Protein Structural Motifs Research Articles

Stereochemistry of Schellman motifs in peptides: Crystal structure of a hexapeptide with a C-terminus 6 ? 1 hydrogen bond

The Schellman motif is a widely observed helix terminating structural motif in proteins, which is generated when the C-terminus residue adopts a left-handed helical (αL) conformation. The resulting hydrogen-bonding pattern involves the formation of an intramolecular 6 → 1 interaction. This helix terminating motif is readily mimicked in synthetic helical peptides by placing an achiral residue at the penultimate position of the sequence. Thus far, the Schellman motif has been characterized crystallographically only in peptide helices of length 7 residues or greater. The structure of the hexapeptide Boc–Pro–Aib–Gly–Leu–Aib–Leu–OMe in crystals reveal a short helical stretch terminated by a Schellman motif, with the formation of 6 → 1 C-terminus hydrogen bond. The crystals are in the space group P212121 with a = 18.155(3) Å, b = 18.864(8) Å, c = 11.834(4) Å, and Z = 4 . The final R1 and wR2 values are 7.68 and 14.6%, respectively , for 1524 observed reflections [Fo ≥ 3ς(Fo)]. A 6 → 1 hydrogen bond between Pro(1)CO · · · Leu(6)NH and a 5 → 2 hydrogen bond between Aib(2)CO · · · Aib(5)NH are observed. An analysis of the available oligopeptides having an achiral Aib residue at the penultimate position suggests that chain length and sequence effects may be the other determining factors in formation of Schellman motifs. © 1999 John Wiley & Sons, Inc. Biopoly 50: 13–22, 1999

Discovery of local packing motifs in protein structures

We present a language for describing structural patterns of residues in protein structures and a method for the discovery of such patterns that recur in a set of protein structures. The patterns impose restrictions on the spatial position of each residue, their order along the amino acid chain, and which amino acids are allowed in each position. Unlike other methods for comparing sets of protein structures, our method is not based on the use of pairwise structure comparisons which is often time consuming and can produce inconsistent results. Instead, the method simultaneously takes into account information from all structures in the search for conserved structure patterns which are potential structure motifs. The method is based on describing the spatial neighborhoods of each residue in each structure as a string and applying a sequence pattern discovery method to find patterns common to subsets of these strings. Finally it is checked whether the similarities between the neighborhood strings correspond to spatially similar substructures. We apply the method to analyze sets of very disparate proteins from the four different protein families: serine proteases, cuprodoxins, cysteine proteinases, and ferredoxins. The motifs found by the method correspond well to the site and motif information given in the annotation of these proteins in PDB, Swiss-Prot, and PROSITE. Furthermore, the motifs are confirmed by using the motif data to constrain the structural alignment of the proteins obtained with the program SAP. This gave the best superposition/alignment of the proteins given the motif assignment. Proteins 1999;34:206–219. © 1999 Wiley-Liss, Inc.

Discovery of local packing motifs in protein structures

We present a language for describing structural patterns of residues in protein structures and a method for the discovery of such patterns that recur in a set of protein structures. The patterns impose restrictions on the spatial position of each residue, their order along the amino acid chain, and which amino acids are allowed in each position. Unlike other methods for comparing sets of protein structures, our method is not based on the use of pairwise structure comparisons which is often time consuming and can produce inconsistent results. Instead, the method simultaneously takes into account information from all structures in the search for conserved structure patterns which are potential structure motifs. The method is based on describing the spatial neighborhoods of each residue in each structure as a string and applying a sequence pattern discovery method to find patterns common to subsets of these strings. Finally it is checked whether the similarities between the neighborhood strings correspond to spatially similar substructures. We apply the method to analyze sets of very disparate proteins from the four different protein families: serine proteases, cuprodoxins, cysteine proteinases, and ferredoxins. The motifs found by the method correspond well to the site and motif information given in the annotation of these proteins in PDB, Swiss-Prot, and PROSITE. Furthermore, the motifs are confirmed by using the motif data to constrain the structural alignment of the proteins obtained with the program SAP. This gave the best superposition/alignment of the proteins given the motif assignment.

