Supersecondary Structure Research Articles

Stable Substructures in Proteins and How to Find Them Using Single-Molecule Force Spectroscopy.

Three-dimensional structures of proteins are a source of fascination for scientists, due to the beauty of their sequence-encoded architectures and their highly diverse range of functions. These functions include acting as powerful catalysts, signal receptors, and versatile molecular motors as well as being building blocks for macroscopic structures, thus defining the shape of multicellular organisms. How protein structure is organized and assembled at the sub-nanometer scale is of great current interest. Specifically, the discovery of stable substructures and supersecondary structures has inspired research into their potential use in rationally engineered proteins with tailor-made properties. Here, we show how the search for stable substructures in large proteins can benefit from recent advances in single-molecule force spectroscopy using highly sensitive dual-beam optical tweezers. Our chapter provides a step-by-step description of the experimental workflow for (1) preparing proteins for mechanical interrogation, (2) interpreting the data, and (3) avoiding the most commonly occurring mistakes.

Beyond Supersecondary Structure: Physics-Based Sequence Alignment.

Traditional approaches to sequence alignment are based on evolutionary ideas. As a result, they are prebiased toward results which are in accord with initial expectations. We present here a method of sequence alignment which is based entirely on the physical properties of the amino acids. This approach has no inherent bias, eliminates much of the computational complexity associated with methods currently in use, and has been shown to give good results for structures which were poorly predicted by traditional methods in recent CASP competitions and to identify sequence differences which correlate with structural and dynamic differences not detectable by traditional methods.

Exploring Protein Supersecondary Structure Through Changes in Protein Folding, Stability, and Flexibility.

The ability to predict how mutations affect protein structure, folding, and flexibility can elucidate the molecular mechanisms leading to disruption of supersecondary structures, the emergence of phenotypes, as well guiding rational protein engineering. The advent of fast and accurate computational tools has enabled us to comprehensively explore the landscape of mutation effects on protein structures, prioritizing mutations for rational experimental validation.Here we describe the use of two complementary web-based in silico methods, DUET and DynaMut, developed to infer the effects of mutations on folding, stability, and flexibility and how they can be used to explore and interpret these effects on protein supersecondary structures.

βαβ Super-Secondary Motifs: Sequence, Structural Overview, and Pursuit of Potential Autonomously Folding βαβ Sequences from (β/α)8/TIM Barrels.

βαβ super-secondary structures constitute the basic building blocks of (β/α)8 class of proteins. Despite the success in designing super-secondary structures, till date, there is not a single example of a natural βαβ sequence known to fold in isolation. In this chapter, to address the finding the "needles" in the haystack scenario, we have combined the sequence preferences and structural features of independent βαβ motifs, dictated by natural selection, with rationally derived parameters from a designed βαβ motif adopting stable fold in solution. Guided by this approach, a set of potential βαβ sequences from (β/α)8/TIM barrels are proposed as likely candidates for autonomously folding based on the assessment of their foldability.

Formation of Cross-Beta Supersecondary Structure by Soft-Amyloid Cores: Strategies for Their Prediction and Characterization.

Proteins with prion-like behavior are attracting an increasing interest, since accumulating evidences indicate that they play relevant roles both in health and disease. The self-assembly of these proteins into insoluble aggregates is associated with severe neuropathological processes such as amyotrophic lateral sclerosis (ALS). However, in normal conditions, they are known to accomplish a wide range of functional roles. The conformational duality of prion-like proteins is often encoded in specific protein regions, named prion-like domains (PrLDs). PrLDs are usually long and disordered regions of low complexity. We have shown that PrLDs might contain soft-amyloid cores that contribute significantly to trigger their aggregation, as well as to support their propagation. Further exploration of the role of these sequences in the conformational conversion of prion-like proteins might provide novel insights into the mechanism of action and regulation of these polypeptides, enabling the future development of therapeutic strategies. Here, we describe a set of methodologies aimed to identify and characterize these short amyloid stretches in a protein or proteome of interest, ranging from in silico detection to in vitro and in vivo evaluation and validation.

Secondary and Supersecondary Structure of Proteins in Light of the Structure of Hydrophobic Cores.

