Supersecondary Structural Elements Research Articles

Levels of the Hierarchical Organization of Protein Sequences: An Analysis of Entropy Characteristics

A set of 24 647 nonhomologous protein sequences was examined. A pentapeptide occurrence profile was constructed for each sequence and hierarchically organized elements of various sizes were detected in each profile by a special mathematical method. Correlations between the hierarchical elements were studied. A total of 11 levels of protein organization with elements ranging from 7 to 56 amino-acid residues were found in the protein sequence set. The organization levels were assumed to correspond to super-secondary structure elements of different topologies.

Folding Mechanism of a Helix-Turn-Helix Protein from Combined 13C-Edited IR and Mutational Studies

Helix-turn-helix motifs are important super-secondary protein structural elements and excellent models for studying the mechanism of protein folding. We have been investigating folding of a de novo designed 38-residue helix-turn-helix motif alpha-t-alpha using IR spectroscopy with site-specific 13C isotopic editing. Our preliminary site-specific thermal unfolding data revealed that alpha-t-alpha is most stable near the centers of both alpha-helices, and likely unfolds from the helical termini and the loose turn region. To obtain more detail about the distribution of thermodynamic stabilities along the structure, additional six isotopically labeled proteins were synthesized and investigated using isotopically-edited IR. The new site-specific thermal unfolding data support the proposed folding mechanism, but reveal additional detail about the folding mechanism. The roles of the individual residue-residue stabilizing interactions were further studied by mutations, designed to destabilize the hydrophobic core near the helical centers and to stabilize the helical termini. Additional mutations both stabilized and destabilized the turn/loop sequence. The effect of the mutations on the overall thermodynamic stability was measured using CD and IR spectroscopies. Five isotopically labeled variants of a selected stabilized and destabilized mutant were synthesized to further study the influence of the mutations on the structural stability of the labeled segments. The effects of the mutations on the global and, especially, local unfolding provide important clues about the stabilization of the helix-turn-helix motif by specific interactions. Our results support and refine the proposed folding mechanism and point out the significance of tertiary interactions for the stability of alpha-t-alpha.

Structural Characteristics of Novel Protein Folds

Folds are the basic building blocks of protein structures. Understanding the emergence of novel protein folds is an important step towards understanding the rules governing the evolution of protein structure and function and for developing tools for protein structure modeling and design. We explored the frequency of occurrences of an exhaustively classified library of supersecondary structural elements (Smotifs), in protein structures, in order to identify features that would define a fold as novel compared to previously known structures. We found that a surprisingly small set of Smotifs is sufficient to describe all known folds. Furthermore, novel folds do not require novel Smotifs, but rather are a new combination of existing ones. Novel folds can be typified by the inclusion of a relatively higher number of rarely occurring Smotifs in their structures and, to a lesser extent, by a novel topological combination of commonly occurring Smotifs. When investigating the structural features of Smotifs, we found that the top 10% of most frequent ones have a higher fraction of internal contacts, while some of the most rare motifs are larger, and contain a longer loop region.

Effects of Mutations on Side-Specific Folding Mechanism of a Helix-Turn-Helix Protein

Helix-turn-helix motifs are important super-secondary protein structural elements and excellent models for studying the mechanism of protein folding. We have been investigating folding of a de novo designed 38-residue helix-turn-helix motif α-t-α using IR spectroscopy with site-specific 13C isotopic editing. Our preliminary site-specific thermal unfolding data revealed that α-t-α is most stable near the centers of both α-helices, and likely unfolds from the helical termini and the loose turn region. To obtain further insights into the folding mechanism, and to investigate the roles of the individual residue-residue stabilizing interactions, we have begun mutational studies of the α-t-α protein. The mutations were designed to both destabilize and further stabilize the hydrophobic core near the helical centers. Additional mutations were designed to stabilize the helical termini and the turn/loop sequence. The overall thermodynamic stability of the α-t-α was measured using CD and IR spectroscopies. The core mutations appreciably decreased or increased the overall folding stability as intended, however, stabilizing the turn and helical termini proved to be a rather challenging task. Site-specific thermal unfolding of the mutated α-t-α were probed with IR on multiple 13C isotopically labeled variants of each mutant. The effects of the mutations on both the global and, in particular, local site-specific unfolding provide important clues about the stabilization of the helix-turn-helix motif by specific interactions. Although additional mutational studies are underway, thus far all the data are consistent with the proposed folding mechanism.

