Folding Nucleus Research Articles

The Folding of a Family of Three-Helix Bundle Proteins: Spectrin R15 Has a Robust Folding Nucleus, Unlike Its Homologous Neighbours

Three homologous spectrin domains have remarkably different folding characteristics. We have previously shown that the slow-folding R16 and R17 spectrin domains can be altered to resemble the fast folding R15, in terms of speed of folding (and unfolding), landscape roughness and folding mechanism, simply by substituting five residues in the core. Here we show that, by contrast, R15 cannot be engineered to resemble R16 and R17. It is possible to engineer a slow-folding version of R15, but our analysis shows that this protein neither has a rougher energy landscape nor does change its folding mechanism. Quite remarkably, R15 appears to be a rare example of a protein with a folding nucleus that does not change in position or in size when its folding nucleus is disrupted. Thus, while two members of this protein family are remarkably plastic, the third has apparently a restricted folding landscape.

Structural consequences of chromophore formation and exploration of conserved lid residues amongst naturally occurring fluorescent proteins

Computational methods were used to generate the lowest energy conformations of the immature precyclized forms of the 28 naturally occurring GFP-like proteins deposited in the pdb. In all 28 GFP-like proteins, the beta-barrel contracts upon chromophore formation and becomes more rigid. Our prior analysis of over 260 distinct naturally occurring GFP-like proteins revealed that most of the conserved residues are located in the top and bottom of the barrel in the turns between the β-sheets (Ong et al. 2011) [1]. Structural analyses, molecular dynamics simulations and the Anisotropic Network Model were used to explore the role of these conserved lid residues as possible folding nuclei. Our results are internally consistent and show that the conserved residues in the top and bottom lids undergo relatively less translational movement than other lid residues, and a number of these residues may play an important role as hinges or folding nuclei in the fluorescent proteins.

Alternative Folding Nuclei Definitions Facilitate the Evolution of a Symmetric Protein Fold from a Smaller Peptide Motif

Protein 3° structure symmetry is a defining feature of nearly one-third of protein folds and is generally thought to result from a combination of gene duplication, fusion, and truncation events. Such events represent major replication errors, involving substantial alteration of protein 3° structure and causing regions of exact repeating 1° structure, both of which are generally considered deleterious to protein folding. Thus, the prevalence of symmetric protein folds is counterintuitive and suggests a specific, yet unexplained, robustness. Using a designed β-trefoil protein, we show that purely symmetric 1° structure enables utilization of alternative definitions of the critical folding nucleus in response to gross structural rearrangement. Thus, major replication errors producing 1° structure symmetry can conserve foldability. The results provide an explanation for the prevalence of symmetric protein folds and highlight a critical role for 1° structure symmetry in protein evolution.

The loop hypothesis: contribution of early formed specific non-local interactions to the determination of protein folding pathways.

The extremely fast and efficient folding transition (in seconds) of globular proteins led to the search for some unifying principles embedded in the physics of the folding polypeptides. Most of the proposed mechanisms highlight the role of local interactions that stabilize secondary structure elements or a folding nucleus as the starting point of the folding pathways, i.e., a "bottom-up" mechanism. Non-local interactions were assumed either to stabilize the nucleus or lead to the later steps of coalescence of the secondary structure elements. An alternative mechanism was proposed, an "up-down" mechanism in which it was assumed that folding starts with the formation of very few non-local interactions which form closed long loops at the initiation of folding. The possible biological advantage of this mechanism, the "loop hypothesis", is that the hydrophobic collapse is associated with ordered compactization which reduces the chance for degradation and misfolding. In the present review the experiments, simulations and theoretical consideration that either directly or indirectly support this mechanism are summarized. It is argued that experiments monitoring the time-dependent development of the formation of specifically targeted early-formed sub-domain structural elements, either long loops or secondary structure elements, are necessary. This can be achieved by the time-resolved FRET-based "double kinetics" method in combination with mutational studies. Yet, attempts to improve the time resolution of the folding initiation should be extended down to the sub-microsecond time regime in order to design experiments that would resolve the classes of proteins which first fold by local or non-local interactions.