The traditional classification of protein structures (with regard to their supersecondary and tertiary conformation) is based on an assessment of conformational similarities between various polypeptide chains and particularly on the presence of specific secondary structural motifs. Mutual relations between secondary folds determine the overall shape of the protein and may be used to assign proteins to specific families (such as the immunoglobulin-like family). An alternative means of conducting structural assessment focuses on the structure of the protein's hydrophobic core. In this case, the protein is treated as a quasi-micelle, which exposes hydrophilic residues on its surface while internalizing hydrophobic residues. The accordance between the actual distribution of hydrophobicity in a protein and its corresponding theoretical ("idealized") distribution can be determined quantitatively, which, in turn, enables comparative analysis of structures regarded as geometrically similar (as well as geometrically divergent structures which are nevertheless regarded as similar in the sense of the fuzzy oil drop model). In this scope, the protein may be compared to an "intelligent micelle," where local disorder is often intentional and related to biological function-unlike traditional surfactant micelles which remain highly symmetrical throughout and do not carry any encoded information.

Formation of Secondary and Supersecondary Structure of Proteins as a Result of Coupling Between Local and Backbone-Electrostatic Interactions: A View Through Cluster-Cumulant Scope.

The secondary structure of proteins results from both local and long-range interactions, the latter being primarily backbone hydrogen bonding. In this chapter, based on our recent work, we suggest that the striking regularity of secondary structure can be described, in a semi-analytical manner, in terms of Kubo cluster cumulants (corresponding to the expansion of the protein's potential of mean force) that originate from the coupling between the backbone-local and backbone-electrostatic interactions. This finding is illustrated by the analysis of the Protein Data Bank statistics. Examples demonstrating the importance of the coupling terms in coarse-grained treatment of proteins are also presented.

Asymmetric protein design from conserved supersecondary structures

Computational design with supersecondary structures as building blocks has proven effective in the construction of new proteins with controlled geometries. So far, this approach has primarily exploited amplification, effectively harnessing the internal folding propensity of self-compatible fragments to achieve sufficient enthalpy for folding. Here we exploit an interface-driven strategy to depart from the repeat design realm, constructing an asymmetric, globular domain from heterologous supersecondary structures. We report the successful design of a dRP lyase domain fold, which agrees with the experimental NMR structure at atomic accuracy (backbone RMSD of 0.94 Å). Our results show that the residual folding information within conserved fragments, combined with efficient interface-directed sampling, can effectively yield globular proteins with novel sequences and biophysical properties.

Designed peptide with a flexible central motif from ranatuerins adapts its conformation to bacterial membranes

The long-standing goal in the field of peptide antibiotics has been to design lead compounds that have a wide spectrum of excellent antibacterial activity but are nontoxic to human cells. Gram-negative and Gram-positive bacteria have very different membranes, which are additionally modified in some drug-resistant species, presenting a challenge for the design of a single membrane-active peptide able to adapt its conformation to various physical properties of membrane microenvironments. In this paper, we describe how a peptide sequence can be constructed starting from an adaptable dynamic turn tandem motif in a central location. The peptide, named flexampin, has been examined firstly by molecular dynamics simulations. It uses a flexible central motif and designed helix-forming cationic amphipathic arms to form a boomerang-like, L-shape, V-shape, and hairpin, super-secondary structures, whichever is the best in matching amphipathic and hydrophobic microenvironments it encounters. Secondly, activity measurements showed that flexampin is bactericidal at low micromolar concentrations against Gram-positive and Gram-negative strains including some multidrug resistant clinical isolates, while it is nontoxic for human circulating blood cells, does not cause DNA damage, and has good selectivity for bacterial cells in comparison to human cells. It is the first membrane-active peptide designed with the ability to self-adjust the orientation of its two cationic helical arms, 3D-hydrophobic moment, and dipole moment for obtaining a better grasp of anionic polar head groups at bacterial membrane surfaces.

Using random forest algorithm to predict super-secondary structure in proteins

A new super-secondary structure dataset with sequence identity < 25%, resolution better than 2.0 A, which contained 2805 non-redundant protein chains and could be classified into four motifs, is built for prediction. The matrix scoring values and hydropathy distribution are extracted from the protein sequences, which are then input to the random forest algorithm to predict the super-secondary structure in proteins. The predictive overall accuracy is 72.71% and 68.54% by fivefold cross-validation and independent dataset test, respectively. The proposed method is also tested on a previous independent test dataset and the predictive overall accuracy 84.89%, which is better than the performance of the previous predictions.

