Diversity in the circumsporozoite protein of Plasmodium vivax: Does it matter?

Since the gene encoding the malarial circumsporozoite surface protein was characterized a decade ago, the corresponding protein has been considered an important vaccine candidate. Victoria Mann, Michael Good and Allan Saul here discuss molecular variation in the circumsporozoite surface protein of Plasmodium vivax in this context. There is still doubt about the degree and importance of polymorphisms in non-repetitive regions of the molecule. The degree of polymorphisms and data on naturally occurring protective responses suggests there has been minimal immunological pressure; the authors contend that antigenic diversity is unlikely to be a major factor in the use of this antigen as a vaccine for P. vivax.     

Analysis of secondary structural and physicochemical changes in protein–protein complexes

Conformation switching in protein–protein complexes is considered important for the molecular recognition process. Overall analysis of 123 protein–protein complexes in a benchmark data-set showed that 6.8% of residues switched over their secondary structure conformation upon complex formation. Amino acid residue-wise preference for conformation change has been analyzed in binding and non-binding site residues separately. In this analysis, residues such as Ser, Leu, Glu, and Lys had higher frequency of secondary structural conformation change. The change of helix to coil and sheet to coil conformation and vice versa has been observed frequently, whereas the conformation change of helix to extended sheet occurred rarely in the studied complexes. Influence of conformation change toward the N and C terminal on either side of the binding site residues has been analyzed. Further, analysis on φ and ψ angle variation, conservation, stability, and solvent accessibility have been performed on binding site residues. Knowledge obtained from the present study could be effectively employed in the protein–protein modeling and docking studies.     

Does the type of matrix matter? A quantitative review of the evidence

It has been increasingly recognized that the type of matrix surrounding habitat patches can affect biodiversity in landscapes, but there were only qualitative reviews of the subject focused on particular taxonomic groups. We present a quantitative review of studies from 1985 to 2008 that compared effects of different matrix types on individuals, populations and communities. We compiled 104 studies, most on animals, covering a broad range of landscape types and spatial scales. Most studies were empirical, focused on individuals and communities, and evaluated abundance/richness in the patch as the dependent variable. The type of matrix surrounding habitat patches influenced the studied parameters in 95% of the studies, but such effects were overall smaller compared to patch size or isolation effects. Matrix type effects were strongly species-specific, with different species responding differently to matrix type in 96% of studies comparing species or group of species. In 88% of studies, matrix types more similar in structure to the patch had higher quality for the studied organisms from the point of view of functional connectivity. Overall, the type of matrix is important, but patch size and isolation are the main determinants of ecological parameters in landscapes. Matrix quality generally increases with increasing structural similarity with habitat patches, a pattern that could be used as a general guideline for management of the matrix in fragmented landscapes.     

A structural perspective of the protein–RNA interactions involved in virus-induced RNA silencing and its suppression

Small RNAs, including small interfering RNAs (siRNAs), microRNAs (miRNAs) and Piwi-associated interfering RNAs (piRNAs), are powerful gene expression regulators. This RNA-mediated regulation results in sequence-specific inhibition of gene expression by translational repression and/or mRNA degradation. siRNAs and miRNAs are generated by RNase III enzymes and subsequently loaded into Argonaute protein, a key component of the RNA induced silencing complex (RISC), to form the core of the RNA silencing machinery. RNA silencing acts as an ancient cell defense system against molecular parasites, such as transgenes, viruses and transposons. RNA silencing also plays an important role in the control of development. In plants, RNA silencing serves as a potent antiviral defense system. In response, many viruses have developed strategies to suppress RNA silencing. The striking sequence diversity among viral suppressors suggests that different viral suppressors could target different components of the RNA silencing machinery at different steps in different suppressing modes. Significant progresses have been made in this field for the past 5 years on the basis of structural information derived from RNase III family proteins, Dicer fragments and homologs, Argonaute homologs and viral suppressors. In this paper, we will review the current progress on the understanding of molecular mechanisms of RNA silencing; highlight the structural principles determining the protein–RNA recognition events along the RNA silencing pathways and the suppression mechanisms displayed by viral suppressors.     

