Tertiary Structure Contacts Research Articles

RNA sequence to structure analysis from comprehensive pairwise mutagenesis of multiple self-cleaving ribozymes.

Self-cleaving ribozymes are RNA molecules that catalyze the cleavage of their own phosphodiester backbones. These ribozymes are found in all domains of life and are also a tool for biotechnical and synthetic biology applications. Self-cleaving ribozymes are also an important model of sequence-to-function relationships for RNA because their small size simplifies synthesis of genetic variants and self-cleaving activity is an accessible readout of the functional consequence of the mutation. Here, we used a high-throughput experimental approach to determine the relative activity for every possible single and double mutant of five self-cleaving ribozymes. From this data, we comprehensively identified non-additive effects between pairs of mutations (epistasis) for all five ribozymes. We analyzed how changes in activity and trends in epistasis map to the ribozyme structures. The variety of structures studied provided opportunities to observe several examples of common structural elements, and the data was collected under identical experimental conditions to enable direct comparison. Heatmap-based visualization of the data revealed patterns indicating structural features of the ribozymes including paired regions, unpaired loops, non-canonical structures, and tertiary structural contacts. The data also revealed signatures of functionally critical nucleotides involved in catalysis. The results demonstrate that the data sets provide structural information similar to chemical or enzymatic probing experiments, but with additional quantitative functional information. The large-scale data sets can be used for models predicting structure and function and for efforts to engineer self-cleaving ribozymes.

A deep dilated convolutional residual network for predicting interchain contacts of protein homodimers.

MotivationDeep learning has revolutionized protein tertiary structure prediction recently. The cutting-edge deep learning methods such as AlphaFold can predict high-accuracy tertiary structures for most individual protein chains. However, the accuracy of predicting quaternary structures of protein complexes consisting of multiple chains is still relatively low due to lack of advanced deep learning methods in the field. Because interchain residue–residue contacts can be used as distance restraints to guide quaternary structure modeling, here we develop a deep dilated convolutional residual network method (DRCon) to predict interchain residue–residue contacts in homodimers from residue–residue co-evolutionary signals derived from multiple sequence alignments of monomers, intrachain residue–residue contacts of monomers extracted from true/predicted tertiary structures or predicted by deep learning, and other sequence and structural features.ResultsTested on three homodimer test datasets (Homo_std dataset, DeepHomo dataset and CASP-CAPRI dataset), the precision of DRCon for top L/5 interchain contact predictions (L: length of monomer in a homodimer) is 43.46%, 47.10% and 33.50% respectively at 6 Å contact threshold, which is substantially better than DeepHomo and DNCON2_inter and similar to Glinter. Moreover, our experiments demonstrate that using predicted tertiary structure or intrachain contacts of monomers in the unbound state as input, DRCon still performs well, even though its accuracy is lower than using true tertiary structures in the bound state are used as input. Finally, our case study shows that good interchain contact predictions can be used to build high-accuracy quaternary structure models of homodimers.Availability and implementationThe source code of DRCon is available at https://github.com/jianlin-cheng/DRCon. The datasets are available at https://zenodo.org/record/5998532#.YgF70vXMKsB.Supplementary information Supplementary data are available at Bioinformatics online.

Interactions, induced by heating, of whey protein isolate (WPI) with sugar beet pectin (SBP) in solution: Comparisons with a dry-state Maillard reaction

We investigate changes in the molecular structures of whey proteins (WPI) caused by heat-induced interaction and reaction with varying concentrations of sugar beet pectin (SBP) in aqueous solution at neutral pH. The resulting complexes were examined using circular dichroism (CD) and steady-state and time-resolved fluorescence spectroscopic techniques. Far-UV CD spectroscopy combined with spectral deconvolution revealed noticeable reduction (up to 5%) in the amount of antiparallel β-sheet element of WPI upon interacting with SBP. Analyses from the UV-VIS and steady-state fluorescence spectroscopies showed changes in the molecular electronic states of both WPI and the feruloyl moieties of SBP upon heat-induced interaction and conjugation. The tertiary structural contacts in WPI, studied by the intrinsic Trp fluorescence, were also disrupted in the presence of SBP in the form of FRET (Fluorescence Resonance Energy Transfer) complexes, most noticeably at a high level of SBP (3%), contrasting to the results from dry-heating. The thermal stability of WPI, at both secondary and tertiary structural levels, did not increase considerably by interacting and conjugating with SBP, especially at an equal concentration of SBP (3%). However, the stability of feruloyl groups of SBP saw moderate improvement and was less pronounced than that of the dry-state reactions.

