Folding and Stability of Ankyrin Repeats Control Biological Protein Function.
Ontology highlight
ABSTRACT:
SUBMITTER: Kumar A
PROVIDER: S-EPMC8229355 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:BackgroundTandem repetition of structural motifs in proteins is frequently observed across all forms of life. Topology of repeating unit and its frequency of occurrence are associated to a wide range of structural and functional roles in diverse proteins, and defects in repeat proteins have been associated with a number of diseases. It is thus desirable to accurately identify specific repeat type and its copy number. Weak evolutionary constraints on repeat units and insertions/deletions between them make their identification difficult at the sequence level and structure based approaches are desired. The proposed graph spectral approach is based on protein structure represented as a graph for detecting one of the most frequently observed structural repeats, Ankyrin repeat.ResultsIt has been shown in a large number of studies that 3-dimensional topology of a protein structure is well captured by a graph, making it possible to analyze a complex protein structure as a mathematical entity. In this study we show that eigen spectra profile of a protein structure graph exhibits a unique repetitive profile for contiguous repeating units enabling the detection of the repeat region and the repeat type. The proposed approach uses a non-redundant set of 58 Ankyrin proteins to define rules for the detection of Ankyrin repeat motifs. It is evaluated on a set of 370 proteins comprising 125 known Ankyrin proteins and remaining non-solenoid proteins and the prediction compared with UniProt annotation, sequence-based approach, RADAR, and structure-based approach, ConSole. To show the efficacy of the approach, we analyzed the complete PDB structural database and identified 641 previously unrecognized Ankyrin repeat proteins. We observe a unique eigen spectra profile for different repeat types and show that the method can be easily extended to detect other repeat types. It is implemented as a web server, AnkPred. It is freely available at 'bioinf.iiit.ac.in/AnkPred'.ConclusionsAnkPred provides an elegant and computationally efficient graph-based approach for detecting Ankyrin structural repeats in proteins. By analyzing the eigen spectra of the protein structure graph and secondary structure information, characteristic features of a known repeat family are identified. This method is especially useful in correctly identifying new members of a repeat family.
| S-EPMC4307672 | biostudies-literature
Project description:Previous studies demonstrated importance of C-mannosylation for efficient protein secretion. To study its impact on protein folding and stability, we analyzed both C-mannosylated and non-C-mannosylated thrombospondin type 1 repeats (TSRs) of netrin receptor UNC-5. In absence of C-mannosylation, UNC-5 TSRs could only be obtained at low temperature and a significant proportion displayed incorrect intermolecular disulfide bridging, which was hardly observed when C-mannosylated. Glycosylated TSRs exhibited higher resistance to thermal and reductive denaturation processes, and the presence of C-mannoses promoted the oxidative folding of a reduced and denatured TSR in vitro. Molecular dynamics simulations supported the experimental studies and showed that C-mannoses can be involved in intramolecular hydrogen bonding and limit the flexibility of the TSR tryptophan-arginine ladder. We propose that in the endoplasmic reticulum folding process, C-mannoses orient the underlying tryptophan residues and facilitate the formation of the tryptophan-arginine ladder, thereby influencing the positioning of cysteines and disulfide bridging.
| S-EPMC6954052 | biostudies-literature
Project description:Repeat proteins are constructed from a linear array of modular units, giving rise to an overall topology lacking long-range interactions. This suggests that stabilizing repeat modules based on consensus information might be added to a repeat protein domain, allowing it to be extended without altering its overall topology. Here we add consensus modules the ankyrin repeat domain from the Drosophila Notch receptor to investigate the structural tolerance to these modules, the relative thermodynamic stability of these hybrid proteins, and how alterations in the energy landscape influence folding kinetics. Insertions of consensus modules between repeats five and six of the Notch ankyrin domain have little effect on the far and near-UV CD spectra, indicating that neither secondary nor tertiary structure is dramatically altered. Furthermore, stable structure is maintained at increased denaturant concentrations in the polypeptides containing the consensus repeats, indicating that the consensus modules are capable of stabilizing much of the domain. However, insertion of the consensus repeats appears to disrupt cooperativity, producing a two-stage (three-state) unfolding transition in which the C-terminal repeats unfold at moderate urea concentrations. Removing the C-terminal repeats (Notch ankyrin repeats six and seven) restores equilibrium two-state folding and demonstrates that the high stability of the consensus repeats is propagated into the N-terminal, naturally occurring Notch ankyrin repeats. This stability increase greatly increases the folding rate, and suggests that the transition state ensemble may be repositioned in the chimeric consensus-stabilized proteins in response to local stability.