Dynamics of the intrinsically disordered protein NUPR1 in isolation and in its fuzzy complexes with DNA and prothymosin α

Intrinsically disordered proteins (IDPs) explore diverse conformations in their free states and, a few of them, also in their molecular complexes. This functional plasticity is essential for the function of IDPs, although their dynamics in both free and bound states is poorly understood. NUPR1 is a protumoral multifunctional IDP, activated during the acute phases of pancreatitis. It interacts with DNA and other IDPs, such as prothymosin α (ProTα), with dissociation constants of ~0.5 μM, and a 1:1 stoichiometry. We studied the structure and picosecond-to-nanosecond (ps-ns) dynamics by using both NMR and SAXS in: (i) isolated NUPR1; (ii) the NUPR1/ProTα complex; and (iii) the NUPR1/double stranded (ds) GGGCGCGCCC complex. Our SAXS findings show that NUPR1 remained disordered when bound to either partner, adopting a worm-like conformation; the fuzziness of bound NUPR1 was also pinpointed by NMR. Residues with the largest values of the relaxation rates (R₁, R_1ρ, R₂ and η_xy), in the free and bound species, were mainly clustered around the 30s region of the sequence, which agree with one of the protein hot-spots already identified by site-directed mutagenesis. Not only residues in this region had larger relaxation rates, but they also moved slower than the rest of the molecule, as indicated by the reduced spectral density approach (RSDA). Upon binding, the energy landscape of NUPR1 was not funneled down to a specific, well-folded conformation, but rather its backbone flexibility was kept, with distinct motions occurring at the hot-spot region.     

A biological cosmos of parallel universes: Does protein structural plasticity facilitate evolution?

While Darwin pictured organismal evolution as "descent with modification" more than 150 years ago, a detailed reconstruction of the basic evolutionary transitions at the molecular level is only emerging now. In particular, the evolution of today's protein structures and their concurrent functions has remained largely mysterious, as the destruction of these structures by mutation seems far easier than their construction. While the accumulation of genomic and structural data has indicated that proteins are related via common ancestors, naturally occurring protein structures are often considered to be evolutionarily robust, thus leaving open the question of how protein structures can be remodelled while selective pressure forces them to function. New information on the proteome, however, increasingly explains the nature of local and global conformational diversity in protein evolution, which allows the acquisition of novel functions via molecular transition forms containing ancestral and novel structures in dynamic equilibrium. Such structural plasticity may permit the evolution of new protein folds and help account for both the origins of new biological functions and the nature of molecular defects.     

Does the order of second-line treatment in rheumatoid arthritis matter?

In a prospective study 88 patients, with rheumatoid arthritis who had stopped taking gold, penicillamine, or levamisole were randomly allocated to one of the alternative drugs and followed up for a minimum of one year. Concurrent studies of the effects of gold, penicillamine, and levamisole prescribed in 123 patients as the first second-line drug were used for comparison. No difference in toxicity or efficacy between primary and secondary use of gold or penicillamine was identified. Variation in the toxicity of levamisole could in part be accounted for by changes in the dose regimen over the four years of study. The length of the treatment-free interval between drugs did not influence subsequent development of toxicity. These results suggest that an adverse reaction to one of the three second-line drugs studied should not prejudice the selection of another.     

Does the examiner's experience matter in evaluation of the kinematics of the upper limb?

This study aimed to evaluate test and retest reliability according to examiner experience with the three-dimensional kinematics of the trunk, scapula, and arm segments during flexion and unilateral abduction of the arm. Ten men and 10 women (mean age, 25.1 [1.1] years) participated in this study. Each volunteer participated in six test sessions, four on the first day (two for each examiner) and two on the second day (one for each examiner). A 48-h interval was given between test days. The assessments were made by one examiner with movement analysis experience and a second examiner without experience. For each session (intra-day), the volunteers performed five repetitions of unilateral arm flexions and abductions using their dominant arms. After 1 h, the data were re-collected and all markers were replaced. Data from the trunk, scapula, and arm were analysed at 30°, 60°, 90°, and 120° of arm flexion and abduction using intraclass coefficient correlation, standard error of the measurement, and analysis of variance. The results did not differ between the experienced and inexperienced examiners except for trunk axial rotation at all studied angles and for arm rotation at 120° of abduction. The examiner previously trained in movement analysis marker placement demonstrated the same intra-tester reliability as the inexperienced tester when marker placement accuracy was the variable of interest.     

Does protein synthesis occur in the nucleus?

Although it is universally accepted that protein synthesis occurs in the cytoplasm, the possibility that translation can also take place in the nucleus has been hotly debated. Reports have been published claiming to demonstrate nuclear translation, but alternative explanations for these results have not been excluded, and other experiments argue against it. Much of the appeal of nuclear translation is that functional proofreading of newly made mRNAs in the nucleus would provide an efficient way to monitor mRNAs for the presence of premature termination codons, thereby avoiding the synthesis of deleterious proteins. mRNAs that are still in the nucleus-associated fraction of cells are subject to translational proofreading resulting in nonsense-mediated mRNA decay and perhaps nonsense-associated alternate splicing. However, these mRNAs are likely to be in the perinuclear cytoplasm rather than within the nucleus. Therefore, in the absence of additional evidence, we conclude that nuclear translation is unlikely to occur.     