Stabilization of whey protein isolate (WPI) through interactions with sugar beet pectin (SBP) induced by controlled dry-heating

It has been shown previously that reacting whey protein isolate (WPI) with sugar beet pectin (SBP) through a Maillard type reaction in a dry-state led to noticeable improvement in the physical, chemical and functional properties of the end products. Studies on the changes in the molecular structure of whey protein as a result of the reaction were carried out in this work to provide a molecular understanding of these observed improvements. The effect of interacting and conjugating with SBP on the apparent secondary structure of WPI was examined by far-UV circular dichroism (CD) spectroscopy and followed by spectral deconvolution. Results showed that the structure of WPI, especially the antiparallel β-sheet element, was preserved by reacting with SBP at the weight ratios of WPI:SBP = 3:1, 2:1, and 1:1. The analysis of difference UV-VIS spectra indicated significant changes occurred in the molecular electronic states of both WPI and the feruloyl moieties of SBP. Initial Maillard reaction products were detected in the form of the FRET complexes with WPI as the donor, and SBP, the acceptor by the studies of the steady-state fluorescence and the lifetime of the excited states. The tertiary structural contacts in WPI, probed by the intrinsic Trp fluorescence, were disrupted significantly upon reacting with SBP at an equal ratio (1:1). The thermal stability of WPI, however, at both secondary and tertiary structural levels, was greatly improved by the dry-heating induced reaction with SBP at all levels. The thermal stability of the feruloyl groups of SBP was also enhanced by reacting with WPI. Furthermore, the fluorescence lifetime studies definitively identified the distinctive advanced Maillard reaction end products (AMREPs) formed between WPI and SBP at varying ratios.

Direct-Coupling Analysis of nucleotide coevolution facilitates RNA secondary and tertiary structure prediction.

Despite the biological importance of non-coding RNA, their structural characterization remains challenging. Making use of the rapidly growing sequence databases, we analyze nucleotide coevolution across homologous sequences via Direct-Coupling Analysis to detect nucleotide-nucleotide contacts. For a representative set of riboswitches, we show that the results of Direct-Coupling Analysis in combination with a generalized Nussinov algorithm systematically improve the results of RNA secondary structure prediction beyond traditional covariance approaches based on mutual information. Even more importantly, we show that the results of Direct-Coupling Analysis are enriched in tertiary structure contacts. By integrating these predictions into molecular modeling tools, systematically improved tertiary structure predictions can be obtained, as compared to using secondary structure information alone.

A widespread self-cleaving ribozyme class is revealed by bioinformatics.

Ribozymes are noncoding RNAs that promote chemical transformations with rate enhancements approaching those of protein enzymes. Although ribozymes are likely to have been abundant during the RNA world era, only ten classes are known to exist among contemporary organisms. We report the discovery and analysis of an additional self-cleaving ribozyme class, called twister, which is present in many species of bacteria and eukarya. Nearly 2700 twister ribozymes were identified that conform to a secondary structure consensus that is small yet complex, with three stems conjoined by internal and terminal loops. Two pseudoknots provide tertiary structure contacts that are critical for catalytic activity. The twister ribozyme motif provides another example of a natural RNA catalyst and calls attention to the potentially varied biological roles of this and other classes of widely distributed self-cleaving RNAs.

Spatial distribution of radiation damage to crystalline proteins at 25-300 K.