| S-EPMC1851695 | biostudies-literature
Project description:Red blood cells are frequently deformed and their cytoskeletal proteins such as spectrin and ankyrin-R are repeatedly subjected to mechanical forces. While the mechanics of spectrin was thoroughly investigated in vitro and in vivo, little is known about the mechanical behavior of ankyrin-R. In this study, we combine coarse-grained steered molecular dynamics simulations and atomic force spectroscopy to examine the mechanical response of ankyrin repeats (ARs) in a model synthetic AR protein NI6C, and in the D34 fragment of native ankyrin-R when these proteins are subjected to various stretching geometry conditions. Our steered molecular dynamics results, supported by AFM measurements, reveal an unusual mechanical anisotropy of ARs: their mechanical stability is greater when their unfolding is forced to propagate from the N-terminus toward the C-terminus (repeats unfold at ~60 pN), as compared to the unfolding in the opposite direction (unfolding force ∼ 30 pN). This anisotropy is also reflected in the complex refolding behavior of ARs. The origin of this unfolding and refolding anisotropy is in the various numbers of native contacts that are broken and formed at the interfaces between neighboring repeats depending on the unfolding/refolding propagation directions. Finally, we discuss how these complex mechanical properties of ARs in D34 may affect its behavior in vivo.
| S-EPMC3296030 | biostudies-literature
Project description:An N-terminally truncated and cooperatively folded version (residues 6-39) of the human Pin1 WW domain (hPin1 WW hereafter) has served as an excellent model system for understanding triple-stranded beta-sheet folding energetics. Here we report that the negatively charged N-terminal sequence (Met1-Ala-Asp-Glu-Glu5) previously deleted, and which is not conserved in highly homologous WW domain family members from yeast or certain fungi, significantly increases the stability of hPin1 WW (approximately 4 kJ mol(-1) at 65 degrees C), in the context of the 1-39 sequence based on equilibrium measurements. N-terminal truncations and mutations in conjunction with a double mutant cycle analysis and a recently published high-resolution X-ray structure of the hPin1 cis/trans-isomerase suggest that the increase in stability is due to an energetically favorable ionic interaction between the negatively charged side chains in the N terminus of full-length hPin1 WW and the positively charged epsilon-ammonium group of residue Lys13 in beta-strand 1. Our data therefore suggest that the ionic interaction between Lys13 and the charged N terminus is the optimal solution for enhanced stability without compromising function, as ascertained by ligand binding studies. Kinetic laser temperature-jump relaxation studies reveal that this stabilizing interaction has not formed to a significant extent in the folding transition state at near physiological temperature, suggesting a differential contribution of the negatively charged N-terminal sequence to protein stability and folding rate. As neither the N-terminal sequence nor Lys13 are highly conserved among WW domains, our data further suggest that caution must be exercised when selecting domain boundaries for WW domains for structural, functional, or thermodynamic studies.
| S-EPMC2206688 | biostudies-literature
Project description:Human granulocytic ehrlichiosis (HGE) is a potentially fatal, tick-borne disease caused by a bacterium related or identical to Ehrlichia phagocytophila. To identify and characterize E. phagocytophila group-specific protein antigen genes, we prepared and screened HGE agent and Ehrlichia equi genomic DNA expression libraries using polyclonal equine E. equi antibodies. Two clones, one each from HGE agent and E. equi, that were recognized specifically by antibodies to the E. phagocytophila group ehrlichiae had complete open reading frames of 3,693 and 3,615 nucleotides, respectively. The two clones were 96.6% identical and predicted a protein with at least 11 tandemly repeated ankyrin motifs. Thus, the gene was named ank (for ankyrin). When the encoded protein, named AnkA, was expressed in Escherichia coli, it was recognized by antibodies from rabbits and mice immunized with the HGE agent, sera from humans convalescent from HGE, and sera from horses convalescent from HGE and E. equi infection. Monospecific AnkA antibodies reacted with proteins in HGE agent immunoblots, and AnkA monoclonal antibodies detected cytoplasmic antigen in E. phagocytophila group bacteria and also detected antigen associated with chromatin in infected but not uninfected HL-60 cell cultures. These results suggest that this Ehrlichia protein may influence host cell gene expression.
