Ordered conformational change in the protein backbone: prediction of conformationally variable positions from sequence and low-resolution structural data

Ordered conformational changes are an important structural property of proteins and are involved in a variety of fundamental biological activities. Large-scale analyses of the implications of such changes for protein function and dysfunction require efficient methods for automated recognition of conformationally variable residue positions. The goal of this work was to study sequence and low-resolution structural properties of residue positions that change backbone conformation upon changes in protein environment and the utility of these properties for automated recognition of such conformationally variable positions. This study was performed using a large nonredundant set of experimentally characterized proteins that undergo ordered conformational transitions obtained from the Database of Macromolecular Movements. The results of this study show that ordered changes in backbone conformation are not limited to solvent accessible loop regions. A considerable fraction of conformationally variable positions is observed in helices and strands, and in buried positions. Conformationally variable positions are less conserved in evolution. Local patterns of (a) sequence neighbors, (b) evolutionary conservation, and (c) solvent accessibility can be used to predict conformationally variable positions with balanced sensitivity and specificity, albeit with large variance at the level of individual proteins. However, including a pattern of secondary structure into the prediction scheme results in a highly unbalanced performance when all conformationally variable positions located in regular secondary structure are misclassified. Application of the present methodology to the prion protein (PrP) shows that conformationally variable positions predicted in its ordered C-terminal domain are located within segments presumed to be involved in refolding of PrP.     

An automatic search for similar spatial arrangements of alpha-helices and beta-strands in globular proteins

A fast search algorithm to reveal similar polypeptide backbone structural motifs in proteins is proposed. It is based on the vector representation of a polypeptide chain fold in which the elements of regular secondary structures are approximated by linear segments (Abagyan and Maiorov, J. Biomol. Struct. Dyn. 5, 1267-1279 (1988)). The algorithm permits insertions and deletions in the polypeptide chain fragments to be compared. The fast search algorithm implemented in FASEAR program is used for collecting beta alpha beta supersecondary structure units in a number of alpha/beta proteins of Brookhaven Data Bank. Variation of geometrical parameters specifying backbone chain fold is estimated. It appears that the conformation of the majority of the fragments, although almost all of them are right-handed, is quite different from that of standard beta alpha beta units. Apart from searching for specific type of secondary structure motif, the algorithm allows automatically to identify new recurrent folding patterns in proteins. It may be of particular interest for the development of tertiary template approach for prediction of protein three-dimensional structure as well for constructing artificial polypeptides with goal-oriented conformation.     

Raman studies of nucleic acids. XII. Conformations of oligonucleotides and deuterated polynucleotides

Laser Raman spectra of the trinucleoside diphoshate ApApA and dinucleoside phosphates ApU, UpA, GpC, CpG, and GpU are reported and discussed. Assignments of conformationally sensitive frequencies are-facilitated by comparison with spectra reported here of poly(rA), poly(rC), and poly(rU) in deuterium oxide solutions. The significant spectral differences between ApU and UpA, and between GpC and CpG, reveal that the sequence isomers have nonidentical conformations in aqueous solution. In UpA at low temperature the bases are stacked and the backbone conformation is similar to that found in ordered polynucleotide structures and RNA. In ApU no base stacking can be detected and the backbone conformation differs from that found in UpA, both in the orientation of phosphodiester linkages and in the internal conformation of ribose. At the conditions employed neither ApU nor UpA exhibits base pairing in aqueous solutions. In both GpC and CpG the bases are stacked and the phosphodiester conformations are similar to those encountered for UpA and RNA. However, major differences between spectra of GpC and CpG indicate that the geometries of stacking and ribosyl conformations are different. In GpC the Raman data favor the formation of hydrogen bonded dimers containing GC pairs. Protonation of C in GpC is sufficient to eliminate the ordered conformation detected by Raman spectroscopy. Despite the ordered backbone conformation evident in GpU, this dinucleoside apparently contains neither stacked nor hydrogen bonded bases at the conditions employed here. The Raman data also confirm the stacking interactions in ApApA, poly(rA), and poly(rC) but suggest that the backbone conformation in poly(rC) differs qualitatively from that found in most ordered polynucleotide structures and is thermally more stable. The present results demonstrate the sensitivity of the Raman technique to sequence-related structural differences in oligonucleotides and provide additional spectra–structure correlations for future conformational studies of RNA by laser Raman spectroscopy.     