Recognition of spatial motifs in protein structures

As the structural database continues to expand, new methods are required to analyse and compare protein structures. Whereas the recognition, comparison, and classification of folds is now more or less a solved problem, tools for the study of constellations of small numbers of residues are few and far between. In this paper, two programs are described for the analysis of spatial motifs in protein structures. The first, SPASM, can be used to find the occurrence of a motif consisting of arbitrary main-chain and/or side-chains in a database of protein structures. The program also has a unique capability to carry out “fuzzy pattern matching” with relaxed requirements on the types of some or all of the matching residues. The second program, RIGOR, scans a single protein structure for the occurrence of any of a set of pre-defined motifs from a database. In one application, spatial motif recognition combined with profile analysis enabled the assignment of the structural and functional class of an uncharacterised hypothetical protein in the sequence database. In another application, the occurrence of short left-handed helical segments in protein structures was investigated, and such segments were found to be fairly common. Potential applications of the techniques presented here lie in the analysis of (newly determined) structures, in comparative structural analysis, in the design and engineering of novel functional sites, and in the prediction of structure and function of uncharacterised proteins.

Induction of protein-like molecular architecture by self-assembly processes

One of the most intriguing self-assembly processes is the folding of peptide chains into native protein structures. We have developed a method for building protein-like structural motifs that incorporate sequences of biological interest. A lipophilic moiety is attached onto an Nα-amino group of a peptide chain, resulting in a ‘peptide-amphiphile’. The alignment of amphiphilic compounds at the lipid–solvent interface is used to facilitate peptide alignment and structure initiation and propagation. Peptide-amphiphiles containing potentially triple-helical structural motifs have been synthesized. The resultant head group structures have been characterized by circular dichroism and NMR spectroscopies. Evidence for a self-assembly process of peptide-amphiphiles has been obtained from: (a) circular dichroism spectra and melting curves characteristic of triple-helices, (b) one- and two-dimensional NMR spectra indicative of stable triple-helical structure at low temperatures and melted triple-helices at high temperatures, and (c) pulsed-field gradient NMR experiments demonstrating different self-diffusion coefficients between proposed triple-helical and non-triple-helical species. The peptide-amphiphiles described here provide a simple approach for building stable protein structural motifs using peptide head groups.

Classification of protein structure motif conformations

Similar to the concept of scene analysis in vision, molecular scene analysis is concerned with the processes of reconstruction and classification of complex molecular images. Such analyses rely on the availability of a priori information in the form of protein structure motifs. Research in this project has involved the study, development and implementation of data mining tools for obtaining such structural motifs. The rapid growth of protein databases has created a demand for novel and efficient techniques for the classification of motif conformations which can be used to anticipate or predict potential substructures in protein images based on primary sequence information.

Generalized pathway model to compute and analyze tunneling matrix elements in proteins

An iterative method is developed to generalize the original pathway model which identifies not simply the strongest pathway involved in the protein-mediation of electron transfer matrix elements but all the relevant ones. The generalized pathway model is a semianalytical, nonperturbative, and computationally tractable method that allows detail investigation of the electron tunneling propagation in the protein medium for any one-electron Hamiltonian, and to examine structure-function relationships in multiple pathway regimes that may be induced by hydrogen bond and through-space contacts in the folded protein structure. This method enables an understanding of how details of the 3-D protein structures control the tunneling process, and whether pathway interference in the protein structural motifs causes substantial deviation from standard pathway analysis.

Characterization of Helix Terminating Schellman Motifs in Peptides. Crystal Structure and Nuclear Overhauser Effect Analysis of a Synthetic Heptapeptide Helix

The Schellman motif is a widely observed, helix terminating structural motif in proteins, which is achieved by the adoption of a left-handed helical (alphaL) conformation by a C-terminus residue. The resulting hydrogen bonding pattern involves an intramolecular 6 f 1 interaction. This helix terminating motif is readily mimicked in synthetic helical peptides by placing an achiral residue at the penultimate position of the helix. The crystal structure of the heptapeptide Boc-Leu-Aib-Val-Gly-Leu-Aib-Val-OMe (1) reveals a 310-helix terminated by a Schellman motif with Aib(6) adopting an alphaL - conformation. The crystals are in the space group P21 with a = 9.958(3)A, b = 20.447-(3)A, c = 11.723 (2)A, beta = 99.74(2)°, and Z = 2. The final R1 and wR2 values are 7.2% and 18.9%, respectively, for 1731 observed reflections [I g 2U(I)]. A 6 - 1 hydrogen bond between Aib(2)CO- - - -Val(7)NH and a 5 - 2 hydrogen bond between Val(3)CO- - - -Aib(6)NH are observed. An analysis of several alphaL terminated helical peptides in crystals suggests that the medium range CialphaH- - - -Ni+3H [dRN(i,i+3)] and CialphaH- - - -Ni+4H [dRN(i,i+4)] interproton distances are indeed characteristic of the Schellman motif. NMR studies in CDCl3 establish retention of the 310-helical conformation with key NOEs demonstrating the persistence of the conformation determined in crystals. The present study demonstrates the identification of the Schellman motif in solution in a synthetic helical peptide.