| S-EPMC101789 | biostudies-literature
Project description:Using single-molecule atomic force microscopy, we find that a protein consisting of six identical ankyrin repeat units flanked by N- and C-terminal modules (N6C) unfolds in a stepwise, unit-by-unit fashion under a mechanical force. Stretching a N6C molecule results in a sawtooth pattern fingerprint, with as many as six peaks separated by approximately 10 nm and an average unfolding force of 50 +/- 20 pN. Our results demonstrate that a stretching force can unfold multiple repeat units individually in a single protein molecule, despite extensive hydrophobic interactions between adjacent units.
| S-EPMC1367297 | biostudies-literature
Project description:Repeat proteins are widespread in nature, with many of them functioning as binding molecules in protein-protein recognition. Their simple structural architecture is used in biotechnology for generating proteins with high affinities to target proteins. Recent folding studies of ankyrin repeat (AR) proteins revealed a new mechanism of protein folding. The formation of an intermediate state is rate limiting in the folding reaction, suggesting a scaffold function of this transient state for intrinsically less stable ARs. To investigate a possible common mechanism of AR folding, we studied the structure and folding of a new thermophilic AR protein (tANK) identified in the archaeon Thermoplasma volcanium. The x-ray structure of the evolutionary much older tANK revealed high homology to the human CDK inhibitor p19(INK4d), whose sequence was used for homology search. As for p19(INK4d), equilibrium and kinetic folding analyses classify tANK to the family of sequential three-state folding proteins, with an unusual fast equilibrium between native and intermediate state. Under equilibrium conditions, the intermediate can be populated to >90%, allowing characterization on a residue-by-residue level using NMR spectroscopy. These data clearly show that the three C-terminal ARs are natively folded in the intermediate state, whereas native cross-peaks for the rest of the molecule are missing. Therefore, the formation of a stable folding unit consisting of three ARs is the necessary rate-limiting step before AR 1 and 2 can assemble to form the native state.
| S-EPMC2268769 | biostudies-literature
Project description:Ankyrin protein repeats bind to a wide range of substrates and are one of the most common protein motifs in nature. Here, we collate a high-quality alignment of 7,407 ankyrin repeats and examine for the first time, the distribution of human population variants from large-scale sequencing of healthy individuals across this family. Population variants are not randomly distributed across the genome but are constrained by gene essentiality and function. Accordingly, we interpret the population variants in context with evolutionary constraint and structural features including secondary structure, accessibility and protein-protein interactions across 383 three-dimensional structures of ankyrin repeats. We find five positions that are highly conserved across homologues and also depleted in missense variants within the human population. These positions are significantly enriched in intra-domain contacts and so likely to be key for repeat packing. In contrast, a group of evolutionarily divergent positions are found to be depleted in missense variants in human and significantly enriched in protein-protein interactions. Our analysis also suggests the domain has three, not two surfaces, each with different patterns of enrichment in protein-substrate interactions and missense variants. Our findings will be of interest to those studying or engineering ankyrin-repeat containing proteins as well as those interpreting the significance of disease variants.
| S-EPMC8415598 | biostudies-literature
Project description:Conditional control of protein function in vivo offers great potential for deconvoluting the roles of individual proteins in complicated systems. We recently developed a method in which a small protein domain, termed a destabilizing domain, confers instability to fusion protein partners in cultured cells. Instability is reversed when a cell-permeable small molecule binds this domain. Here we describe the use of this system to regulate protein function in living mammals. We show regulation of secreted proteins and their biological activity with conditional secretion of an immunomodulatory cytokine, resulting in tumor burden reduction in mouse models. Additionally, we use this approach to control the function of a specific protein after systemic delivery of the gene that encodes it to a tumor, suggesting uses for enhancing the specificity and efficacy of targeted gene-based therapies. This method represents a new strategy to regulate protein function in living organisms with a high level of control.
| S-EPMC2605277 | biostudies-literature