Super Secondary Structure Consisting of a Polyproline II Helix and a \u03b2-Turn in Leucine Rich Repeats in Bacterial Type III Secretion System Effectors

Leucine rich repeats (LRRs) are present in over 100,000 proteins from viruses to eukaryotes. The LRRs are 20–30 residues long and occur in tandem. LRRs form parallel stacks of short β-strands and then assume a super helical arrangement called a solenoid structure. Individual LRRs are separated into highly conserved segment (HCS) with the consensus of LxxLxLxxNxL and variable segment (VS). Eight classes have been recognized. Bacterial LRRs are short and characterized by two prolines in the VS; the consensus is xxLPxLPxx with Nine residues (N-subtype) and xxLPxxLPxx with Ten residues (T-subtype). Bacterial LRRs are contained in type III secretion system effectors such as YopM, IpaH3/9.8, SspH1/2, and SlrP from bacteria. Some LRRs in decorin, fribromodulin, TLR8/9, and FLRT2/3 from vertebrate also contain the motifs. In order to understand structural features of bacterial LRRs, we performed both secondary structures assignments using four programs—DSSP-PPII, PROSS, SEGNO, and XTLSSTR—and HELFIT analyses (calculating helix axis, pitch, radius, residues per turn, and handedness), based on the atomic coordinates of their crystal structures. The N-subtype VS adopts a left handed polyproline II helix (PPII) with four, five or six residues and a type I β-turn at the C-terminal side. Thus, the N-subtype is characterized by a super secondary structure consisting of a PPII and a β-turn. In contrast, the T-subtype VS prefers two separate PPIIs with two or three and two residues. The HELFIT analysis indicates that the type I β-turn is a right handed helix. The HELFIT analysis determines three unit vectors of the helix axes of PPII (P), β-turn (B), and LRR domain (A). Three structural parameters using these three helix axes are suggested to characterize the super secondary structure and the LRR domain.

Molecular cloning, bioinformatics analysis, and expression of small heat shock protein beta-1 from Camelus dromedarius, Arabian camel.

Small heat shock protein beta-1 (HSPB-1) plays an essential role in the protection of cells against environmental stress.Elucidation of its molecular, structural, and biological characteristics in a naturally wild-type model is essential. Although the sequence information of the HSPB-1 gene is available for many mammalian species, the HSPB-1 gene of Arabian camel (Arabian camel HSPB-1) has not yet been structurally characterized. We cloned and functionally characterized a full-length of Arabian camel HSPB-1 cDNA. It is 791 bp long, with a 5′-untranslated region (UTR) of 34 bp, a 3′-UTR of 151 bp with a poly(A) tail, and an open reading frame (ORF) of 606 bp encoding a protein of 201 amino acids (accession number: MF278354). The tissue-specific expression analysis of Arabian camel HSPB-1 mRNA was examined using quantitative real-time PCR (qRT-PCR); which suggested that Arabian camel HSPB-1 mRNA was constitutionally expressed in all examined tissues of Arabian camel, with the predominately level in the esophagus tissue. Peptide mass fingerprint-mass spectrometry (PMF-MS) analysis of the purified Arabian camel HSPB-1 protein confirmed the identity of this protein. Phylogenetic analysis showed that the HSPB-1 protein of Arabian camel is grouped together with those of Bactrian camel and Alpaca. Comparing the modelled 3D structure of Arabian camel HSPB-1 protein with the available protein 3D structure of HSPB-1 from human confirmed the presence of α-crystallin domain, and high similarities were noted between the two structures by using super secondary structure prediction.

Rapid and reliable protein structure determination via chemical shift threading.

Protein structure determination using nuclear magnetic resonance (NMR) spectroscopy can be both time-consuming and labor intensive. Here we demonstrate how chemical shift threading can permit rapid, robust, and accurate protein structure determination using only chemical shift data. Threading is a relatively old bioinformatics technique that uses a combination of sequence information and predicted (or experimentally acquired) low-resolution structural data to generate high-resolution 3D protein structures. The key motivations behind using NMR chemical shifts for protein threading lie in the fact that they are easy to measure, they are available prior to 3D structure determination, and they contain vital structural information. The method we have developed uses not only sequence and chemical shift similarity but also chemical shift-derived secondary structure, shift-derived super-secondary structure, and shift-derived accessible surface area to generate a high quality protein structure regardless of the sequence similarity (or lack thereof) to a known structure already in the PDB. The method (called E-Thrifty) was found to be very fast (often < 10min/structure) and to significantly outperform other shift-based or threading-based structure determination methods (in terms of top template model accuracy)-with an average TM-score performance of 0.68 (vs. 0.50-0.62 for other methods). Coupled with recent developments in chemical shift refinement, these results suggest that protein structure determination, using only NMR chemical shifts, is becoming increasingly practical and reliable. E-Thrifty is available as a web server at http://ethrifty.ca .