Effects of Confinement on the Structure and Dynamics of an Intrinsically Disordered Peptide: A Molecular-Dynamics Study

In vivo, proteins and peptides are exposed to radically different environments than those in bulk. Because of the abundance of other cellular components, proteins perform their function in crowded and confined spaces. Confinement has been shown to alter the structure, dynamics, and folding of proteins that possess a native fold. Little is known, however, of the effects of confinement on biologically important intrinsically disordered proteins or peptides (IDP). Here, we use extensive molecular dynamics simulations to investigate the effects of confinement in an IDP, the Aβ21-30, a central folding nucleus of the full length amyloid β-protein. In this study, we report results derived from 107 μs of molecular dynamics simulations that subjected the Aβ21-30 to two types of confinement: hydrophilic and hydrophobic pores. Results show that turn structures are enhanced as a function of decreasing pore size (increasing confinement) over other structures, including coils, β-hairpins, and bridges. However, the percentage occurrence of the dominant hydrogen bond between amino acids Asp23 and Ser26 shown to stabilize the turn in bulk simulations does not increase as a function of confinement signifying a disconnect between structure and internal hydrogen bonding. Differences in structure and dynamics of the decapeptide due to hydrophilic and hydrophobic confinement are more apparent at the extreme confinement conditions, where a reduction of the available phase space in hydrophilic confinement is explained in terms of interactions between the decapeptide and a layer of water at the interface between the decapeptide and the surface of the pore, and a smaller size of the decapeptide in the hydrophobic pores is rationalized in terms of peptide-surface interactions.

3P263 beta-Trefoilタンパクのフォールディングコアの残基間平均距離統計に基づく解析(22A.生命情報科学:構造ゲノミクス,ポスター,日本生物物理学会年会第51回(2013年度))

3P263 beta-Trefoilタンパクのフォールディングコアの残基間平均距離統計に基づく解析(22A.生命情報科学:構造ゲノミクス,ポスター,日本生物物理学会年会第51回(2013年度))

Structural Dynamics of Iowa, Dutch, and Arctic Mutations of the 21-30 Fragment of Amyloid Beta under Aqueous Salt Environments

The amyloid β protein (Aβ) has been implicated in the pathogenesis of Alzheimer's disease. Previous in vitro experiments have shown that the central Aβ(21-30) fragment may have special importance because it may act as a folding nucleus of the full-length protein. Recently, experiments of the full-length wild type (WT) peptide under aqueous salt environments have revealed varied responses in both the structure and growth of aggregates of the full-length peptide under these environments. Here we use all-atom molecular dynamics simulations of monomeric Aβ(21-30) to examine pre-aggregate structural alterations under similar dissolved salt conditions (CaCl2 and KCl). Further, we make use of the wild-type and three common mutations of the decapeptide (Arctic[E22/G22], Dutch[E22/Q22], and Iowa[D23/N23]) to explore the possible dependence of charged side-chain and salt-ion interactions in driving structural changes under aqueous salt environments. Our results indicate that the production and stability of open (random-coil) structures is enhanced under CaCl2 for the wild-type decapeptide, and both Dutch and Iowa mutations; while KCl environments enhanced the production of turn structures for wild-type and β-structures for the Iowa and Dutch mutations. Additionally, we present a possible explanation for these differences in structural response as a combination of volume exclusion, ion-residue interactions, and ion effects on the hydration of the decapeptide.

Tolerance of Protein Folding to a Circular Permutation in a PDZ Domain

Circular permutation is a common molecular mechanism for evolution of proteins. However, such re-arrangement of secondary structure connectivity may interfere with the folding mechanism causing accumulation of folding intermediates, which in turn can lead to misfolding. We solved the crystal structure and investigated the folding pathway of a circularly permuted variant of a PDZ domain, SAP97 PDZ2. Our data illustrate how well circular permutation may work as a mechanism for molecular evolution. The circular permutant retains the overall structure and function of the native protein domain. Further, unlike most examples in the literature, this circular permutant displays a folding mechanism that is virtually identical to that of the wild type. This observation contrasts with previous data on the circularly permuted PDZ2 domain from PTP-BL, for which the folding pathway was remarkably affected by the same mutation in sequence connectivity. The different effects of this circular permutation in two homologous proteins show the strong influence of sequence as compared to topology. Circular permutation, when peripheral to the major folding nucleus, may have little effect on folding pathways and could explain why, despite the dramatic change in primary structure, it is frequently tolerated by different protein folds.

