Do Intrinsically Disordered Proteins Possess High Specificity in Protein–Protein Interactions?

Specific protein–protein interactions are critical to cellular function. Structural flexibility and disorder‐to‐order transitions upon binding enable intrinsically disordered proteins (IDPs) to overcome steric restrictions and form complementary binding interfaces, and thus, IDPs are widely considered to have high specificity and low affinity for molecular recognition. However, flexibility may also enable IDPs to form complementary binding interfaces with misbinding partners, resulting in a great number of nonspecific interactions. Consequently, it is questionable whether IDPs really possess high specificity. In this work, we investigated this question from a thermodynamic viewpoint. We collected mutant thermodynamic data for 35 ordered protein complexes and 43 disordered protein complexes. We found that the enthalpy–entropy compensation for disordered protein complexes was more complete than that for ordered protein complexes. We further simulated the binding processes of ordered and disordered protein complexes under mutations. Simulation data confirmed the observation of experimental data analyses and further revealed that disordered protein complexes possessed smaller changes in binding free energy than ordered protein complexes under the same mutation perturbations. Therefore, interactions of IDPs are more malleable than those of ordered proteins due to their structural flexibility in the complex. Our results provide new clues for exploring the relationship between protein flexibility, adaptability, and specificity.     

Monitoring the Interaction of α-Synuclein with Calcium Ions through Exclusively Heteronuclear Nuclear Magnetic Resonance Experiments

Many properties of intrinsically disordered proteins (IDPs), or protein regions (IDRs), are modulated by the nature of amino acid side chains as well as by local solvent exposure. We propose a set of exclusively heteronuclear NMR experiments to investigate these features in different experimental conditions that are relevant for physiological function. The proposed approach is generally applicable to many IDPs/IDRs whose assignment is available in the Biological Magnetic Resonance Bank (BMRB) to investigate how their properties are modulated by different, physiologically relevant conditions. The experiments, tested on α-synuclein, are then used to investigate how α-synuclein senses Ca²⁺ concentration jumps associated with the transmission of nerve signals. Novel modules in the primary sequence of α-synuclein optimized for calcium sensing in highly flexible, disordered protein segments are identified.     

The importance of the compact disordered state in the fuzzy interactions between intrinsically disordered proteins

The intrinsically disordered C-terminal domain (CTD) of protein 4.1G is able to specifically bind a 26-residue intrinsically disordered region of NuMA, forming a dynamic fuzzy complex. As one of a few cases of extremely fuzzy interactions between two intrinsically disordered proteins/regions (IDPs/IDRs) without induced folding, the principle of the binding is unknown. Here, we combined experimental and computational methods to explore the detailed mechanism of the interaction between 4.1G-CTD and NuMA. MD simulations suggest that the kinetic hub states in the structure ensemble of 4.1G-CTD are favorable in the fuzzy complex. The feature of these hub states is that the binding ‘hot spot’ motifs βA and βB exhibit β strand propensities and are well packed to each other. The binding between 4.1G-CTD and NuMA is disrupted at low pH, which changes the intramolecular packing of 4.1G-CTD and weakens the packing between βA and βB motifs. Low pH conditions also lead to increased hydrodynamic radius and acceleration of backbone dynamics of 4.1G-CTD. All these results underscore the importance of tertiary structural arrangements and overall compactness of 4.1G-CTD in its binding to NuMA, i.e. the compact disordered state of 4.1G-CTD is crucial for binding. Different from the short linear motifs (SLiMs) that are often found to mediate IDP interactions, 4.1G-CTD functions as an intrinsically disordered domain (IDD), which is a functional and structural unit similar to conventional protein domains. This work sheds light on the molecular recognition mechanism of IDPs/IDRs and expands the conventional structure-function paradigm in protein biochemistry.

Tertiary structural arrangements and overall compactness is important for interactions between IDPs.     