The spatial distribution of radiation damage (assayed by increases in atomic B factors) to thaumatin and urease crystals at temperatures ranging from 25 to 300 K is reported. The nature of the damage changes dramatically at approximately 180 K. Above this temperature the role of solvent diffusion is apparent in thaumatin crystals, as solvent-exposed turns and loops are especially sensitive. In urease, a flap covering the active site is the most sensitive part of the molecule and nearby loops show enhanced sensitivity. Below 180 K sensitivity is correlated with poor local packing, especially in thaumatin. At all temperatures, the component of the damage that is spatially uniform within the unit cell accounts for more than half of the total increase in the atomic B factors and correlates with changes in mosaicity. This component may arise from lattice-level, rather than local, disorder. The effects of primary structure on radiation sensitivity are small compared with those of tertiary structure, local packing, solvent accessibility and crystal contacts.

MPlot--a server to analyze and visualize tertiary structure contacts and geometrical features of helical membrane proteins

MPlot is a webserver that provides a quick and easy way for structural biologists to analyze, visualize and plot tertiary structure contacts of helical membrane proteins. As input, experimentally determined or computationally modeled protein structures in PDB format are required. The automatic analysis concatenates in house tools to calculate cut-off dependent van der Waals contacts or crossing angles of transmembrane helices with third party tools to compute main chain or side chain hydrogen bonds or membrane planes. Moreover, MPlot allows new features and tools to be added on a regular basis. For that purpose, MPlot was embedded in a framework that facilitates advanced users to compose new workflows from existing tools, or to substitute intermediate results with results from their (own) tools. The outputs can be viewed online in a Jmol based protein viewer, or via automatically generated scripts in PyMOL. For further illustration, the results can be downloaded as a 2D graph, representing the spatial arrangement of transmembrane helices true to scale. For analysis and statistics, all results can be downloaded as text files that may serve as inputs for or as standard data to validate the output of knowledge based tertiary structure prediction tools.URL: http://proteinformatics.charite.de/mplot/.

Predicting loop-helix tertiary structural contacts in RNA pseudoknots.

Tertiary interactions between loops and helical stems play critical roles in the biological function of many RNA pseudoknots. However, quantitative predictions for RNA tertiary interactions remain elusive. Here we report a statistical mechanical model for the prediction of noncanonical loop-stem base-pairing interactions in RNA pseudoknots. Central to the model is the evaluation of the conformational entropy for the pseudoknotted folds with defined loop-stem tertiary structural contacts. We develop an RNA virtual bond-based conformational model (Vfold model), which permits a rigorous computation of the conformational entropy for a given fold that contains loop-stem tertiary contacts. With the entropy parameters predicted from the Vfold model and the energy parameters for the tertiary contacts as inserted parameters, we can then predict the RNA folding thermodynamics, from which we can extract the tertiary contact thermodynamic parameters from theory-experimental comparisons. These comparisons reveal a contact enthalpy (DeltaH) of -14 kcal/mol and a contact entropy (DeltaS) of -38 cal/mol/K for a protonated C(+)*(G-C) base triple at pH 7.0, and (DeltaH = -7 kcal/mol, DeltaS = -19 cal/mol/K) for an unprotonated base triple. Tests of the model for a series of pseudoknots show good theory-experiment agreement. Based on the extracted energy parameters for the tertiary structural contacts, the model enables predictions for the structure, stability, and folding pathways for RNA pseudoknots with known or postulated loop-stem tertiary contacts from the nucleotide sequence alone.

How general is the nucleation-condensation mechanism?

We investigate the structures of the major folding transition states of nine proteins by correlation of published Phi-values with inter-residue contact maps. Combined with previous studies on six proteins, the analysis suggests that at least 10 of the 15 small globular proteins fold via a nucleation-condensation mechanism with a concurrent build-up of secondary and tertiary structure contacts, but a structural consolidation that is clearly nonuniformly distributed over the molecule and most intense in a single structural region suggesting the occurrence of a single folding nucleus. However, on average helix- and sheet-forming residues show somewhat larger Phi-values in the major transition state, suggesting that secondary structure formation is one important driving force in the nucleation-condensation in many proteins and that secondary-structure forming residues tend to be more prominent in folding nuclei. We synthesize the combined information on these 10 of 15 proteins into a unified nucleation-condensation mechanism which also accounts for effects described by the framework, hydrophobic collapse, zipper, and funnel models.