Helix, sheet, and polyproline II frequencies and strong nearest neighbor effects in a restricted coil library

A central issue in protein folding is the degree to which each residue's backbone conformational preferences stabilize the native state. We have studied the conformational preferences of each amino acid when the amino acid is not constrained to be in a regular secondary structure. In this large but highly restricted coil library, the backbone preferentially adopts dihedral angles consistent with the polyproline II conformation rather than alpha or beta conformations. The preference for the polyproline II conformation is independent of the degree of solvation. In conjunction with a new masking procedure, the frequencies in our coil library accurately recapitulate both helix and sheet frequencies for the amino acids in structured regions, as well as polyproline II propensities. Therefore, structural propensities for alpha-helices and beta-sheets and for polyproline II conformations in unfolded peptides can be rationalized solely by local effects. In addition, these propensities are often strongly affected by both the chemical nature and the conformation of neighboring residues, contrary to the Flory isolated residue hypothesis.     

On the properties and sequence context of structurally ambivalent fragments in proteins

The goal of this work is to characterize structurally ambivalent fragments in proteins. We have searched the Protein Data Bank and identified all structurally ambivalent peptides (SAPs) of length five or greater that exist in two different backbone conformations. The SAPs were classified in five distinct categories based on their structure. We propose a novel index that provides a quantitative measure of conformational variability of a sequence fragment. It measures the context-dependent width of the distribution of (phi,xi) dihedral angles associated with each amino acid type. This index was used to analyze the local structural propensity of both SAPs and the sequence fragments contiguous to them. We also analyzed type-specific amino acid composition, solvent accessibility, and overall structural properties of SAPs and their sequence context. We show that each type of SAP has an unusual, type-specific amino acid composition and, as a result, simultaneous intrinsic preferences for two distinct types of backbone conformation. All types of SAPs have lower sequence complexity than average. Fragments that adopt helical conformation in one protein and sheet conformation in another have the lowest sequence complexity and are sampled from a relatively limited repertoire of possible residue combinations. A statistically significant difference between two distinct conformations of the same SAP is observed not only in the overall structural properties of proteins harboring the SAP but also in the properties of its flanking regions and in the pattern of solvent accessibility. These results have implications for protein design and structure prediction.     

Molecular dynamics simulations of polypeptide conformations in water: A comparison of alpha, beta, and poly(pro)II conformations.

A significant fraction of the so-called "random coil" residues in globular proteins exists in the left-handed poly(Pro)II conformation. In order to compare the behavior of this secondary structure with that of the other regular secondary structures, molecular dynamics simulations, with the GROMOS suite of programs, of an alanine octapeptide in water, in alpha-helix, beta-strand, and left-handed poly(Pro)II conformations, have been performed. Our results indicate a limited flexibility for the alpha-helix conformation and a relatively larger flexibility for the beta-strand and poly(Pro)II conformations. The behavior of oligopeptides with a starting configuration of beta-strand and poly(Pro)II conformations, both lacking interchain hydrogen bonds, were similar. The (phi, psi) angles reflect a continuum of structures including both beta and P(II) conformations, but with a preference for local P(II) regions. Differences in the network of water molecules involved in hydrogen bonding with the backbone of the polypeptide were observed in local regions of beta and P(II) conformations. Such water bridges help stabilize the P(II) conformation relative to the beta conformation. Proteins 1999;36:400-406.     

An Automatic Search for Similar Spatial Arrangements of α-Helices and β-Strands in Globular Proteins

Abstract

A fast search algorithm to reveal similar polypeptide backbone structural motifs in proteins is proposed. It is based on the vector representation of a polypeptide chain fold in which the elements of regular secondary structures are approximated by linear segments (Abagyan and Maiorov, J. Biomol. Struct. Dyn. 5, 1267–1279 (1988)). The algorithm permits insertions and deletions in the polypeptide chain fragments to be compared. The fast search algorithm implemented in FASEAR program is used for collecting βαβ supersecondary structure units in a number of α/β proteins of Brookhaven Data Bank. Variation of geometrical parameters specifying backbone chain fold is estimated. It appears that the conformation of the majority of the fragments, although almost all of them are right-handed, is quite different from that of standard βαβ units. Apart from searching for specific type of secondary structure motif, the algorithm allows automatically to identify new recurrent folding patterns in proteins. It may be of particular interest for the development of tertiary template approach for prediction of protein three-dimensional structure as well for constructing artificial polypeptides with goal-oriented conformation.     