Evidence for an Ordering Transition near 120 K in an Intrinsically Disordered Protein,Casein

Intrinsically disordered proteins (IDPs) are proteins that possess large unstructured regions. Their importance is increasingly recognized in biology but their characterization remains a challenging task. We employed field swept Electron Spin Echoes in pulsed EPR to investigate low-temperature stochastic molecular librations in a spin-labeled IDP, casein (the main protein of milk). For comparison, a spin-labeled globular protein, hen egg white lysozyme, is also investigated. For casein these motions were found to start at 100 K while for lysozyme only above 130 K, which was ascribed to a denser and more ordered molecular packing in lysozyme. However, above 120 K, the motions in casein were found to depend on temperature much slower than those in lysozyme. This abrupt change in casein was assigned to an ordering transition in which peptide residues rearrange making the molecular packing more rigid and/or more cohesive. The found features of molecular motions in these two proteins turned out to be very similar to those known for gel-phase lipid bilayers composed of conformationally ordered and conformationally disordered lipids. This analogy with a simpler molecular system may appear helpful for elucidation properties of molecular packing in IDPs.     

Molecular evolution of the plant ECERIFERUM1 and ECERIFERUM3 genes involved in aliphatic hydrocarbon production

The Arabidopsis ECERIFERUM1 (CER1) protein is a decarbonylase that converts fatty acid metabolites into alkanes. Alkanes are components of waxes in the plant cuticle, a waterproof barrier serving to protect land plants from both biotic and abiotic stimuli. CER1 enzymes can be used to produce alternative and sustainable hydrocarbons in eukaryotic systems. In this report we identified 193 CER1 and 128 CER3 sequences from 56 land plants respectively. CER1 and CER3 proteins have high amino acid similarity and both are involved in alkane synthesis in Arabidopsis. The common homologues of CER1 and CER3 genes were identified in three species of chlorophytes, which may be one of the earliest plant taxa that possess CER1 and CER3 genes. To facilitate potential applications, the 3-dimensional structure and conserved motifs of CER1 proteins were also characterized. CER1 and CER3 proteins are structurally similar, but CER1 proteins have more conserved histidine-containing motifs common to fatty acid hydroxylases and stearoyl-CoA desaturases. There was no significant loss or gain of protein motifs after ancient and recent duplications, suggesting that varied properties of CER1 proteins may be associated with less-conserved regions. Among 56 land plants, the codon-based assessments of selection modes revealed that neither entire proteins nor individual amino acids of CER1 proteins were significantly subjected to positive selection, indicating that CER1 proteins are highly conserved throughout evolution.     

Detection of discriminative sequence motifs in proteins obtained from prokaryotes grown at various temperatures

Recent investigations on the stability of proteins have demonstrated various structural factors, but few have considered sequence factors such as protein motifs. These motifs represent highly conserved regions and describe critical regions that may only exist on proteins that remain functional at high temperatures. This investigation presents a method for identifying and comparing corresponding mesophilic and thermophilic sequence motifs between protein families. Discriminative motifs that are conserved only in the mesophilic or thermophilic subfamily are identified. Analysis of the results shows that, although the subfamilies of most protein families share similar motifs, some discriminative motifs are present in particular thermophilic/mesophilic subfamilies. The thermophilic discriminative motifs are conserved only in thermophilic organisms, revealing that physiochemical principles support thermostability.     

Rate constants and mechanisms of intrinsically disordered proteins binding to structured targets

The binding of intrinsically disordered proteins (IDPs) to structured targets is gaining increasing attention. Here we review experimental and computational studies on the binding kinetics of IDPs. Experiments have yielded both the binding rate constants and the binding mechanisms, the latter via mutation and deletion studies and NMR techniques. Most computational studies have aimed at qualitative understanding of the binding rate constants or at mapping the free energy surfaces after the IDPs are engaged with their targets. The experiments and computation show that IDPs generally gain structures after they are engaged with their targets; that is, interactions with the targets facilitate the IDPs' folding. It also seems clear that the initial contact of an IDP with the target is formed by just a segment, not the entire IDP. The docking of one segment to its sub-site followed by coalescing of other segments around the corresponding sub-sites emerges as a recurring feature in the binding of IDPs. Such a dock-and-coalesce model forms the basis for quantitative calculation of binding rate constants. For both disordered and ordered proteins, strong electrostatic attraction with their targets can enhance the binding rate constants by several orders of magnitude. There are now tremendous opportunities in narrowing the gap in our understanding of IDPs relative to ordered proteins with regard to binding kinetics.     