Experimental support for the foldability–function tradeoff hypothesis: Segregation of the folding nucleus and functional regions in fibroblast growth factor‐1

The acquisition of function is often associated with destabilizing mutations, giving rise to the stability-function tradeoff hypothesis. To test whether function is also accommodated at the expense of foldability, fibroblast growth factor-1 (FGF-1) was subjected to a comprehensive φ-value analysis at each of the 11 turn regions. FGF-1, a β-trefoil fold, represents an excellent model system with which to evaluate the influence of function on foldability: because of its threefold symmetric structure, analysis of FGF-1 allows for direct comparisons between symmetry-related regions of the protein that are associated with function to those that are not; thus, a structural basis for regions of foldability can potentially be identified. The resulting φ-value distribution of FGF-1 is highly polarized, with the majority of positions described as either folded-like or denatured-like in the folding transition state. Regions important for folding are shown to be asymmetrically distributed within the protein architecture; furthermore, regions associated with function (i.e., heparin-binding affinity and receptor-binding affinity) are localized to regions of the protein that fold after barrier crossing (late in the folding pathway). These results provide experimental support for the foldability-function tradeoff hypothesis in the evolution of FGF-1. Notably, the results identify the potential for folding redundancy in symmetric protein architecture with important implications for protein evolution and design.

Nucleation‐based prediction of the protein folding rate and its correlation with the folding nucleus size

The problem of protein self-organization is in the focus of current molecular biology studies. Although the general principles are understood, many details remain unclear. Specifically, protein folding rates are of interest because they dictate the rate of protein aggregation which underlies many human diseases. Here we offer predictions of protein folding rates and their correlation with folding nucleus sizes. We calculated free energies of the transition state and sizes of folding nuclei for 84 proteins and peptides whose other parameters were measured at the point of thermodynamic equilibrium between their unfolded and native states. We used the dynamic programming method where each residue was considered to be either as folded as in its native state or completely disordered. The calculated and measured folding rates showed a good correlation at the temperature mid-transition point (the correlation coefficient was 0.75). Also, we pioneered in demonstrating a moderate (-0.57) correlation coefficient between the calculated sizes of folding nuclei and the folding rates. Predictions made by different methods were compared. The established good correlation between the estimated free energy barrier and the experimentally found folding rate of each studied protein/peptide indicates that our model gives reliable results for the considered data set.

Dynamics of metastable β-hairpin structures in the folding nucleus of amyloid β-protein.

The amyloid β-protein (Aβ), which is present predominately as a 40- or 42-residue peptide, is postulated to play a seminal role in the pathogenesis of Alzheimer's disease (AD). Folding of the Aβ(21-30) decapeptide region is a critical step in the aggregation of Aβ. We report results of constant temperature all-atom molecular dynamics simulations in explicit water of the dynamics of monomeric Aβ(21-30) and its Dutch [Glu22Gln], Arctic [Glu22Gly], and Iowa [Asp23Asn] isoforms that are associated with familial forms of cerebral amyloid angiopathy and AD. The simulations revealed a variety of loop conformers that exhibited a hydrogen bond network involving the Asp23 and Ser26 amino acids. A population of conformers, not part of the loop population, was found to form metastable β-hairpin structures with the highest probability in the Iowa mutant. At least three β-hairpin structures were found that differed in their hydrogen bonding register, average number of backbone hydrogen bonds, and lifetimes. Analysis revealed that the Dutch mutant had the longest β-hairpin lifetime (≥500 ns), closely followed by the Iowa mutant (≈500 ns). Aβ(21-30) and the Arctic mutant had significantly lower lifetimes (≈200 ns). Hydrophobic packing of side chains was responsible for enhanced β-hairpin lifetimes in the Dutch and Iowa mutants, whereas lifetimes in Aβ(21-30) and its Arctic mutant were influenced by the backbone hydrogen bonding. The data suggest that prolonged β-hairpin lifetimes may impact peptide pathogenicity in vivo.

Structural Dynamics of the Amyloid β-Protein Monomer Folding Nucleus

Alzheimer's disease (AD) is linked to the aberrant assembly of the amyloid β-protein (Aβ). The (21)AEDVGSNKGA(30) segment, Aβ(21-30), forms a turn that acts as a monomer folding nucleus. Amino acid substitutions within this nucleus cause familial forms of AD. To determine the biophysical characteristics of the folding nucleus, we studied the biologically relevant acetyl-Aβ(21-30)-amide peptide using experimental techniques (limited proteolysis, thermal denaturation, urea denaturation followed by pulse proteolysis, and electron microscopy) and computational methods (molecular dynamics). Our results reveal a highly stable foldon and suggest new strategies for therapeutic drug development.