Structure of the C-terminal domain of TRADD reveals a novel fold in the death domain superfamily

The TNFR1-associated death domain protein (TRADD) is an intracellular adaptor protein involved in various signaling pathways, such as antiapoptosis. Its C-terminal death domain (DD) is responsible for binding other DD-containing proteins including the p75 neurotrophin receptor (p75NTR). Here we present a solution structure of TRADD DD derived from high-resolution NMR spectroscopy. The TRADD DD comprises two super-secondary structures, an all-helix Greek key motif and a β-hairpin motif flanked by two α helices, which make it unique among all known DD structures. The β-hairpin motif is essential for TRADD DD to fold into a functional globular domain. The highly-charged surface suggests a critical role of electrostatic interactions in TRADD DD-mediated signaling. This novel structure represents a new class within the DD superfamily and provides a structural basis for studying homotypic DD interactions. NMR titration revealed a direct weak interaction between TRADD DD and p75NTR DD monomers. A binding site next to the p75NTR DD homodimerization interface indicates that TRADD DD recruitment to p75NTR requires separation of the p75NTR DD homodimer, explaining the mechanism of NGF-dependent activation of p75NTR-TRADD-mediated antiapoptotic pathway in breast cancer cell.

Ribosomal proteins as documents of the transition from unstructured (poly)peptides to folded proteins.

For the most part, contemporary proteins can be traced back to a basic set of a few thousand domain prototypes, many of which were already established in the Last Universal Common Ancestor of life on Earth, around 3.5 billion years ago. The origin of these domain prototypes, however, remains poorly understood. One hypothesis posits that they arose from an ancestral set of peptides, which acted as cofactors of RNA-mediated catalysis and replication. Initially, these peptides were entirely dependent on the RNA scaffold for their structure, but as their complexity increased, they became able to form structures by excluding water through hydrophobic contacts, making them independent of the RNA scaffold. Their ability to fold was thus an emergent property of peptide-RNA coevolution. The ribosome is the main survivor of this primordial RNA world and offers an excellent model system for retracing the steps that led to the folded proteins of today, due to its very slow rate of change. Close to the peptidyl transferase center, which is the oldest part of the ribosome, proteins are extended and largely devoid of secondary structure; further from the center, their secondary structure content increases and supersecondary topologies become common, although the proteins still largely lack a hydrophobic core; at the ribosomal periphery, supersecondary structures coalesce around hydrophobic cores, forming folds that resemble those seen in proteins of the cytosol. Collectively, ribosomal proteins thus offer a window onto the time when proteins were acquiring the ability to fold.

Protein Structure Determination by Assembling Super-Secondary Structure Motifs Using Pseudocontact Shifts

Computational and nuclear magnetic resonance hybrid approaches provide efficient tools for 3D structure determination of small proteins, but currently available algorithms struggle to perform with larger proteins. Here we demonstrate a new computational algorithm that assembles the 3D structure of a protein from its constituent super-secondary structural motifs (Smotifs) with the help of pseudocontact shift (PCS) restraints for backbone amide protons, where the PCSs are produced from different metal centers. The algorithm, DINGO-PCS (3D assembly of Individual Smotifs to Near-native Geometry as Orchestrated by PCSs), employs the PCSs to recognize, orient, and assemble the constituent Smotifs of the target protein without any other experimental data or computational force fields. Using a universal Smotif database, the DINGO-PCS algorithm exhaustively enumerates any given Smotif. We benchmarked the program against ten different protein targets ranging from 100 to 220 residues with different topologies. For nine of these targets, the method was able to identify near-native Smotifs.

Frozen in Time: The History of Proteins.