From cofactor to enzymes. The molecular evolution of pyridoxal-5'-phosphate-dependent enzymes

The pyridoxal-5'-phosphate (vitamin B(6))-dependent enzymes that act on amino acid substrates have multiple evolutionary origins. Thus, the common mechanistic features of B(6) enzymes are not accidental historical traits but reflect evolutionary or chemical necessities. The B(6) enzymes belong to four independent evolutionary lineages of paralogous proteins, of which the alpha family (with aspartate aminotransferase as the prototype enzyme) is by far the largest and most diverse. The considerably smaller beta family (tryptophan synthase beta as the prototype enzyme) is structurally and functionally more homogenous. Both the D-alanine aminotransferase family and the alanine racemase family consist of only a few enzymes. The primordial pyridoxal-5'-phosphate-dependent protein catalysts apparently first diverged into reaction-specific protoenzymes, which then diverged further by specializing for substrate specificity. Aminotransferases as well as amino acid decarboxylases are found in two different evolutionary lineages, providing examples of convergent enzyme evolution. The functional specialization of most B(6) enzymes seems to have already occurred in the universal ancestor cell before the divergence of eukaryotes, archebacteria, and eubacteria 1500 million years ago. Pyridoxal-5'-phosphate must have emerged very early in biological evolution; conceivably, metal ions and organic cofactors were the first biological catalysts. To simulate particular steps of molecular evolution, both the substrate and reaction specificity of existent B(6) enzymes were changed by substitution of active-site residues, and monoclonal pyridoxal-5'-phosphate-dependent catalytic antibodies were produced with selection criteria that might have been operative in the evolution of protein-assisted pyridoxal catalysis.     

Potential of fragment recombination for rational design of proteins

It is hypothesized that protein domains evolved from smaller intrinsically stable subunits via combinatorial assembly. Illegitimate recombination of fragments that encode protein subunits could have quickly led to diversification of protein folds and their functionality. This evolutionary concept presents an attractive strategy to protein engineering, e.g., to create new scaffolds for enzyme design. We previously combined structurally similar parts from two ancient protein folds, the (βα)(8)-barrel and the flavodoxin-like fold. The resulting "hopeful monster" differed significantly from the intended (βα)(8)-barrel fold by an extra β-strand in the core. In this study, we ask what modifications are necessary to form the intended structure and what potential this approach has for the rational design of functional proteins. Guided by computational design, we optimized the interface between the fragments with five targeted mutations yielding a stable, monomeric protein whose predicted structure was verified experimentally. We further tested binding of a phosphorylated compound and detected that some affinity was already present due to an intact phosphate-binding site provided by one fragment. The affinity could be improved quickly to the level of natural proteins by introducing two additional mutations. The study illustrates the potential of recombining protein fragments with unique properties to design new and functional proteins, offering both a possible pathway of protein evolution and a protocol to rapidly engineer proteins for new applications.     

Proteus: a random forest classifier to predict disorder-to-order transitioning binding regions in intrinsically disordered proteins

The focus of the computational structural biology community has taken a dramatic shift over the past one-and-a-half decades from the classical protein structure prediction problem to the possible understanding of intrinsically disordered proteins (IDP) or proteins containing regions of disorder (IDPR). The current interest lies in the unraveling of a disorder-to-order transitioning code embedded in the amino acid sequences of IDPs/IDPRs. Disordered proteins are characterized by an enormous amount of structural plasticity which makes them promiscuous in binding to different partners, multi-functional in cellular activity and atypical in folding energy landscapes resembling partially folded molten globules. Also, their involvement in several deadly human diseases (e.g. cancer, cardiovascular and neurodegenerative diseases) makes them attractive drug targets, and important for a biochemical understanding of the disease(s). The study of the structural ensemble of IDPs is rather difficult, in particular for transient interactions. When bound to a structured partner, an IDPR adapts an ordered conformation in the complex. The residues that undergo this disorder-to-order transition are called protean residues, generally found in short contiguous stretches and the first step in understanding the modus operandi of an IDP/IDPR would be to predict these residues. There are a few available methods which predict these protean segments from their amino acid sequences; however, their performance reported in the literature leaves clear room for improvement. With this background, the current study presents ‘Proteus’, a random forest classifier that predicts the likelihood of a residue undergoing a disorder-to-order transition upon binding to a potential partner protein. The prediction is based on features that can be calculated using the amino acid sequence alone. Proteus compares favorably with existing methods predicting twice as many true positives as the second best method (55 vs. 27%) with a much higher precision on an independent data set. The current study also sheds some light on a possible ‘disorder-to-order’ transitioning consensus, untangled, yet embedded in the amino acid sequence of IDPs. Some guidelines have also been suggested for proceeding with a real-life structural modeling involving an IDPR using Proteus.     