Analysis of protein structures reveals regions of rare backbone conformation at functional sites

Regions of rare conformation were located in 300 protein crystal structures representing seven major protein folds. A distance matrix algorithm was used to search rapidly for 9-residue fragments of rare backbone conformation using a comparison to a relational database of encoded fragments derived from the database of nonredundant structures. Rare fragments were found in 61% of the analyzed protein structures. Detailed analysis was performed for 78 proteins of different folds. The rare fragments were located near functional sites in 72% of the protein structures. The rare fragments often formed parts of ligand-binding sites (59%), protein-protein interfaces (8%), and domain-domain contacts (5%). Of the remaining structures, 5% had a high average B-factor or high local B-factors. Statistical analysis suggests that the association between ligands and rare regions does not occur by chance alone. The present study is likely to underestimate the number of functional sites, because not all analyzed protein structures contained a ligand. The results suggest that rapid searches for regions with rare local backbone conformations can assist in prediction of functional sites in novel proteins.     

Conformational mapping of the N-terminal segment of surfactant protein B in lipid using 13C-enhanced Fourier transform infrared spectroscopy.

Synthetic peptides based on the N-terminal domain of human surfactant protein B (SP-B1-25; 25 amino acid residues; NH2-FPIPLPYCWLCRALIKRIQAMIPKG) retain important lung activities of the full-length, 79-residue protein. Here, we used physical techniques to examine the secondary conformation of SP-B1-25 in aqueous, lipid and structure-promoting environments. Circular dichroism and conventional, 12C-Fourier transform infrared (FTIR) spectroscopy each indicated a predominate alpha-helical conformation for SP-B1-25 in phosphate-buffered saline, liposomes of 1-palmitoyl-2-oleoyl phosphatidylglycerol and the structure-promoting solvent hexafluoroisopropanol; FTIR spectra also showed significant beta- and random conformations for peptide in these three environments. In further experiments designed to map secondary structure to specific residues, isotope-enhanced FTIR spectroscopy was performed with 1-palmitoyl-2-oleoyl phosphatidylglycerol liposomes and a suite of SP-B1-25 peptides labeled with 13C-carbonyl groups at either single or multiple sites. Combining these 13C-enhanced FTIR results with energy minimizations and molecular simulations indicated the following model for SP-B1-25 in 1-palmitoyl-2-oleoyl phosphatidylglycerol: beta-sheet (residues 1-6), alpha-helix (residues 8-22) and random (residues 23-25) conformations. Analogous structural motifs are observed in the corresponding homologous N-terminal regions of several proteins that also share the 'saposin-like' (i.e. 5-helix bundle) folding pattern of full-length, human SP-B. In future studies, 13C-enhanced FTIR spectroscopy and energy minimizations may be of general use in defining backbone conformations at amino acid resolution, particularly for peptides or proteins in membrane environments.     

A Self-Organizing Algorithm for Modeling Protein Loops

Protein loops, the flexible short segments connecting two stable secondary structural units in proteins, play a critical role in protein structure and function. Constructing chemically sensible conformations of protein loops that seamlessly bridge the gap between the anchor points without introducing any steric collisions remains an open challenge. A variety of algorithms have been developed to tackle the loop closure problem, ranging from inverse kinematics to knowledge-based approaches that utilize pre-existing fragments extracted from known protein structures. However, many of these approaches focus on the generation of conformations that mainly satisfy the fixed end point condition, leaving the steric constraints to be resolved in subsequent post-processing steps. In the present work, we describe a simple solution that simultaneously satisfies not only the end point and steric conditions, but also chirality and planarity constraints. Starting from random initial atomic coordinates, each individual conformation is generated independently by using a simple alternating scheme of pairwise distance adjustments of randomly chosen atoms, followed by fast geometric matching of the conformationally rigid components of the constituent amino acids. The method is conceptually simple, numerically stable and computationally efficient. Very importantly, additional constraints, such as those derived from NMR experiments, hydrogen bonds or salt bridges, can be incorporated into the algorithm in a straightforward and inexpensive way, making the method ideal for solving more complex multi-loop problems. The remarkable performance and robustness of the algorithm are demonstrated on a set of protein loops of length 4, 8, and 12 that have been used in previous studies.     