Folding Kinetics of Staphylococcal Nuclease Studied by Tryptophan Engineering and Rapid Mixing Methods

To monitor the development of tertiary structural contacts during folding, a unique tryptophan residue was introduced at seven partially buried locations (residues 15, 27, 61, 76, 91, 102 and 121) of a tryptophan-free variant of staphylococcal nuclease (P47G/P117G/H124L/W140H). Thermal unfolding measurements by circular dichroism indicate that the variants are destabilized, but maintain the ability to fold into a native-like structure. For the variants with Trp at positions 15, 27 and 61, the intrinsic fluorescence is significantly quenched in the native state due to close contact with polar side-chains that act as intramolecular quenchers. All other variants exhibit enhanced fluorescence under native conditions consistent with burial of the tryptophan residues in an apolar environment. The kinetics of folding was observed by continuous and stopped-flow fluorescence measurements over refolding times ranging from 100 μs to 10 s. The folding kinetics of all variants is quantitatively described by a mechanism involving a major pathway with a series of intermediate states and a minor parallel channel. The engineered tryptophan residues in the β-barrel and the N-terminal part of the α-helical domain become partially shielded from the solvent at an early stage (<1 ms), indicating that this region undergoes a rapid collapse. For some variants, a major increase in fluorescence coincides with the rate-limiting step of folding on the 100 ms time scale, indicating that these tryptophan residues are buried only during the late stages of folding. Other variants exhibit a transient increase in fluorescence during the 10 ms phase followed by a decrease during the rate-limiting phase. These observations are consistent with burial of these probes in a collapsed, but loosely packed intermediate, followed by the rate-limiting formation of the densely packed native core, which brings the tryptophan residues into close contact with intramolecular quenchers.

Mg2+-Induced Compaction of Single RNA Molecules Monitored by Tethered Particle Microscopy

We have applied tethered particle microscopy (TPM) as a single molecule analysis tool to studies of the conformational dynamics of poly-uridine(U) messenger (m)RNA and 16S ribosomal (r)RNA molecules. Using stroboscopic total internal reflection illumination and rigorous selection criteria to distinguish from nonspecific tethering, we have tracked the nanometer-scale Brownian motion of RNA-tethered fluorescent microspheres in all three dimensions at pH 7.5, 22°C, in 10mM or 100mM NaCl in the absence or presence of 10mM MgCl2. The addition of Mg2+ to low-ionic strength buffer results in significant compaction and stiffening of poly(U) mRNA, but not of 16S rRNA. Furthermore, the motion of poly(U)-tethered microspheres is more heterogeneous than that of 16S rRNA-tethered microspheres. Analysis of in-plane bead motion suggests that poly(U) RNA, but less so 16S rRNA, can be modeled both in the presence and absence of Mg2+ by a statistical Gaussian polymer model. We attribute these differences to the Mg2+-induced compaction of the relatively weakly structured and structurally disperse poly(U) mRNA, in contrast to Mg2+-induced reinforcement of existing secondary and tertiary structure contacts in the highly structured 16S rRNA. Both effects are nonspecific, however, as they are dampened in the presence of higher concentrations of monovalent cations.

Hydroxyl radical footprinting in vivo: mapping macromolecular structures with synchrotron radiation

We used a high flux synchrotron X-ray beam to map the structure of 16S rRNA and RNase P in viable bacteria in situ. A 300 ms exposure to the X-ray beam was sufficient for optimal cleavage of the phosphodiester backbone. The in vivo footprints of the 16S rRNA in frozen cells were similar to those obtained in vitro and were consistent with the predicted accessibility of the RNA backbone to hydroxyl radical. Protection or enhanced cleavage of certain nucleotides in vivo can be explained by interactions with tRNA and perturbation of the subunit interface. Thus, short exposures to a synchrotron X-ray beam can footprint the tertiary structure and protein contacts of RNA–protein complexes with nucleotide resolution in living cells.