The GD box: A widespread noncontiguous supersecondary structural element

Identification and characterization of recurrent supersecondary structural elements is central to understanding the rules governing protein tertiary structure. Here, we describe the GD box, a widespread noncontiguous supersecondary element, which we initially found in a group of topologically distinct but homologous beta-barrels--the cradle-loop barrels. The GD box is similar both in sequence and structure and comprises two short unpaired beta-strands connected by an orthogonal type-II beta-turn and a noncontiguous beta-strand forming hydrogen bonds with the beta-turn. Using structure-based analysis, we have detected 518 instances of the GD box in a nonredundant subset of the SCOP database comprising 3771 domains. Apart from the cradle-loop barrels, this motif is also found in a diverse set of nonhomologous folds including other topologically related beta-barrels. Since nonlocal interactions are fundamental in the formation of protein structure, systematic identification and characterization of other noncontiguous supersecondary structural elements is likely to prove valuable to protein structure modeling, validation, and prediction.

Mining super-secondary structure motifs from 3d protein structures: a sequence order independent approach.

Super-Secondary structure elements (super-SSEs) are the structurally conserved ensembles of secondary structure elements (SSEs) within a protein. They are of great biological interest. In this work, we present a method to formally represent and mine the sequence order independent super-SSE motifs that occur repeatedly in large data sets of protein structures. We represent a protein structure as a graph, and mine the common cliques from a set of protein graphs in order to find the motifs. We mine two categories of super-SSE motifs: the generic motifs that occur frequently across the entire database of protein structures, and the fold-preferential motifs that are concentrated in particular protein fold types. From the experimental data set of 600 proteins belonging to 15 large SCOP Folds, we have discovered 21 generic motifs and 75 fold-preferential motifs that are both statistically significant and biologically relevant. A number of the discovered motifs (both generic and fold-preferential) resemble the well-known super-SSE motifs in the literature such as beta hairpins, Greek keys, zinc fingers, etc. Some of the discovered motifs are of novel shapes that have not been documented yet. Our method is time-efficient where it can discover all the motifs across the 600 proteins in less than 14 minutes on a standalone PC. The discovered motifs are reported in our project webpage: http://www1.i2r.a-star.edu.sg/~azeyar/SuperSSE/

SMALL PEPTIDES WITH A β-HAIRPIN STRUCTURE

In recent years, b-hairpin peptides have been studied in detail. b-Turns are the most common element of non-repetitive structure recognized in proteins.1 They consist of supersecondary structural elements in which two antiparallel adjacent peptide b-sheets are linked by a short loop. The loop size can vary; the majority of b-hairpins have loops of two to six residues,2 the most common loop size being two. Their dihedral angles (Table 1) are used to classify them.3

Water in protein structure prediction.

Proteins have evolved to use water to help guide folding. A physically motivated, nonpairwise-additive model of water-mediated interactions added to a protein structure prediction Hamiltonian yields marked improvement in the quality of structure prediction for larger proteins. Free energy profile analysis suggests that long-range water-mediated potentials guide folding and smooth the underlying folding funnel. Analyzing simulation trajectories gives direct evidence that water-mediated interactions facilitate native-like packing of supersecondary structural elements. Long-range pairing of hydrophilic groups is an integral part of protein architecture. Specific water-mediated interactions are a universal feature of biomolecular recognition landscapes in both folding and binding.

More than the sum of their parts: on the evolution of proteins from peptides.

Despite their seemingly endless diversity, proteins adopt a limited number of structural forms. It has been estimated that 80% of proteins will be found to adopt one of only about 400 folds, most of which are already known. These folds are largely formed by a limited 'vocabulary' of recurring supersecondary structure elements, often by repetition of the same element and, increasingly, elements similar in both structure and sequence are discovered. This suggests that modern proteins evolved by fusion and recombination from a more ancient peptide world and that many of the core folds observed today may contain homologous building blocks. The peptides forming these building blocks would not in themselves have had the ability to fold, but would have emerged as cofactors supporting RNA-based replication and catalysis (the 'RNA world'). Their association into larger structures and eventual fusion into polypeptide chains would have allowed them to become independent of their RNA scaffold, leading to the evolution of a novel type of macromolecule: the folded protein.