Structure-Function Insights of Jaburetox and Soyuretox: Novel Intrinsically Disordered Polypeptides Derived from Plant Ureases

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) do not have a stable 3D structure but still have important biological activities. Jaburetox is a recombinant peptide derived from the jack bean (Canavalia ensiformis) urease and presents entomotoxic and antimicrobial actions. The structure of Jaburetox was elucidated using nuclear magnetic resonance which reveals it is an IDP with small amounts of secondary structure. Different approaches have demonstrated that Jaburetox acquires certain folding upon interaction with lipid membranes, a characteristic commonly found in other IDPs and usually important for their biological functions. Soyuretox, a recombinant peptide derived from the soybean (Glycine max) ubiquitous urease and homologous to Jaburetox, was also characterized for its biological activities and structural properties. Soyuretox is also an IDP, presenting more secondary structure in comparison with Jaburetox and similar entomotoxic and fungitoxic effects. Moreover, Soyuretox was found to be nontoxic to zebra fish, while Jaburetox was innocuous to mice and rats. This profile of toxicity affecting detrimental species without damaging mammals or the environment qualified them to be used in biotechnological applications. Both peptides were employed to develop transgenic crops and these plants were active against insects and nematodes, unveiling their immense potentiality for field applications.     

Novel NMR Assignment Strategy Reveals Structural Heterogeneity in Solution of the nsP3 HVD Domain of Venezuelan Equine Encephalitis Virus

In recent years, intrinsically disordered proteins (IDPs) and disordered domains have attracted great attention. Many of them contain linear motifs that mediate interactions with other factors during formation of multicomponent protein complexes. NMR spectrometry is a valuable tool for characterizing this type of interactions on both amino acid (aa) and atomic levels. Alphaviruses encode a nonstructural protein nsP3, which drives viral replication complex assembly. nsP3 proteins contain over 200-aa-long hypervariable domains (HVDs), which exhibits no homology between different alphavirus species, are predicted to be intrinsically disordered and appear to be critical for alphavirus adaptation to different cells. Previously, we have shown that nsP3 HVD of chikungunya virus (CHIKV) is completely disordered with low tendency to form secondary structures in free form. In this new study, we used novel NMR approaches to assign the spectra for the nsP3 HVD of Venezuelan equine encephalitis virus (VEEV). The HVDs of CHIKV and VEEV have no homology but are both involved in replication complex assembly and function. We have found that VEEV nsP3 HVD is also mostly disordered but contains a short stable α-helix in its C-terminal fragment, which mediates interaction with the members of cellular Fragile X syndrome protein family. Our NMR data also suggest that VEEV HVD has several regions with tendency to form secondary structures.     

Targeting an Interaction Between Two Disordered Domains by Using a Designed Peptide

Intrinsically disordered regions in proteins (IDRs) mediate many disease-related protein–protein interactions. However, the unfolded character and continuous conformational changes of IDRs make them difficult to target for therapeutic purposes. Here, we show that a designed peptide based on the disordered p53 linker domain can be used to target a partner IDR from the anti-apoptotic iASPP protein, promoting apoptosis of cancer cells. The p53 linker forms a hairpin-like structure with its two termini in close proximity. We designed a peptide derived from the disordered termini without the hairpin, designated as p53 LinkTer. The LinkTer peptide binds the disordered RT loop of iASPP with the same affinity as the parent p53 linker peptide, and inhibits the p53–iASPP interaction in vitro. The LinkTer peptide shows increased stability to proteolysis, penetrates cancer cells, causes nuclei shrinkage, and compromises the viability of cells. We conclude that a designed peptide comprising only the IDR from a peptide sequence can serve as an improved inhibitor since it binds its target protein without the need for pre-folding, paving the way for therapeutic targeting of IDRs.     