Molecular dynamics studies of β-hairpin folding with the presence of the sodium ion

Metal ions are ubiquitous in protein systems and play a significant role during their folding processes. Nineteen independent structures were determined for the Na+/β-hairpin interacting systems, and their folding pathways are different. (i) For NaS47, the turn is rapidly shaped with the help of Na+ and acts as the folding nucleus for the rest regions. Two intermediate states are observed and the resulted structure is the most folded. (ii) For NaB41, NaB52, NaB54, NaS55 and NaB56, the inclusive Na+ ions are anchored by β-strands. The local structures around the Na+ ions and the turn regions fold simultaneously and serve as two independent folding nuclei. (iii) The other systems have no folding nuclei and correspond to low-folded structures. Long-range electrostatic interactions contribute a lot to the folding, especially from the four negatively charged residues (Glu42, Asp46, Asp47 and Glu56). The initial positions of the Na+ ions are largely responsible for the different folding behaviors. The interactions with sidechain- rather than backbone-O atoms generally lead to more compact structures. Another factor affecting the folding is whether the O atoms are associated with native H-bonds, and those involved show decreased affinities to metal ions. The addition of water solvent does not induce obvious folding and conformational transitions to the Na+/β-hairpin interacting systems.

Identification of Domains in Protein Structures from the Analysis of Intramolecular Interactions

The subdivision of protein structures into smaller and independent structural domains has a fundamental importance in understanding protein evolution and function and in the development of protein classification methods as well as in the interpretation of experimental data. Due to the rapid growth in the number of solved protein structures, the need for devising new accurate algorithmic methods has become more and more urgent. In this paper, we propose a new computational approach that is based on the concept of domain as a compact and independent folding unit and on the analysis of the residue-residue energy interactions obtainable through classical all-atom force field calculations. In particular, starting from the analysis of the nonbonded interaction energy matrix associated with a protein, our method filters out and selects only those specific subsets of interactions that define possible independent folding nuclei within a complex protein structure. This allows grouping different protein fragments into energy clusters that are found to correspond to structural domains. The strategy has been tested using proper benchmark data sets, and the results have shown that the new approach is fast and reliable in determining the number of domains in a totally ab initio manner and without making use of any training set or knowledge of the systems in exam. Moreover, our method, identifying the most relevant residues for the stabilization of each domain, may complement the results given by other classification techniques and may provide useful information to design and guide new experiments.

Formation of RNA spatial structures

The review considers different experimental and theoretical approaches to the investigation of RNA folding and identification of nucleotides that critically affect the folding of molecules, such as tRNA, and several classes of ribozymes. For instance, it has been shown that nucleotides of the D- and T-loop regions are the last to be involved in the tRNA structure, or, rather, they are not included in the tRNA folding nucleus. A specially developed SHAPE method was used to show that the long-recognized hierarchical folding model does not hold true for tRNA folding. In the second part of the review, algorithms and programs used for the prediction of RNA secondary structures, as well as for modeling RNA folding, are considered.

Trimming Down a Protein Structure to Its Bare Foldons: SPATIAL ORGANIZATION OF THE COOPERATIVE UNIT

Folding of the ribosomal protein S6 is a malleable process controlled by two competing, and partly overlapping, folding nuclei. Together, these nuclei extend over most of the S6 structure, except the edge strand β2, which is consistently missing in the folding transition states; despite being part of the S6 four-stranded sheet, β2 seems not to be part of the cooperative unit of the protein. The question is then whether β2 can be removed from the S6 structure without compromising folding cooperativity or native state integrity. To investigate this, we constructed a truncated variant of S6 lacking β2, reducing the size of the protein from 96 to 76 residues (S6(Δβ2)). The new S6 variant expresses well in Escherichia coli and has a well dispersed heteronuclear single quantum correlation spectrum and a perfectly wild-type-like crystal structure, but with a smaller three-stranded β-sheet. Moreover, S6(Δβ2) displays an archetypical v-shaped chevron plot with decreased slope of the unfolding limb, as expected from a protein with maintained folding cooperativity and reduced size. The results support the notion that foldons, as defined by the structural distribution of the folding nuclei, represent a property-based level of hierarchy in the build-up of larger protein structures and suggest that the role of β2 in S6 is mainly in intermolecular binding, consistent with the position of this strand in the ribosomal assembly.

