 71.6% in a multiple cross-validation test on 126 unique protein chains. A test on a new set of 124 recently solved protein structures that have no significant sequence similarity to the learning set confirms the high level of accuracy. The average cross-validated accuracy for all 250 sequence-unique chains is above 72%. Using various data sets, the method is compared to alternative prediction methods, some of which also use multiple alignments: the performance advantage of the network system is at least 6 percentage points in three-state accuracy. In addition, the network estimates secondary structure content from multiple sequence alignments about as well as circular dichroism spectroscopy on a single protein and classifies 75% of the 250 proteins correctly into one of four protein structural classes. Of particular practical importance is the definition of a position-specific reliability index. For 40% of all residues the method has a sustained three-state accuracy of 88%, as high as the overall average for homology modelling. A further strength of the method is greatly increased accuracy in predicting the placement of secondary structure segments.

Amino Acid Sequence↗
PubMed1994

A study of protein secondary structure by Fourier transform infrared/photoacoustic spectroscopy and its application for recombinant proteins.

FT-IR/PAS (Fourier transform infrared/photoacoustic spectroscopy) was used to evaluate the secondary structure of proteins. Four well-studied proteins, concanavalin A, hemoglobin, lysozyme, and trypsin, which have different distributions of secondary structures, were used for assignments of the infrared bands and evaluating the accuracy of FT-IR/PAS methods. Secondary structure contents estimated from FT-IR/PAS and other physical methods (e.g., X-ray diffraction, CD, and traditional FT-IR) show good agreement. In addition, the secondary structure can be evaluated with as little as 0.5 micrograms of protein (concanavalin A), suggesting that FT-IR/PAS is a sensitive and useful technique that could be applied to studies of the folding of recombinant and mutant proteins where only small amounts of material are available. Recombinant phosphorylase kinase gamma 1-300 subunit expressed in Escherichia coli was found in the inclusion bodies. We found that renatured phosphorylase kinase gamma 1-300 subunit has two kinase forms: one has a 10-fold higher activity than the other one. Both fractions, however, are the same as judged from sodium dodecylsulfate-polyacrylamide gel electrophoresis. Differences in conformation were demonstrated by using the FT-IR/PAS method, which showed that the low-activity form has more beta-sheet structure than the form with high activity. Analysis of these kinase forms by CD confirms the interpretation made by the FT-IR/PAS method.

Protein Structure, Secondary↗
PubMed2003

SSEP: Secondary structural elements of proteins.

SSEP is a comprehensive resource for accessing information related to the secondary structural elements present in the 25 and 90% non-redundant protein chains. The database contains 1771 protein chains from 1670 protein structures and 6182 protein chains from 5425 protein structures in 25 and 90% non-redundant protein chains, respectively. The current version provides information about the alpha-helical segments and beta-strand fragments of varying lengths. In addition, it also contains the information about 3(10)-helix, beta- and nu-turns and hairpin loops. The free graphics program RASMOL has been interfaced with the search engine to visualize the three-dimensional structures of the user queried secondary structural fragment. The database is updated regularly and is available through Bioinformatics web server at http://cluster.physics.iisc.ernet.in/ssep/ or http://144.16.71.148/ssep/.

Databases, Protein↗
PubMed2006

CONTRAfold: RNA secondary structure prediction without physics-based models.

MOTIVATION: For several decades, free energy minimization methods have been the dominant strategy for single sequence RNA secondary structure prediction. More recently, stochastic context-free grammars (SCFGs) have emerged as an alternative probabilistic methodology for modeling RNA structure. Unlike physics-based methods, which rely on thousands of experimentally-measured thermodynamic parameters, SCFGs use fully-automated statistical learning algorithms to derive model parameters. Despite this advantage, however, probabilistic methods have not replaced free energy minimization methods as the tool of choice for secondary structure prediction, as the accuracies of the best current SCFGs have yet to match those of the best physics-based models. RESULTS: In this paper, we present CONTRAfold, a novel secondary structure prediction method based on conditional log-linear models (CLLMs), a flexible class of probabilistic models which generalize upon SCFGs by using discriminative training and feature-rich scoring. In a series of cross-validation experiments, we show that grammar-based secondary structure prediction methods formulated as CLLMs consistently outperform their SCFG analogs. Furthermore, CONTRAfold, a CLLM incorporating most of the features found in typical thermodynamic models, achieves the highest single sequence prediction accuracies to date, outperforming currently available probabilistic and physics-based techniques. Our result thus closes the gap between probabilistic and thermodynamic models, demonstrating that statistical learning procedures provide an effective alternative to empirical measurement of thermodynamic parameters for RNA secondary structure prediction. AVAILABILITY: Source code for CONTRAfold is available at http://contra.stanford.edu/contrafold/.

Algorithms↗