How random are intrinsically disordered proteins? A small angle scattering perspective

While the crucial role of intrinsically disordered proteins (IDPs) in the cell cycle is now recognized, deciphering their molecular mode of action at the structural level still remains highly challenging and requires a combination of many biophysical approaches. Among them, small angle X-ray scattering (SAXS) has been extremely successful in the last decade and has become an indispensable technique for addressing many of the fundamental questions regarding the activities of IDPs. After introducing some experimental issues specific to IDPs and in relation to the latest technical developments, this article presents the interest of the theory of polymer physics to evaluate the flexibility of fully disordered proteins. The different strategies to obtain 3-dimensional models of IDPs, free in solution and associated in a complex, are then reviewed. Indeed, recent computational advances have made it possible to readily extract maximum information from the scattering curve with a special emphasis on highly flexible systems, such as multidomain proteins and IDPs. Furthermore, integrated computational approaches now enable the generation of ensembles of conformers to translate the unique flexible characteristics of IDPs by taking into consideration the constraints of more and more various complementary experiment. In particular, a combination of SAXS with high-resolution techniques, such as x-ray crystallography and NMR, allows us to provide reliable models and to gain unique structural insights about the protein over multiple structural scales. The latest neutron scattering experiments also promise new advances in the study of the conformational changes of macromolecules involving more complex systems.     

Genetic algorithms as a tool for helix design – computational and experimental studies on prion protein helix 1

Summary Evolutionary computing is a general optimization mechanism successfully implemented for a variety of numeric problems in a variety of fields, including structural biology. We here present an evolutionary approach to optimize helix stability in peptides and proteins employing the AGADIR energy function for helix stability as scoring function. With the ability to apply masks determining positions, which are to remain constant or fixed to a certain class of amino acids, our algorithm is capable of developing stable helical scaffolds containing a wide variety of structural and functional amino acid patterns. The algorithm showed good convergence behaviour in all tested cases and can be parameterized in a wide variety of ways. We have applied our algorithm for the optimization of the stability of prion protein helix 1, a structural element of the prion protein which is thought to play a crucial role in the conformational transition from the cellular to the pathogenic form of the prion protein, and which therefore poses an interesting target for pharmacological as well as genetic engineering approaches to counter the as of yet uncurable prion diseases. NMR spectroscopic investigations of selected stabilizing and destabilizing mutations found by our algorithm could demonstrate its ability to create stabilized variants of secondary structure elements.     

Structural,functional, and evolutionary analysis of late embryogenesis abundant proteins (LEA) in Triticum aestivum: A detailed molecular level biochemistry using in silico approach

LEA (Late Embryogenesis Abundant) proteins are abundant in plants and play a crucial role in abiotic stress tolerance. In our work, we primarily focused on the variations in physiochemical properties, conserved domains, secondary structure, gene ontology and evolutionary relationships among 40 LEA proteins of Triticum aestivum (common wheat). Wheat LEA protein belongs to first 6 classes out of the 13 classes present in LEApdB, the comprehensive database for LEA proteins. Proteins belonging to each LEApdB class have structures and functions distinguished from other classes. The study found three different conserved LEA domains in Triticum aestivum. One important domain was dehydrin, present in wheat proteins of classes 1, 2 and 4, though varied in sequence level, have similar biological processes. The study also found sequence level and phylogenetic similarity between dehydrin domains of class 1 and 4, but distinct from that of LEApdB class 2. This study also demonstrated functional diversity in two class 6 proteins occurred due to many destabilizing mutations in the LEA4 domain that caused alteration of ligand binding and conformational shift from 3₁₀-helix → turn within the domain. The LEA4 domains of these proteins also showed functional similarity and evolutionary relatedness with three other proteins of genus Aegilops, denoting that these proteins in Triticum aestivum were derived from its ancestor Aegilops. The study also assigned LEApdB class 4 to an unclassified LEA protein ‘WZY2-1’ based on amino acid composition, conserved domain, motif architecture and phylogenetic relatedness with class 4 proteins. Our study has revealed a detailed analysis of LEA proteins in Triticum aestivum and can serve as a pillar for further investigations and comparative analysis of wheat LEA proteins with other cereal or plant types.     