Expression patterns and transcript processing of ftt-1 and ftt-2, two C. elegans 14-3-3 homologues

A wide diversity of biological functions have been attributed to the highly conserved and ubiquitous 14-3-3 protein family. Yet how much of this diversity is inherent in the basic structure of 14-3-3 and how much is due to isoform specific functions is not yet fully understood. Here, two Caenorhabditis elegans 14-3-3 isoforms whose protein sequences are 90% similar were found to differ significantly in both their genomic structure and expression patterns. The two genes, ftt- 1 (IV) (fourteen-three-three) and ftt- 2 (X), differ in both the position and sequence of their introns. Since the various intron/exon boundaries respect neither functional nor structural protein motifs, the introns appear to be relatively recent evolutionary additions. ftt- 1(IV) encodes three germline enhanced transcripts, two of which are related through the differential use of alternative poly(A) addition sites. RNA in situ hybridization studies reveal high levels of ftt- 1 throughout the gonad with particularly high levels in the distal arm. In contrast, ftt- 2 (X) encodes a single transcript which is expressed somatically. In embryos, high levels of ftt- 1 transcripts appear to be maternally supplied, whereas ftt- 2 is expressed as an early zygotic transcript whose expression pattern later localizes to the posterior region of post-proliferative embryos. These expression pattern differences between ftt- 1 and ftt- 2 suggest that these two 14-3-3 isoforms perform distinct biological roles within the worm.

A Computational Approach to Modeling Nucleic Acid Hairpin Structures

Hairpin is a structural motif frequently observed in both RNA and DNA molecules. This motif is involved specifically in various biological functions (e.g., gene expression and regulation). To understand how these hairpin motifs perform their functions, it is important to study their structures. Compared to protein structural motifs, structures of nucleic acid hairpins are less known. Based on a set of reduced coordinates for describing nucleic acid structures and a sampling algorithm that equilibrates structures using Metropolis Monte Carlo simulation, we developed a method to model nucleic acid hairpin structures. This method was used to predict the structure of a DNA hairpin with a single-guanosine loop. The lowest energy structure from the ensemble of 200 sampled structures has a RMSD of < 1.5 A, from the structure determined using NMR. Additional constraints for the loop bases were introduced for modeling an RNA hairpin with two nucleotides in the loop. The modeled structure of this RNA hairpin has extensive base stacking and an extra hydrogen bond (between the CYT in the loop and a phosphate oxygen), as observed in the NMR structure.

An iterative method for improved protein structural motif recognition.

We present an iterative algorithm that uses randomness and statistical techniques to improve existing methods for recognizing protein structural motifs. Our algorithm is particularly effective in situations where large numbers of sufficiently diverse examples of the motif are not known. These are precisely the situations that pose significant difficulties for previously known methods. We have implemented our algorithm and we demonstrate its performance on the coiled coil motif. We test our program LearnCoil on the domain of 3-stranded coiled coils and subclasses of 2-stranded coiled coils. We show empirically that for these motifs, our method overcomes the problem of limited data.

26] Construction of biologically active protein molecular architecture using self-assembling peptide-amphiphiles

The peptide-amphiphiles described here provide a simple approach for building stable protein structural motifs using peptide head groups. One of the most intriguing features of this system is the possible formation of stable lipid films on solid substrates, or the use of the novel amphiphiles in bilayer membrane systems, where the lipid tail serves not only as a peptide structure-inducing agent but also as an anchor of the functional head group in the lipid assembly. The peptide-amphiphile system potentially offers great versatility with regard to head and tail group composition and overall geometries and macromolecular structures. For building materials with molecular and cellular recognition capacity, it is essential to have a wide repertoire of tools to produce characteristic supersecondary structures at surfaces and interfaces.

Synthesis and properties of a biocompatible analogue for β-turn protein structural motifs based on 5-amino-3-pentynoic acid

5-Amino-3-pentynoic acid, a new ‘lightly’ constrained amino acid surrogate for glycylglycine and model for dipeptide homologues was prepared from propagylamine and methods were devised for its incorporation into linear oligopeptides through both C- and N-terminal extension.