Analyses of Protein Sequences Using Inter-Residue Average Distance Statistics to Study Folding Processes and the Significance of Their Partial Sequences

One of the goals of molecular bioinformatics is decoding amino acid sequences to extract information on the principles of protein folding. However, this is difficult to perform with standard bioinformatics techniques such as multiple sequence alignment and so on. Thus, we propose a technique based on inter-residue average distance statistics to make predictions regarding the protein folding mechanisms of amino acid sequences. Our method involves constructing a kind of predicted contact map called an Average Distance Map (ADM) based on average distance statistics to pinpoint regions of possible folding nuclei for proteins. Only information on the amino acid sequence of a given protein is required for the present method. In this article, we summarize the results of studies using our method to analyze how specific protein sequences affect folding properties. In particular, we present studies on proteins in the phage lysozyme, such as the globin, fatty acid binding protein-like, and the cupredoxin-like fold families. In the present review, we characterize the 3D architectures of these proteins through the properties of the protein ADMs. Furthermore, we combine the information on the conserved residues within the regions predicted by the ADMs with our results obtained so far. Such information may help identify the folding characteristics of each protein. We discuss this possibility in the present review.

Stable folding core in the folding transition state of an α-helical integral membrane protein

Defining the structural features of a transition state is important in understanding a folding reaction. Here, we use Φ-value and double mutant analyses to probe the folding transition state of the membrane protein bacteriorhodopsin. We focus on the final C-terminal helix, helix G, of this seven transmembrane helical protein. Φ-values could be derived for 12 amino acid residues in helix G, most of which have low or intermediate values, suggesting that native structure is disrupted at these amino acid positions in the transition state. Notably, a cluster of residues between E204 and M209 all have Φ-values close to zero. Disruption of helix G is further confirmed by a low Φ-value of 0.2 between residues T170 on helix F and S226 on helix G, suggesting the absence of a native hydrogen bond between helices F and G. Φ-values for paired mutations involved in four interhelical hydrogen bonds revealed that all but one of these bonds is absent in the transition state. The unstructured helix G contrasts with Φ-values along helix B that are generally high, implying native structure in helix B in the transition state. Thus helix B seems to constitute part of a stable folding nucleus while the consolidation of helix G is a relatively late folding event. Polarization of secondary structure correlates with sequence position, with a structured helix B near the N terminus contrasting with an unstructured C-terminal helix G.

Role of glycosylation in structure and stability of Erythrina corallodendron lectin (EcorL): A molecular dynamics study

The effect of glycosylation on structure and stability of glycoproteins has been a topic of considerable interest. In this work, we have investigated the solution conformation of the oligosaccharide and its effect on the structure and stability of the glycoprotein by carrying out a series of long Molecular dynamics (MD) simulations on glycosylated Erythrina corallodendron lectin (EcorL) and nonglycosylated recombinant Erythrina corallodendron lectin (rEcorL). Our results indicate that, despite the similarity in overall three dimensional structures, glycosylated EcorL has lesser nonpolar solvent accessible surface area compared to nonglycosylated EcorL. This might explain the experimental observation of higher thermodynamic stability for glycosylated EcorL compared to nonglycosylated EcorL. Analysis of the simulation results indicates that, dynamic view of interactions between protein residues and oligosaccharide is entirely different from the static picture seen in the crystal structure. The oligosaccharide moiety had dynamically stable interactions with Lys 55 and Tyr 53, both of which are separated in sequence from the site of glycosylation, Asn 17. It is possible that glycosylation helps in forming long-range contacts between amino acids, which are separated in sequence and thus provides a folding nucleus. Thus our simulations not only reveal the conformations sampled by the oligosaccharide, but also provide novel insights into possible molecular mechanisms by which glycosylation can help in folding of the glycoprotein by formation of folding nucleus involving specific contacts with the oligosaccharide moiety.

Prediction of folding nuclei in tRNA molecules

Prediction of folding nuclei in RNA molecules allows one to look in a new way at the problem of possible RNA base sequence folding and at problems associated with incorrect RNA folding, as well as at RNA structure stability. We have chosen a model and energy parameters for description of RNA structure. The algorithm for studying processes including protein folding/unfolding was successfully applied to calculations on tRNA. Four tRNA molecules were considered whose structures were obtained in the free state (tRNA(Phe), tRNA(Asp), tRNA(fMet), and tRNA(Lys)). The calculated Φ-values for tRNA molecules correlate with experimental data showing that nucleotide residues in the D and T hairpin regions are involved in tRNA structure last, or more exactly, they are not included in the tRNA folding nucleus. High Φ-values in the anticodon hairpin region show that the nucleus of tRNA folding is localized just in that place.