Role of supersecondary structural elements in protein G folding

Knowledge on the folding of supersecondary structures is very important for a coherent understanding of protein folding mechanisms. Here, the hairpin 1, hairpin 1-helix and helix-hairpin 2 of protein G have each been subject of 50 folding Monte Carlo simulations and 48 ns unfolding molecular dynamics simulations. We find that the helix-hairpin 2 structure can stabilize itself to some extent independent of the rest of protein G but hairpin 1 cannot. Interestingly, an intermediate characterized by a helix-hairpin 2 packed in a non-native arrangement is observed in 50% of all simulations, independently of the temperature, force field, and exact amino acid sequence. This is in line with the framework model which envisages a secondary-tertiary hierarchical process.

Computer simulations aimed at structure prediction of supersecondary motifs in proteins.

It is well established that protein structures are more conserved than protein sequences. One-third of all known protein structures can be classified into ten protein folds, which themselves are composed mainly of alpha-helical hairpin, beta hairpin, and betaalphabeta supersecondary structural elements. In this study, we explore the ability of a recent Monte Carlo-based procedure to generate the 3D structures of eight polypeptides that correspond to units of supersecondary structure and three-stranded antiparallel beta sheet. Starting from extended or misfolded compact conformations, all Monte Carlo simulations show significant success in predicting the native topology using a simplified chain representation and an energy model optimized on other structures. Preliminary results on model peptides from nucleotide binding proteins suggest that this simple protein folding model can help clarify the relation between sequence and topology.

De novo design and characterization of an apolar helical hairpin peptide at atomic resolution: Compaction mediated by weak interactions.

Design of helical super secondary structural motifs is expected to provide important scaffolds to incorporate functional sites, thus allowing the engineering of novel miniproteins with function. An alpha,beta-dehydrophenylalanine containing 21-residue apolar peptide was designed to mimic the helical hairpin motif by using a simple geometrical design strategy. The synthetic peptide folds into the desired structure as assessed crystallographically at 1.0-A resolution. The two helices of the helical-hairpin motif, connected by a flexible (Gly)(4) linker, are docked to each other by the concerted influence of weak interactions. The folding of the peptide without binary patterning of amino acids, disulfide bonds, or metal ions is a remarkable observation. The results demonstrate that preferred interactions among the hydrophobic residues selectively discriminate their putative partners in space, leading to the unique folding of the peptide, also a hallmark of the unique folding of hydrophobic core in globular proteins. We demonstrate here the engineering of molecules by using weak interactions pointing to their possible further exploitation in the de novo design of protein super secondary structural elements.

Review of the fourth Johns Hopkins Protein Folding Meeting

Review of the fourth Johns Hopkins Protein Folding Meeting

Identifying the structural boundaries of independent folding domains in the alpha subunit of tryptophan synthase, a beta/alpha barrel protein.

Two equilibrium intermediates have previously been observed in the urea denaturation of the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, an eight-stranded beta/alpha barrel protein. In the current study, a series of amino-terminal fragments were characterized to probe the elementary folding units that may be in part responsible for this complex behavior. Stop-codon mutagenesis was used to produce eight fragments ranging in size from 105-214 residues and containing incremental elements of secondary structure. Equilibrium studies by circular dichroism indicate that all of these fragments are capable of adopting secondary structure. All except for the shortest fragment fold cooperatively. The addition of the fourth, sixth, and eighth beta-strands leads to distinct increases in structure, cooperativity, and/or stability, suggesting that folding involves the modular assembly of betaalphabeta supersecondary structural elements. One-dimensional NMR titrations at high concentrations of urea, probing the environment around His92, were also performed to test for the presence of residual structure in the fragments. All fragments that contained the first four betaalpha units of structure exhibited a cooperative unfolding transition at high concentrations of urea with significant but reduced stability relative to the full-length protein. These results suggest that the residual structure in alphaTS requires the participation of hydrophobic residues in multiple beta-strands that span the entire sequence.

