Alternative Protein Structure Research Articles

In proteins, the structural responses of a position to mutation rely on the Goldilocks principle: not too many links, not too few.

A disease has distinct genetic and molecular hallmarks such as sequence variants that are likely to produce the alternative protein structures accountable for individual responses to drugs and disease development. Thus, to set up customized therapies, the structural influences of amino acids on one another need to be tracked down. Using network-based models and classical analysis of amino acid and atomic packing in protein structures, the influence of first shell neighbors on the structural fate of a position upon mutation, is revisited. Regardless of the type and position in a structure, amino acids satisfy on average over their neighbors, a low and similar number of atomic interactions, the average called the neighborhood watch (Nw). The structural tolerance of a position to mutation depends on the modulation of the composition and/or proximity of neighbors to maintain the same Nw, before and after mutation, at every position. Changes, upon mutation of the number of atomic interactions at the level of individual pairs (wij) are structurally tolerated but influence structural dynamics. Robust, fragile and rescue interactions can be identified with Nw and wij, offering a framework to classify sequence variants according to position-dependent structural changes.

Molecular analysis of alternative transcripts of equine AXL receptor tyrosine kinase gene.

ObjectiveSince athletic performance is a most importance trait in horses, most research focused on physiological and physical studies of horse athletic abilities. In contrast, the molecular analysis as well as the regulatory pathway studies remain insufficient for evaluation and prediction of horse athletic abilities. In our previous study, we identified AXL receptor tyrosine kinase (AXL) gene which was expressed as alternative spliced isoforms in skeletal muscle during exercise. In the present study, we validated two AXL alternative splicing transcripts (named as AXLa for long form and AXLb for short form) in equine skeletal muscle to gain insight(s) into the role of each alternative transcript during exercise.MethodsWe validated two isoforms of AXL transcripts in horse tissues by reverse transcriptase polymerase chain reaction (RT-PCR), and then cloned the transcripts to confirm the alternative locus and its sequences. Additionally, we examined the expression patterns of AXLa and AXLb transcripts in horse tissues by quantitative RT-PCR (qRT-PCR).ResultsBoth of AXLa and AXLb transcripts were expressed in horse skeletal muscle and the expression levels were significantly increased after exercise. The sequencing analysis showed that there was an alternative splicing event at exon 11 between AXLa and AXLb transcripts. 3-dimentional (3D) prediction of the alternative protein structures revealed that the structural distance of the connective region between fibronectin type 3 (FN3) and immunoglobin (Ig) domain was different between two alternative isoforms.ConclusionIt is assumed that the expression patterns of AXLa and AXLb transcripts would be involved in regulation of exercise-induced stress in horse muscle possibly through an NF-κB signaling pathway. Further study is necessary to uncover biological function(s) and significance of the alternative splicing isoforms in race horse skeletal muscle.

Automatic Assignment of Methyl-NMR Spectra of Supramolecular Machines Using Graph Theory.

Methyl groups are powerful probes for the analysis of structure, dynamics and function of supramolecular assemblies, using both solution- and solid-state NMR. Widespread application of the methodology has been limited due to the challenges associated with assigning spectral resonances to specific locations within a biomolecule. Here, we present Methyl Assignment by Graph Matching (MAGMA), for the automatic assignment of methyl resonances. A graph matching protocol examines all possibilities for each resonance in order to determine an exact assignment that includes a complete description of any ambiguity. MAGMA gives 100% accuracy in confident assignments when tested against both synthetic data, and 9 cross-validated examples using both solution- and solid-state NMR data. We show that this remarkable accuracy enables a user to distinguish between alternative protein structures. In a drug discovery application on HSP90, we show the method can rapidly and efficiently distinguish between possible ligand binding modes. By providing an exact and robust solution to methyl resonance assignment, MAGMA can facilitate significantly accelerated studies of supramolecular machines using methyl-based NMR spectroscopy.

Alternative splicing acting as a bridge in evolution.