Approaching a complete classification of protein secondary structure

A complete classification of types of the protein secondary structure is developed on the basis of computer analysis of the crystallographic structural data deposited in the protein Data Bank. The majority of amino acid residues fall into five conformation types. A conclusion is drawn that the number of sequence variants of torsion angles phi, psi in globular proteins is limited and is essentially less than the number of possible amino acid sequences for this chain length. Along with alpha-helix and beta-structure, the distribution analysis assigning every maximum of distribution of amino acid conformations on Ramachandran map to a certain type of the secondary structure exposed a third type of the secondary structure that was previously neglected. This type of the structure is extended left-handed helical conformation, designated as mobile (M-) conformation. A full set of M-conformation fragments that seems to play a major role in protein globule dynamics has been obtained, a small radius of correlation for the polypeptide chain in M-conformation is demonstrated. It explains a prevalence of short segments of mobile conformation revealed in globular proteins. For secondary structure types, the frequency of occurrence of amino acid residues has been computed.     

PII structure in the model peptides for unfolded proteins: studies on ubiquitin fragments and several alanine-rich peptides containing QQQ, SSS, FFF, and VVV

A great deal of attention has been paid lately to the structures in unfolded proteins due to the recent discovery of many biologically functional but natively unfolded proteins and the far-reaching implications of order in unfolded states for protein folding. Recently, studies on oligo-Ala, oligo-Lys, oligo-Asp, and oligo-Glu, as well as oligo-Pro, have indicated that the left-handed polyproline II (PII) is the major local structure in these short peptides. Here, we show by NMR and CD studies that ubiquitin fragments, model unfolded peptides composed of nonrepeating amino acids, and four alanine-rich peptides containing QQQ, SSS, FFF, and VVV sequences are all present in aqueous solution predominantly in the extended PII or beta conformation. The results from this and related studies indicate that PII might be a major backbone conformation in unfolded proteins. The presence of defined local backbone structure in unfolded proteins is inconsistent with predictions from random coil models.     

13C-NMR chemical shifts and the conformations of rigid polypeptides

¹³C-nmr chemical shifts of backbone carbonyl and side-chain β-carbons in polypeptides provide structural information. Recent utilization of substituent effects on ¹³C-nmr chemical shifts (principally γ-effects) has permitted the rationalization of their sequence and conformation dependence when observed in linear, flexible polypeptides. In this report, we apply the γ-effect method to study the ¹³C-nmr chemical shifts observed in solution and in the solid state for the backbone carbonyl and side-chain β-carbons in conformationally rigid polypeptides, which are usually cyclic. As found previously for flexible, linear polypetides, the relative ¹³C-nmr chemical shifts observed for the backbone carbonyl and side-chain β-carbons in conformationally rigid polypeptides are predictable from knowledge of their peptide residue sequence (primary structure) and conformation (secondary structure) via the γ-effect method.     

A novel approach to fold recognition using sequence-derived properties from sets of structurally similar local fragments of proteins

Comparative modeling methods can consistently produce reliable structural models for protein sequences with more than 25% sequence identity to proteins with known structure. However, there is a good chance that also sequences with lower sequence identity have their structural components represented in structural databases. To this end, we present a novel fragment-based method using sets of structurally similar local fragments of proteins. The approach differs from other fragment-based methods that use only single backbone fragments. Instead, we use a library of groups containing sets of sequence fragments with geometrically similar local structures and extract sequence related properties to assign these specific geometrical conformations to target sequences. We test the ability of the approach to recognize correct SCOP folds for 273 sequences from the 49 most popular folds. 49% of these sequences have the correct fold as their top prediction, while 82% have the correct fold in one of the top five predictions. Moreover, the approach shows no performance reduction on a subset of sequence targets with less than 10% sequence identity to any protein used to build the library.     

Folding of peptide fragments comprising the complete sequence of proteins. Models for initiation of protein folding. I. Myohemerythrin.