Simulations of reversible protein aggregate and crystal structure

We simulated the structure of reversible protein aggregates as a function of protein surface characteristics, protein-protein interaction energies, and the entropic penalty accompanying the immobilization of protein in a solid phase. These simulations represent an extension of our previous work on kinetically irreversible protein aggregate structure and are based on an explicit accounting of the specific protein-protein interactions that occur within reversible aggregates and crystals. We considered protein monomers with a mixture of hydrophobic and hydrophilic surface regions suspended in a polar solvent; the energetic driving force for aggregation is provided by the burial of solvent-exposed hydrophobic surface area. We analyzed the physical properties of the generated aggregates, including density, protein-protein contact distributions, solvent accessible surface area, porosity, and order, and compared our results with the protein crystallization literature as well as with the kinetically irreversible case. The physical properties of reversible aggregates were consonant with those observed for the irreversible aggregates, although in general, reversible aggregates were more stable energetically and were more crystal-like in their order content than their irreversible counterparts. The reversible aggregates were less dense than the irreversible aggregates, indicating that the increased energetic stability is derived primarily from the optimality rather than the density of the packing in the solid phase. The extent of hydrophobic protein-protein contacts and solvent-exposed surface area within the aggregate phase depended on the aggregation pathway: reversible aggregates tended to have a greater proportion of hydrophobic-hydrophobic contacts and a smaller fraction of hydrophobic solvent-exposed surface area. Furthermore, the arrangement of hydrophobic patches on the protein surface played a major role in the distribution of protein contacts and solvent content. This was readily reflected in the order of the aggregates: the greater the contiguity of the hydrophobic patches on the monomer surface, the less ordered the aggregates became, despite the opportunities for rearrangement offered by a reversible pathway. These simulations have enhanced our understanding of the impact of protein structural motifs on aggregate properties and on the demarcation between aggregation and crystallization.

IDENTIFICATION OF GENES INVOLVED IN THE ADAPTATION OF THE LUNG TO THE AIR ENVIRONMENT. ▴ 354

We are using the technique of differential display RT-PCR in order to identify genes important in lung development. We are particularly interested in genes whose products are essential to mesenchymal-epithelial interactions in late gestation lung. We isolated 38 cDNA probes representing mRNAs whose expression is modulated by glucocorticoid (GC) and dihydro-testosterone (DHT) in days 19-20 fibroblasts (fib) or epithelial cells (epi) of fetal rat lung. Among 18 fib-specific sequences, 16 represented mRNAs whose expression is increased in the presence of GC and decreased in the presence of DHT. This pattern of expression is expected for FPF, an activity generated by fibs which augments epi surfactant production. Twenty sequences were epi-specific and represent mRNAs meeting criteria for GC receptor (GR) stimulating factor (an activity produced by epi which stimulates fib GR). We sequenced 20 (15 epi, 5 fib) subclones (18 novel). Nine showed interesting homologies to sequences in Genbank. The predicted protein product of one epi-clone had 66-70% homology(over 60 residues) to 70 bacterial flagellins but had no identified homologs in eukaryotes. This suggests a novel protein, perhaps a precursor of a lung ciliary protein. The predicted protein products of 3 epi-clones had primary sequence homology or protein structural motifs suggestive of transcriptional regulators. One clone had homology to rat/mouse progesterone receptor (a stage and tissue-specific transcriptional regulator). Another had homology to yeast HPR1 protein (a positive transcriptional regulator). A third clone had a region with a leucine-zipper motif, a motif common to transcriptional activators. Transcriptional regulatory sequences are of interest as regulation of transcription is likely a key mechanism in the modulation of gene expression in late gestation lung development. These findings open new areas for the study of regulation of fetal lung maturation.

Human Skeletal Muscle Nebulin Sequence Encodes a Blueprint for Thin Filament Architecture: SEQUENCE MOTIFS AND AFFINITY PROFILES OF TANDEM REPEATS AND TERMINAL SH3

Analysis of deduced protein sequence and structural motifs of approximately 5500 residues of human fetal skeletal muscle nebulin reveals the design principles of this giant multifunctional protein in the sarcomere. The bulk of the sequence is constructed of approximately 150 tandem copies of approximately 35-residue modules that can be classified into seven types. The majority of these modules form 20 super-repeats, with each super-repeat containing a 7-module set (one of each type in the same order). These super-repeats are further divided into eight segments: with six segments containing adjacent, highly homologous super-repeats, one single repeat segment consisting of 8 nebulin modules of the same type, and a non-repeat segment terminating with a SH3 domain at the C terminus. The interactions of actin, tropomyosin, troponin, and calmodulin with nebulin fragments consisting of either repeating modules or the SH3 domain support its role as a giant actin-binding cofilament of the composite thin filament. Such affinity profiles also suggest that nebulin may bind to tropomyosin and troponin to form a composite calcium-linked regulatory complex on the thin filament. The modular construction, super-repeat structure, and segmental organization of nebulin sequence appear to encode thin filament length, periodicity, insertion, and sarcomere proportion in the resting muscle.