Alternative splicing (AS) regulates diverse cellular and developmental functions through alternative protein structures of different isoforms. Alternative exons dominate AS in vertebrates; however, very little is known about the extent and function of AS in lower eukaryotes. To understand the role of introns in gene evolution, we examined AS from a green algal and five fungal genomes using a novel EST-based gene-modeling algorithm (COMBEST). AS from each genome was classified with COMBEST that maps EST sequences to genomes to build gene models. Various aspects of AS were analyzed through statistical methods. The interplay of intron 3n length, phase, coding property, and intron retention (RI) were examined with Chi-square testing. With 3 to 834 times EST coverage, we identified up to 73% of AS in intron-containing genes and found preponderance of RI among 11 types of AS. The number of exons, expression level, and maximum intron length correlated with number of AS per gene (NAG), and intron-rich genes suppressed AS. Genes with AS were more ancient, and AS was conserved among fungal genomes. Among stopless introns, non-retained introns (NRI) avoided, but major RI preferred 3n length. In contrast, stop-containing introns showed uniform distribution among 3n, 3n+1, and 3n+2 lengths. We found a clue to the intron phase enigma: it was the coding function of introns involved in AS that dictates the intron phase bias. Majority of AS is non-functional, and the extent of AS is suppressed for intron-rich genes. RI through 3n length, stop codon, and phase bias bridges the transition from functionless to functional alternative isoforms.

Alternative NF-κB Isoforms in the Drosophila Neuromuscular Junction and Brain

The Drosophila NF-κB protein Dorsal is expressed at the larval neuromuscular junction, where its expression appears unrelated to known Dorsal functions in embryonic patterning and innate immunity. Using confocal microscopy with domain-specific antisera, we demonstrate that larval muscle expresses only the B isoform of Dorsal, which arises by intron retention. We find that Dorsal B interacts with and stabilizes Cactus at the neuromuscular junction, but exhibits Cactus independent localization and an absence of detectable nuclear translocation. We further find that the Dorsal-related immune factor Dif encodes a B isoform, reflecting a conservation of B domains across a range of insect NF-κB proteins. Carrying out mutagenesis of the Dif locus via a site-specific recombineering approach, we demonstrate that Dif B is the major, if not sole, Dif isoform in the mushroom bodies of the larval brain. The Dorsal and Dif B isoforms thus share a specific association with nervous system tissues as well as an alternative protein structure.

Carbohydrate-binding modules (CBMs) revisited: reduced amount of water counterbalances the need for CBMs

BackgroundA vast number of organisms are known to produce structurally diversified cellulases capable of degrading cellulose, the most abundant biopolymer on earth. The generally accepted paradigm is that the carbohydrate-binding modules (CBMs) of cellulases are required for efficient saccharification of insoluble substrates. Based on sequence data, surprisingly more than 60% of the cellulases identified lack carbohydrate-binding modules or alternative protein structures linked to cellulases (dockerins). This finding poses the question about the role of the CBMs: why would most cellulases lack CBMs, if they are necessary for the efficient hydrolysis of cellulose?ResultsThe advantage of CBMs, which increase the affinity of cellulases to substrates, was found to be diminished by reducing the amount of water in the hydrolytic system, which increases the probability of enzyme-substrate interaction. At low substrate concentration (1% w/w), CBMs were found to be more important in the catalytic performance of the cellobiohydrolases TrCel7A and TrCel6A of Trichoderma reesei as compared to that of the endoglucanases TrCel5A and TrCel7B. Increasing the substrate concentration while maintaining the enzyme-to-substrate ratio enhanced adsorption of TrCel7A, independent of the presence of the CBM. At 20% (w/w) substrate concentration, the hydrolytic performance of cellulases without CBMs caught up with that of cellulases with CBMs. This phenomenon was more noticeable on the lignin-containing pretreated wheat straw as compared to the cellulosic Avicel, presumably due to unproductive adsorption of enzymes to lignin.ConclusionsHere we propose that the water content in the natural environments of carbohydrate-degrading organisms might have led to the evolution of various substrate-binding structures. In addition, some well recognized problems of economical saccharification such as unproductive binding of cellulases, which reduces the hydrolysis rate and prevents recycling of enzymes, could be partially overcome by omitting CBMs. This finding could help solve bottlenecks of enzymatic hydrolysis of lignocelluloses and speed up commercialization of second generation bioethanol.