Measurements of protein sequence-structure correlations.

Correlations between protein structures and amino acid sequences are widely used for protein structure prediction. For example, secondary structure predictors generally use correlations between a secondary structure sequence and corresponding primary structure sequence, whereas threading algorithms and similar tertiary structure predictors typically incorporate interresidue contact potentials. To investigate the relative importance of these sequence-structure interactions, we measured the mutual information among the primary structure, secondary structure and side-chain surface exposure, both for adjacent residues along the amino acid sequence and for tertiary structure contacts between residues distantly separated along the backbone. We found that local interactions along the amino acid chain are far more important than non-local contacts and that correlations between proximate amino acids are essentially uninformative. This suggests that knowledge-based contact potentials may be less important for structure predication than is generally believed.

Structural Context of Exons in Protein Domains: Implications for Protein Modelling and Design

Intron boundaries were extracted from genomic data and mapped onto single-domain human and murine protein structures taken from the Protein Data Bank. A first analysis of this set of proteins shows that intron boundaries prefer to be in non-regular secondary structure elements, while avoiding α-helices and β-strands. This fact alone suggests an evolutionary model in which introns are constrained by protein structure, particularly by tertiary structure contacts. In addition, in silico recombination experiments of a subset of these proteins together with their homologues, including those in different species, show that introns have a tendency to occur away from artificial crossover hot spots. Altogether, these findings support a model in which genes can preferentially harbour introns in less constrained regions of the protein fold they code for. In the light of these findings, we discuss some implications for protein modelling and design.

Folding Pathways of Prion and Doppel

The relevance of various residue positions for the stability and the folding characteristics of the prion protein in its normal cellular form are investigated by using molecular dynamics simulations of models exploiting the topology of the native state. These models allow for reproducing the experimentally validated two-state behavior of the normal prion isoform. Highly significant correlations are found between the most topologically relevant sites in our analysis and the single point mutations known to be associated with the arousal of the genetic forms of prion disease. Insight into the conformational change is provided by comparing the folding process of cellular prion and doppel that share a similar native state topology: the folding pathways of the former can be grouped in two main classes according to which tertiary structure contacts are formed first enroute to the native state. For the latter a single class of pathways leads to the native state again through a two-state process. Our results are consistent and supportive of the recent experimental findings that doppel lacks the scrapie isoform and that such remarkably different behavior involves residues in the region containing the two β-strands and the intervening helix.

Linkage of monovalent and divalent ion binding in the folding of the P4-P6 domain of the Tetrahymena ribozyme.

We have explored the linkage of monovalent and divalent ion binding in the folding of the P4-P6 domain of Tetrahymena thermophila ribozyme by examining the Mg2+-induced folding and the urea-induced denaturation of the folded state as a function of Na+ under equilibrium folding conditions using hydroxyl radical footprinting. These studies allowed a thermodynamic examination of eight discrete protection sites within P4-P6 that are involved in several tertiary structure contacts. Monovalent ions compete with Mg2+ ions in mediating P4-P6 folding. The urea denaturation isotherms demonstrated DeltaDeltaG values of >2 kcal x mol(-1) in experiments conducted in 10 versus 200 mM NaCl at a constant 10 mM MgCl2. However, the individual-site isotherms reported by footprinting revealed that larger than average changes in DeltaG values were localized to specific sites within the Mg2+-rich A-bulge. The competitive effects of monovalent ions were less when K+ rather than Na+ was the monovalent cation present. This result indicates the importance of the specific K+ binding sites that are associated with AA-platform structures to P4-P6 folding and stability. These site-specific footprinting data provide quantitative and site-specific measurements of the ion-linked stability for P4-P6 that are interpreted with respect to crystallographic data.