In an attempt to delineate potential folding initiation sites for different protein structural motifs, we have synthesized series of peptides that span the entire length of the polypeptide chain of two proteins, and examined their conformational preferences in aqueous solution using proton nuclear magnetic resonance and circular dichroism spectroscopy. We describe here the behavior of peptides derived from a simple four-helix bundle protein, myohemerythrin. The peptides correspond to the sequences of the four long helices (the A, B, C and D helices), the N- and C-terminal loops and the connecting sequences between the helices. The peptides corresponding to the helices of the folded protein all exhibit preferences for helix-like conformations in solution. The conformational ensembles of the A- and D-helix peptides contain ordered helical forms, as shown by extensive series of medium-range nuclear Overhauser effect connectivities, while the B- and C-helix peptides exhibit conformational preferences for nascent helix. All four peptides adopt ordered helical conformations in mixtures of trifluoroethanol and water. The terminal and interconnecting loop peptides also appear to contain appreciable populations of conformers with backbone phi and psi angles in the alpha-region and include highly populated hydrophobic cluster and/or turn conformations in some cases. Trifluoroethanol is unable to drive these peptides towards helical conformations. Overall, the peptide fragments of myohemerythrin have a marked preference towards secondary structure formation in aqueous solution. In contrast, peptide fragments derived from the beta-sandwich protein plastocyanin are relatively devoid of secondary structure in aqueous solution (see accompanying paper). These results suggest that the two different protein structural motifs may require different propensities for formation of local elements of secondary structure to initiate folding, and that there is a prepartitioning of conformational space determined by the local amino acid sequence that is different for the helical and beta-sandwich structural motifs.     

Computation of DNA backbone conformations

We present an algorithm for the computation of 2'-deoxyribose-phosphodiester backbone conformations that are stereochemically compatible with a given arrangement of nucleic acid bases in a DNA structure. The algorithm involves the sequential computation of 2'-deoxyribose and phosphodiester conformers (collectively referred to as a backbone 'segment'), beginning at the 5'-end of a DNA strand. Computation of the possible segment conformations is achieved by the initial creation of a fragment library, with each fragment representing a set of bond lengths, bond angles and torsion angles. Following exhaustive searching of sugar conformations, each segment conformation is reduced to a single vector, defined by a specific distance, angle and torsion angle, that allows calculation of the O(1)' position. A given 'allowed' conformation of a backbone segment is determined based on its compatibility with the base positions and with the position of the preceding backbone segment. Initial computation of allowable segment conformations of a strand is followed by the determination of continuous backbone solutions for the strand, beginning at the 3'-end. The algorithm is also able to detect repeating segment conformations that arise in structures containing geometrically repeating dinucleotide steps. To illustrate the utility and properties of the algorithm, we have applied it to a series of experimental DNA structures. Regardless of the conformational complexity of these structures, we are able to compute backbone conformations for each structure. Hence, the algorithm, which is currently implemented within a new computer program NASDAC (Nucleic Acids: Structure, Dynamics and Conformation), should have generally applicability to the computation of DNA structures.     

Predicting conformational switches in proteins.

We describe a new computational technique to predict conformationally switching elements in proteins from their amino acid sequences. The method, called ASP (Ambivalent Structure Predictor), analyzes results from a secondary structure prediction algorithm to identify regions of conformational ambivalence. ASP identifies ambivalent regions in 16 test protein sequences for which function involves substantial backbone rearrangements. In the test set, all sites previously described as conformational switches are correctly predicted to be structurally ambivalent regions. No such regions are predicted in three negative control protein sequences. ASP may be useful as a guide for experimental studies on protein function and motion in the absence of detailed three-dimensional structural data.     

Reconstruction of protein backbones from the BriX collection of canonical protein fragments

As modeling of changes in backbone conformation still lacks a computationally efficient solution, we developed a discretisation of the conformational states accessible to the protein backbone similar to the successful rotamer approach in side chains. The BriX fragment database, consisting of fragments from 4 to 14 residues long, was realized through identification of recurrent backbone fragments from a non-redundant set of high-resolution protein structures. BriX contains an alphabet of more than 1,000 frequently observed conformations per peptide length for 6 different variation levels. Analysis of the performance of BriX revealed an average structural coverage of protein structures of more than 99% within a root mean square distance (RMSD) of 1 Angstrom. Globally, we are able to reconstruct protein structures with an average accuracy of 0.48 Angstrom RMSD. As expected, regular structures are well covered, but, interestingly, many loop regions that appear irregular at first glance are also found to form a recurrent structural motif, albeit with lower frequency of occurrence than regular secondary structures. Larger loop regions could be completely reconstructed from smaller recurrent elements, between 4 and 8 residues long. Finally, we observed that a significant amount of short sequences tend to display strong structural ambiguity between alpha helix and extended conformations. When the sequence length increases, this so-called sequence plasticity is no longer observed, illustrating the context dependency of polypeptide structures.