Does urinary taurine reflect changes in protein metabolism? A study with cycloheximide in rats

Abstract

We have previously reported on the changes in urinary taurine levels in rats following treatment with some hepatotoxic agents and compounds reported to affect protein synthesis. This study follows the time course of the elevation of urinary taurine after treatment of rats with cycloheximide which was maximal 8–12 h alter dosing and was dose related. [³H]-leucine incorporation into proteins was used as an indicator of protein synthesis. There was a significant reduction in [³H]-leucine incorporation into acid precipitable proteins 8 h but not 24 h after dosing. The reduction in incorporation was negatively correlated with the raised levels of both serum and urinary taurine 8 h after dosing. Liver glutathione was raised both 8 and 24 h after dosing rats and liver taurine was significantly reduced at 8 h. It is suggested that measuring urinary taurine in collections made continuously might provide a simple, non-invasive biomarker for monitoring the effects of xenobiotics or other external stimuli on the status of protein synthesis.     

A mutant α-amylase with only part of the catalytic domain and its structural implication

A truncated mutant α-amylase, Xa-S2, was obtained from Xanthomonas campestris wild type α-amylases (Xa-WT) through random mutagenesis that contained 167 amino acid residues (approx 65% shorter than that of Xa-WT). Secondary structure prediction implied that Xa-S2, would be unable to form the whole (β/α)₈-barrel catalytic domain and did not have the three conserved catalytic residues of wild type α-amylase, but it still displays the starch-hydrolyzing activity. Xa-S2 was prepared, characterized and compared to the recombinant wild-type enzymes. The K _m for starch was 32 mg/ml; activity was optimal at pH 6.2 and 30°C. In contrast, the K _m for starch of Xa-WT was 8 mg/ml and optimal enzyme activity was at pH 6.0–6.2 and 45–50°C. Our results suggested that Xa-S2 is a new amylase with a minimal catalytic domain for hydrolyzing substrates with of α-1,4-glucosidic bonds. T. Ke and X. D. Ma contributed equally to this work     

Is protein unfolding the reverse of protein folding? A lattice simulation analysis.

Simulations and experiments that monitor protein unfolding under denaturing conditions are commonly employed to study the mechanism by which a protein folds to its native state in a physiological environment. Due to the differences in conditions and the complexity of the reaction, unfolding is not necessarily the reverse of folding. To assess the relevance of temperature initiated unfolding studies to the folding problem, we compare the folding and unfolding of a 125-residue protein model by Monte Carlo dynamics at two temperatures; the lower one corresponds to the range used in T -jump experiments and the higher one to the range used in unfolding simulations of all-atom models. The trajectories that lead from the native state to the denatured state at these elevated temperatures are less diverse than those observed in the folding simulations. At the lower temperature, the system unfolds through a mandatory intermediate that corresponds to a local free energy minimum. At the higher temperature, no such intermediate is observed, but a similar pathway is followed. The structures contributing to the unfolding pathways resemble most closely those that make up the "fast track" of folding. The transition state for unfolding at the lower temperature (above Tm) is determined and is found to be more structured than the transition state for folding below the melting temperature. This shift towards the native state is consistent with the Hammond postulate. The implications for unfolding simulations of higher resolution models and for unfolding experiments of proteins are discussed.     

Do mRNA and rRNA binding sites of E.coli ribosomal protein S15 share common structural determinants?

Escherichia coli ribosomal protein S15 recognizes two RNA targets: a three-way junction in 16S rRNA and a pseudoknot structure on its own mRNA. Binding to mRNA occurs when S15 is expressed in excess over its rRNA target, resulting in an inhibition of translation start. The sole apparent similarity between the rRNA and mRNA targets is the presence of a G-U/G-C motif that contributes only modestly to rRNA binding but is essential for mRNA. To get more information on the structural determinants used by S15 to bind its mRNA target as compared to its rRNA site, we used site-directed mutagenesis, substitution by nucleotide analogs, footprinting experiments on both RNA and protein, and graphic modeling. The size of the mRNA-binding site could be reduced to 45 nucleotides, without loss of affinity. This short RNA preferentially folds into a pseudoknot, the formation of which depends on magnesium concentration and temperature. The size of the loop L2 that bridges the two stems of the pseudoknot through the minor groove could not be reduced below nine nucleotides. Then we showed that the pseudoknot recognizes the same side of S15 as 16S rRNA, although shielding a smaller surface area. It turned out that the G-U/G-C motif is recognized from the minor groove in both cases, and that the G-C pair is recognized in a very similar manner. However, the wobble G-U pair of the mRNA is not directly contacted by S15, as in rRNA, but is most likely involved in building a precise conformation of the RNA, essential for binding. Otherwise, unique specific features are utilized, such as the three-way junction in the case of 16S rRNA and the looped out A(-46) for the mRNA pseudoknot.     