Thermodynamic analysis of a designed three-stranded coiled coil.

The study and successful design of coiled-coil protein structural motifs have provided much insight into the rules governing protein folding and stability. In this work we use a thermodynamic approach to quantitate the rules that govern the specific oligomerization of coiled coils. We have designed a highly stable trimeric coiled coil by placing valine residues at each a position and leucine residues at each d position of the heptad repeating unit. The peptide forms a very stable trimer as determined by sedimentation equilibrium, and the concentration dependence of its circular dichroism spectrum follows a cooperative monomer/dimer/trimer equilibrium with the dimer state as a highly unstable intermediate. Its guanidinium chloride denaturation curve was collected at several peptide concentrations, and analysis of the data confirms the cooperativity of the trimerization process and provides a free energy of stabilization of - 18.4 kcal mol-1 for the trimer. The heat capacity, delta Cp, was measured by global analysis of thermal unfolding data collected at a number of guanidinium chloride concentrations. Guanidinium chloride induces cold denaturation in the thermal unfolding curves, providing a reasonably well-determined value for delta Cp of 750 cal deg-1 mol-1. This translates to a delta Cp of 8.6 cal deg-1 mol-1 per residue and corresponds well to that expected of a coiled coil with a well-defined tertiary structure.

Fluctuations in molecular size, entanglement complexity, and anisometry along molecular dynamics trajectories of short α‐helices

AbstractWe present a detailed study of the intrinsic shape stability of short flexible polypeptide chains by using a number of distinct descriptors. Some large‐scale structural features found in macromolecular folding—unfolding dynamics can be monitored in terms of molecular size and compactness. Similarly, one could employ descriptors of anisometry to that purpose. In addition, we have recently proposed a new family of global, absolute shape descriptors to quantify the complexity of the self‐entanglements in a polymer, or, loosely, its “degree of folding.” This latter aspect of the molecular shape is characterized by combining simple geometrical and topological properties of the α‐carbon backbone. The resulting descriptor, the probability of observing a rigid backbone as a projected curve exhibiting N overcrossings, has been employed already for the analysis of molecular dynamics of linear hydrocarbons and aliphatic carboxylic acids. In this work, three distinct properties of a molecular chain are monitored simultaneously, for the first lime, along computer‐simulated dynamic trajectories: size, anisotropy, and entanglements. The systems studied are a series of 3 polypeptide helices: 10‐polyalanine, 10‐polyvaline, and 10‐polyglycine. Constant temperature trajectories have been computed for these helices, at thermal equilibrium with a simulated external bath, with temperatures ranging from 300 to 700 K. The differential structural stability of the helices is assessed from the various viewpoints. Even though the systems are simple, they contain some of the essential features expected in polypeptides of relevance to structural biology. The results show that the, descriptors of anisometry and entanglements are rather independent. For instance, it is found that, contrary to intuition the unfolding of helices may involve nonhelical “coil” configurations that are both self‐entangled and prolate. The simultaneous study of independent shape descriptors provides a new picture of the nature of conformations found during dynamics trajectories. The resulting description characterizes a polymer conformation in a absolute terms, and not relative to an ad hoc reference structure. The proposed approach gives some insights into the stability of secondary structural motifs in proteins and the mechanisms involved into their formation or unfolding. © 1995 John Wiley &amp; Sons, Inc.

Thioredoxin--a fold for all reasons.

The thioredoxin fold is a characteristic protein structural motif that has been found in five distinct classes of proteins that have the common property of interacting with cysteine-containing substrates.

Algorithms for protein structural motif recognition.

The identification of protein sequences that fold into certain known three-dimensional (3D) structures, or motifs, is evaluated through a probabilistic analysis of their one-dimensional (1D) sequences. We present a correlation method that runs in linear time and incorporates pairwise dependencies between amino acid residues at multiple distances to assess the conditional probability that a given residue is part of a given 3D structure. This method is generalized to multiple motifs, where a dynamic programming approach leads to an efficient algorithm that runs in linear time for practical problems. By this approach, we were able to distinguish (2-stranded) coiled-coil from non-coiled-coil domains and globins from nonglobins. When tested on the Brookhaven X-ray crystal structure database, the method does not produce any false-positive or false-negative predictions of coiled coils.