Proteolytic Sensitivity and Helper T-cell Epitope Immunodominance Associated with the Mobile Loop in Hsp10s

Antigen three-dimensional structure potentially limits antigen processing and presentation to helper T-cell epitopes. The association of helper T-cell epitopes with the mobile loop in Hsp10s from mycobacteria and bacteriophage T4 suggests that the mobile loop facilitates proteolytic processing and presentation of adjacent sequences. Sites of initial proteolytic cleavage were mapped in divergent Hsp10s after treatment with a variety of proteases including cathepsin S. Each protease preferentially cleaved the Hsp10s in the mobile loop. Flexibility in the 22-residue mobile loop most probably allows it to conform to protease active sites. Three variants of the bacteriophage T4 Hsp10 were constructed with deletions in the mobile loop to test the hypothesis that shorter loops would be less sensitive to proteolysis. The two largest deletions effectively inhibited proteolysis by several proteases. Circular dichroism spectra and chemical cross-linking of the deletion variants indicate that the secondary and quaternary structures of the variants are native-like, and all three variants were more thermostable than the wild-type Hsp10. Local structural flexibility appears to be a general requirement for proteolytic sensitivity, and thus, it could be an important factor in antigen processing and helper T-cell epitope immunogenicity.

Physical aspects of the interaction of pyrimidine and purine bases with proteins

The study of complexation of purine and pyrimidine derivatives with two kind of serum albumin using 1H NMR and spectrofluorescence technique are performed in order to obtain model interactions nucleic acid bases–protein.The order of binding strength is as follows: 5mC &gt; 5mU(T) &gt; 3mU &gt; U for pyrimidines and N6mAde &gt; N6Ado &gt; 1mAde &gt; ATP &gt; Ado &gt; Ade for adenine derivatives.The effect of temperature on the hydrophobic interaction between the nitrogen bases and protein has been studied. The destabilization of protein structure causes loss of tertiary structural contacts, indicating that protein conformation is crucial for binding.

Conformational changes of an exchangeable apolipoprotein, apolipophorin III from Locusta migratoria, at low pH: correlation with lipid binding.

Locusta migratoria apolipophorin III (apoLp-III) is a helix bundle exchangeable apolipoprotein that reversibly binds to lipoprotein surfaces. Structural reorganization of its five amphipathic alpha-helices enables the transition from the lipid-free to lipid-bound state. ApoLp-III-induced transformation of dimyristoylphosphatidylcholine (DMPC) bilayer vesicles into smaller discoidal complexes is enhanced as a function of decreasing pH, with maximal transformation occurring at pH 3.5. Over the entire pH range studied, apoLp-III retains nearly all of its secondary structure content. Whereas no changes in fluorescence emission maximum of the two Trp residues in apoLp-III were observed in the pH range from 7.0 to 4.0, a further decrease in pH resulted in a strong red shift. Near-UV circular dichroism spectra of apoLp-III showed well-defined extrema (at 286 and 292 nm) between pH 7.0 and pH 4.0, which were attributed to Trp115. Below pH 4.0, these extrema collapsed, indicating a less rigid environment for Trp115. Similarly, the fluorescence intensity of 8-anilinonaphthalene-1-sulfonate in the presence of apoLp-III increased 4-fold below pH 4.0, indicating exposure of hydrophobic sites in the protein in this pH range. Taken together, the data suggest two conformational states of the protein. In the first state between pH 7.0 and pH 4.0, apoLp-III retains a nativelike helix bundle structure. The second state, found between pH 3.0 and pH 4.0, is reminiscent of a molten globule, wherein tertiary structure contacts are disrupted without a significant loss of secondary structure content. In both states DMPC vesicle transformation is enhanced by lowering the solution pH, reaching an optimum in the second state. The correlation between tertiary structure and lipid binding activity suggests that helix bundle organization is a determinant of apoLp-III lipid binding activity.