Modifying the determinants of α-ketoacid substrate selectivity in mycobacterium tuberculosis α-isopropylmalate synthase

α-Isopropylmalate synthase (IPMS) catalyses the reaction between α-ketoisovalerate and acetyl coenzyme A (AcCoA) in the first step of leucine biosynthesis. IPMS is closely related to homocitrate synthase, which catalyses the reaction between AcCoA and the unbranched α-ketoacid α-ketoglutarate. Analysis of these enzymes suggests that several differently conserved key residues are responsible for the different substrate selectivity. These residues were systematically substituted in the Mycobacterium tuberculosis IPMS, resulting in changes in substrate specificity. A variant of IPMS was constructed with a preference for the unbranched α-ketoacids α-ketobutyrate and pyruvate over the natural branched substrate α-ketoisovalerate.     

How noise in force fields can affect the structural refinement of protein models?

Structural refinement of predicted models of biological macromolecules using atomistic or coarse‐grained molecular force fields having various degree of error is investigated. The goal of this analysis is to estimate what is the probability for designing an effective structural refinement based on computations of conformational energies using force field, and starting from a structure predicted from the sequence (using template‐based or template‐free modeling), and refining it to bring the structure into closer proximity to the native state. It is widely believed that it should be possible to develop such a successful structure refinement algorithm by applying an iterative procedure with stochastic sampling and appropriate energy function, which assesses the quality (correctness) of protein decoys. Here, an analysis of noise in an artificially introduced scoring function is investigated for a model of an ideal sampling scheme, where the underlying distribution of RMSDs is assumed to be Gaussian. Sampling of the conformational space is performed by random generation of RMSD values. We demonstrate that whenever the random noise in a force field exceeds some level, it is impossible to obtain reliable structural refinement. The magnitude of the noise, above which a structural refinement, on average is impossible, depends strongly on the quality of sampling scheme and a size of the protein. Finally, possible strategies to overcome the intrinsic limitations in the force fields for impacting the development of successful refinement algorithms are discussed. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

On the functional and structural characterization of hubs in protein–protein interaction networks

A number of interesting issues have been addressed on biological networks about their global and local properties. The connection between the topological properties of proteins in Protein–Protein Interaction (PPI) networks and their biological relevance has been investigated focusing on hubs, i.e. proteins with a large number of interacting partners. We will survey the literature trying to answer the following questions: Do hub proteins have special biological properties? Do they tend to be more essential than non-hub proteins? Are they more evolutionarily conserved? Do they play a central role in modular organization of the protein interaction network? Are there structural properties that characterize hub proteins?     

Molecular characterization and phylogenetic relationships of a protein with potential oxygen-binding capabilities in the grasshopper embryo. A hemocyanin in insects?

Arthropodan hemocyanins, prophenoloxidases (PPOs), and insect hexamerins form a superfamily of hemolymph proteins that we propose to call the AHPH superfamily. The evolutionary and functional relationships of these proteins are illuminated by a new embryonic hemolymph protein (EHP) that is expressed during early stages of development in the grasshopper embryo. EHP is a 78-kDa soluble protein present initially in the yolk sac content, and later in the embryonic hemolymph. Protein purification and peptide sequencing were used to identify an embryonic cDNA clone coding for EHP. In situ hybridization identifies hemocytes as EHP-expressing cells. As deduced from the cDNA clone, EHP is a secreted protein with two potential glycosylation sites. Sequence analysis defines EHP as a member of the AHPH superfamily. Phylogenetic analyses with all the currently available AHPH proteins, including EHP, were performed to ascertain the evolutionary history of this protein superfamily. We used both the entire protein sequence and each of the three domains present in the AHPH proteins. The phylogenies inferred for each of the domains suggest a mosaic evolution of these protein modules. Phylogenetic and multivariate analyses consistently group EHP with crustacean hemocyanins and, less closely, with insect hexamerins, relative to cheliceratan hemocyanins and PPOs. The grasshopper protein rigorously preserves the residues involved in oxygen binding, oligomerization, and allosteric regulation of the oxygen transport proteins. Although insects were thought not to have hemocyanins, we propose that EHP functions as an oxygen transport or storage protein during embryonic development.