Analysis of sequence repeats of proteins in the PDB

Internal repeats in protein sequences play a significant role in the evolution of protein structure and function. Applications of different bioinformatics tools help in the identification and characterization of these repeats. In the present study, we analyzed sequence repeats in a non-redundant set of proteins available in the Protein Data Bank (PDB). We used RADAR for detecting internal repeats in a protein, PDBeFOLD for assessing structural similarity, PDBsum for finding functional involvement and Pfam for domain assignment of the repeats in a protein. Through the analysis of sequence repeats, we found that identity of the sequence repeats falls in the range of 20–40% and, the superimposed structures of the most of the sequence repeats maintain similar overall folding. Analysis sequence repeats at the functional level reveals that most of the sequence repeats are involved in the function of the protein through functionally involved residues in the repeat regions. We also found that sequence repeats in single and two domain proteins often contained conserved sequence motifs for the function of the domain.     

Molecular phylogenetic study and expression analysis of ATP-binding cassette transporter gene family in Oryza sativa in response to salt stress

ATP-binding cassette (ABC) transporter is a large gene superfamily that utilizes the energy released from ATP hydrolysis for transporting myriad of substrates across the biological membranes. Although many investigations have been done on the structural and functional analysis of the ABC transporters in Oryza sativa, much less is known about molecular phylogenetic and global expression pattern of the complete ABC family in rice. In this study, we have carried out a comprehensive phylogenetic analysis constructing neighbor-joining and maximum-likelihood trees based on various statistical methods of different ABC protein subfamily of five plant lineages including Chlamydomonas reinhardtii (green algae), Physcomitrella patens (moss), Selaginella moellendorffii (lycophyte), Arabidopsis thaliana (dicot) and O. sativa (monocot) to explore the origin and evolutionary patterns of these ABC genes. We have identified several conserved motifs in nucleotide binding domain (NBD) of ABC proteins among all plant lineages during evolution. Amongst the different ABC protein subfamilies, ‘ABCE’ has not yet been identified in lower plant genomes (algae, moss and lycophytes). The result indicated that gene duplication and diversification process acted upon these genes as a major operative force creating new groups and subgroups and functional divergence during evolution. We have demonstrated that rice ABCI subfamily consists of only half size transporters that represented highly dynamic members showing maximum sequence variations among the other rice ABC subfamilies. The evolutionary and the expression analysis contribute to a deep insight into the evolution and diversity of rice ABC proteins and their roles in response to salt stress that facilitate our further understanding on rice ABC transporters.     

Forced Folding of a Disordered Protein Accesses an Alternative Folding Landscape

Intrinsically disordered proteins (IDPs) are involved in diverse cellular functions. Many IDPs can interact with multiple binding partners, resulting in their folding into alternative ligand‐specific functional structures. For such multi‐structural IDPs, a key question is whether these multiple structures are fully encoded in the protein sequence, as is the case in many globular proteins. To answer this question, here we employed a combination of single‐molecule and ensemble techniques to compare ligand‐induced and osmolyte‐forced folding of α‐synuclein. Our results reveal context‐dependent modulation of the protein′s folding landscape, suggesting that the codes for the protein′s native folds are partially encoded in its primary sequence, and are completed only upon interaction with binding partners. Our findings suggest a critical role for cellular interactions in expanding the repertoire of folds and functions available to disordered proteins.     

Mapping the potential energy landscape of intrinsically disordered proteins at amino Acid resolution

Intrinsically disordered regions are predicted to exist in a significant fraction of proteins encoded in eukaryotic genomes. The high levels of conformational plasticity of this class of proteins endows them with unique capacities to act in functional modes not achievable by folded proteins, but also places their molecular characterization beyond the reach of classical structural biology. New techniques are therefore required to understand the relationship between primary sequence and biological function in this class of proteins. Although dependences of some NMR parameters such as chemical shifts (CSs) or residual dipolar couplings (RDCs) on structural propensity are known, so that sampling regimes are often inferred from experimental observation, there is currently no framework that allows for a statistical mapping of the available Ramachandran space of each amino acid in terms of conformational propensity. In this study we develop such an approach, combining highly efficient conformational sampling with ensemble selection to map the backbone conformational sampling of IDPs on a residue specific level. By systematically analyzing the ability of NMR data to map the conformational landscape of disordered proteins, we identify combinations of RDCs and CSs that can be used to raise conformational degeneracies inherent to different data types, and apply these approaches to characterize the conformational behavior of two intrinsically disordered proteins, the K18 domain from Tau protein and N(TAIL) from measles virus nucleoprotein. In both cases, we identify the enhanced populations of turn and helical regions in key regions of the proteins, as well as contiguous strands that show clear and enhanced polyproline II sampling.