The ribosome is imprinted with a detailed molecular chronology of the origins and early evolution of proteins. Here we show that when arranged by evolutionary phase of ribosomal evolution, ribosomal protein (rProtein) segments reveal an atomic level history of protein folding. The data support a model in which aboriginal oligomers evolved into globular proteins in a hierarchical step-wise process. Complexity of assembly and folding of polypeptide increased incrementally in concert with expansion of rRNA. (i) Short random coil proto-peptides bound to rRNA, and (ii) lengthened over time and coalesced into β–β secondary elements. These secondary elements (iii) accreted and collapsed, primarily into β-domains. Domains (iv) accumulated and gained complex super-secondary structures composed of mixtures of α-helices and β-strands. Early protein evolution was guided and accelerated by interactions with rRNA. rRNA and proto-peptide provided mutual protection from chemical degradation and disassembly. rRNA stabilized polypeptide assemblies, which evolved in a stepwise process into globular domains, bypassing the immense space of random unproductive sequences. Coded proteins originated as oligomers and polymers created by the ribosome, on the ribosome and for the ribosome. Synthesis of increasingly longer products was iteratively coupled with lengthening and maturation of the ribosomal exit tunnel. Protein catalysis appears to be a late byproduct of selection for sophisticated and finely controlled assembly.

Formation of toroids by self-assembly of an α-α corner mimetic: supramolecular cyclization.

An α-α corner mimetic self-assembles to form a rod-like supramolecular structure which bends and closes end-to-end like a cyclization reaction to form uniform toroids. Each peptide fragment containing l-leucine, α-aminoisobutyric acid (Aib) and l-tyrosine forms rigid 310 helical structures stabilized by multiple intramolecular N-HO hydrogen bonds. Two 310 helices are connected by the spacer 3-aminomethyl-benzylamine and maintain an angular distance of 120° and therefore mimic the α-α corner motif of a protein super secondary structure. The individual α-α corner subunits are themselves regularly interlinked through multiple water mediated intermolecular hydrogen-bonding interactions to form the rod-like supramolecular structure and toroids. The formation of the supramolecular structure has been proven with X-ray crystallography and other spectroscopic techniques. The cyclization of the supramolecular structure and toroid formation were studied by optical microscope, AFM and FE-SEM experiments. Despite other assignments such as exfoliation of graphene from graphite, the compound exhibits significant memory to finally produce the toroids.

From the Evolution of Protein Sequences Able to Resist Self-Assembly to the Prediction of Aggregation Propensity.

Folding of polypeptide chains into biologically active entities is an astonishingly complex process, determined by the nature and the sequence of residues emerging from ribosomes. While it has been long believed that evolution has pressed genomes so that specific sequences could adopt unique, functional three-dimensional folds, it is now clear that complex protein machineries act as quality control system and supervise folding. Notwithstanding that, events such as erroneous folding, partial folding, or misfolding are frequent during the life of a cell or a whole organism, and they can escape controls. One of the possible outcomes of this misbehavior is cross-β aggregation, a super secondary structure which represents the hallmark of self-assembled, well organized, and extremely ordered structures termed amyloid fibrils. What if evolution would have not taken into account such possibilities? Twenty years of research point toward the idea that, in fact, evolution has constantly supervised the risk of errors and minimized their impact. In this review we tried to survey the major findings in the amyloid field, trying to describe what the real pitfalls of protein folding are-from an evolutionary perspective-and how sequence and structural features have evolved to balance the need for perfect, dynamic, functionally efficient structures, and the detrimental effects implicit in the dangerous process of folding. We will discuss how the knowledge obtained from these studies has been employed to produce computational methods able to assess, predict, and discriminate the aggregation properties of protein sequences.

Conformational Shift of a β-Hairpin Peptide upon Complex Formation with an Oligo-proline Peptide Studied by Mass Spectrometry.

So-called super-secondary structures such as the β-hairpin, studied here, form an intermediate hierarchy between secondary and tertiary structures of proteins. Their sequence-derived 'pure' peptide backbone conformation is combined with 'remote' interstrand or interresidue contacts reminiscent of the 3D-structure of full-length proteins. This renders them ideally suited for studying potential nucleation sites of protein folding reactions as well as intermolecular interactions. But β-hairpins do not merely serve as model systems; their unique structure characteristics warrant a central role in structural studies on their own. In this study we applied photo cross-linking in combination with high-resolution mass spectrometry and computational modeling as well as with ion mobility-mass spectrometry to elucidate these structural properties. Using variants of a known β-hairpin representative, the so-called trpzip peptide and its ligands, we found evidence for a conformational transition of the β-hairpin and its impact on ligand binding.