Protein folding pathways and state transitions described by classical equations of motion of an elastic network model

Protein topology defined by the matrix of residue contacts has proved to be a fruitful basis for the study of protein dynamics. The widely implemented coarse-grained elastic network model of backbone fluctuations has been used to describe crystallographic temperature factors, allosteric couplings, and some aspects of the folding pathway. In the present study, we develop a model of protein dynamics based on the classical equations of motion of a damped network model (DNM) that describes the folding path from a completely unfolded state to the native conformation through a single-well potential derived purely from the native conformation. The kinetic energy gained through the collapse of the protein chain is dissipated through a friction term in the equations of motion that models the water bath. This approach is completely general and sufficiently fast that it can be applied to large proteins. Folding pathways for various proteins of different classes are described and shown to correlate with experimental observations and molecular dynamics and Monte Carlo simulations. Allosteric transitions between alternative protein structures are also modeled within the DNM through an asymmetric double-well potential.

Use of Relaxation Enhancements in a Paramagnetic Environment for the Structure Determination of Proteins Using NMR Spectroscopy

More information: Relaxation enhancements induced by an inert paramagnetic environment (PREs) are used together with a limited NOE data set in a model-free alternative protein structure refinement procedure. Structures of two model systems (8 kDa ubiquitin and the 42 kDa maltodextrin-binding protein in complex with β-cyclodextrin) were obtained employing PREs and NOEs of exchangeable protons.

A designed protein as experimental model of primordial folding

How do proteins accomplish folding during early evolution? Theoretically the mechanism involves the selective stabilization of the native structure against all other competing compact conformations in a process that involves cumulative changes in the amino acid sequence along geological timescales. Thus, an evolved protein folds into a single structure at physiological temperature, but the conformational competition remains latent. For natural proteins such competition should emerge only near cryogenic temperatures, which places it beyond experimental testing. Here, we introduce a designed monomeric miniprotein (FSD-1ss) that within biological temperatures (330-280 K) switches between simple fast folding and highly complex conformational dynamics in a structurally degenerate compact ensemble. Our findings demonstrate the physical basis for protein folding evolution in a designed protein, which exhibits poorly evolved or primordial folding. Furthermore, these results open the door to the experimental exploration of primitive folding and the switching between alternative protein structures that takes place in evolutionary branching points and prion diseases, as well as the benchmarking of de novo design methods.

Conformational analysis of alternative protein structures

Alternative structural models determined experimentally are available for an increasing number of proteins. Structural and functional studies of these proteins need to take these models into consideration as they can present considerable structural differences. The characterization of the structural differences and similarities between these models is a fundamental task in structural biology requiring appropriate methods. We propose a method for characterizing sets of alternative structural models. Three types of analysis are performed: grouping according to structural similarity, visualization and detection of structural variation and comparison of subsets for identifying and locating distinct conformational states. The alpha carbon atoms are used in order to analyse the backbone conformations. Alternatively, side-chain atoms are used for detailed conformational analysis of specific sites. The method takes into account estimates of atom coordinate uncertainty. The invariant regions are used to generate optimal superpositions of these models. We present the results obtained for three proteins showing different degrees of conformational variability: relative motion of two structurally conserved subdomains, a disordered subdomain and flexibility in the functional site associated with ligand binding. The method has been applied in the analysis of the alternative models available in SCOP. Considerable structural variability can be observed for most proteins. The results of the analysis of the SCOP alternative models, the estimates of coordinate uncertainty as well as the source code of the implementation are available in the STRuster web site: http://struster.bioinf.mpi-inf.mpg.de.