Escher: A new docking procedure applied to the reconstruction of protein tertiary structure

Evaluation of Surface Complementarity, Hydrogen bonding, and Electrostatic interaction in molecular Recognition (ESCHER) is a new docking procedure consisting of three modules that work in series. The first module evaluates the geometric complementarity and produces a set of rough solutions for the docking problem. The second module identifies molecular collisions within those solutions, and the third evaluates their electrostatic complementarity. We describe the algorithm and its application to the docking of cocrystallized protein domains and unbound components of protein-protein complexes. Furthermore, ESCHER has been applied to the reassociation of secondary and supersecondary structure elements. The possibility of applying a docking method to the problem of protein structure prediction is discussed. Proteins 28:556–567, 1997. © 1997 Wiley-Liss, Inc.

Escher: A new docking procedure applied to the reconstruction of protein tertiary structure

Evaluation of Surface Complementarity, Hydrogen bonding, and Electrostatic interaction in molecular Recognition (ESCHER) is a new docking procedure consisting of three modules that work in series. The first module evaluates the geometric complementarity and produces a set of rough solutions for the docking problem. The second module identifies molecular collisions within those solutions, and the third evaluates their electrostatic complementarity. We describe the algorithm and its application to the docking of cocrystallized protein domains and unbound components of protein-protein complexes. Furthermore, ESCHER has been applied to the reassociation of secondary and supersecondary structure elements. The possibility of applying a docking method to the problem of protein structure prediction is discussed.

A consensus prediction of the secondary structure for the 6‐phospho‐β‐D‐galactosidase superfamily

Two separate unrefined models for the secondary structure of two subfamilies of the 6-phospho-beta-D-galactosidase superfamily were independently constructed by examining patterns of variation and conservation within homologous protein sequences, assigning surface, interior, parsing, and active site residues to positions in the alignment, and identifying periodicities in these. A consensus model for the secondary structure of the entire superfamily was then built. The prediction tests the limits of an unrefined prediction made using this approach in a large protein with substantial functional and sequence divergence within the family. The protein belongs to the (alpha-beta class), with the core beta strands aligned parallel. The supersecondary structural elements that are readily identified in this model is a parallel beta sheet built by strands C, D, and E, with helices 2 and 3 connecting strands (C+D) and (D+E), respectively, and an analogous beta-alpha unit (strand G and helix 7) toward the end of the sequence. The resemblance of the supersecondary model to the tertiary structure formed by 8-fold alpha-beta barrel proteins is almost certainly not coincidental.

Knowledge-based prediction of protein structures

We propose a knowledge-based approach to the prediction of protein structures in cases where there is no sequence-homology to proteins with known spatial structure. Using methods from Artificial Intelligence we attempt to take into account long-range interactions within the prediction process. This allows not only the assignment of secondary but also of supersecondary structure elements. In particular, the patterns used as conditions of prediction rules are generated by learning methods from information contained in the Protein Data Base. Patterns on higher levels of the protein structure hierarchy are used as constraints to reduce the combinatorial search space. These patterns may also be used to search for specified structure motifs by interactive retrieval.

Escherichia coli DNA polymerase I. Sequence characterization and secondary structure prediction.

The primary sequence of DNA polymerase I from Escherichia coli K12 as derived from the DNA sequence (Joyce, C. M., Kelley, W. S., and Grindley, N. D. F. (1982) J. Biol. Chem. 257, 1958-1964) has been verified. Protein sequencing through eight cycles of the Klenow large fragment yields a unique sequence corresponding to residues 324 to 331 from the translated DNA sequence and defines the subtilisin cleavage site for formation of the large and small fragments as Thr323-Val324. Site-specific cleavage of whole enzyme and large fragment at cysteines and sizing of the resulting fragments verify the location of the two cysteines at residues 262 and 907 as assigned by the DNA sequencing. Isolation of tryptic peptides derived from DNA polymerase I yielded unique peptides whose composition exactly corresponded to theoretical tryptic peptides derived from the translated DNA sequence. Identification of the expected carboxyl-terminal tryptic peptide and carboxypeptidase digestion of whole enzyme and large fragment confirm histidine-928 as the carboxylterminus. A secondary structure prediction is made using the available primary sequence data. The model contains 43% alpha helix, 17% beta-structure, 58 beta-turns, and several interesting super-secondary structure elements.