Properties of polyproline II, a secondary structure element implicated in protein-protein interactions

The polyproline II (PPII) conformation of protein backbone is an important secondary structure type. It is unusual in that, due to steric constraints, its main-chain hydrogen-bond donors and acceptors cannot easily be satisfied. It is unable to make local hydrogen bonds, in a manner similar to that of alpha-helices, and it cannot easily satisfy the hydrogen-bonding potential of neighboring residues in polyproline conformation in a manner analogous to beta-strands. Here we describe an analysis of polyproline conformations using the HOMSTRAD database of structurally aligned proteins. This allows us not only to determine amino acid propensities from a much larger database than previously but also to investigate conservation of amino acids in polyproline conformations, and the conservation of the conformation itself. Although proline is common in polyproline helices, helices without proline represent 46% of the total. No other amino acid appears to be greatly preferred; glycine and aromatic amino acids have low propensities for PPII. Accordingly, the hydrogen-bonding potential of PPII main-chain is mainly satisfied by water molecules and by other parts of the main-chain. Side-chain to main-chain interactions are mostly nonlocal. Interestingly, the increased number of nonsatisfied H-bond donors and acceptors (as compared with alpha-helices and beta-strands) makes PPII conformers well suited to take part in protein-protein interactions.     

A propensity scale for type II polyproline helices (PPII): aromatic amino acids in proline-rich sequences strongly disfavor PPII due to proline-aromatic interactions

Type II polyproline helices (PPII) are a fundamental secondary structure of proteins, common in globular and nonglobular regions and important in cellular signaling. We developed a propensity scale for PPII using a host-guest system with sequence Ac-GPPXPPGY-NH(2), where X represents any amino acid. We found that proline has the highest PPII propensity, but most other amino acids display significant PPII propensities. The PPII propensity of leucine was the highest of all propensities of non-proline residues. Alanine and residues with linear side chains displayed the next highest PPII propensities. Three classes of residues displayed lower PPII propensities: β-branched amino acids (Thr, Val, and Ile), short amino acids with polar side chains (Asn, protonated Asp, Ser, Thr, and Cys), and aromatic amino acids (Phe, Tyr, and Trp). tert-Leucine particularly disfavored PPII. The basis of the low PPII propensities of aromatic amino acids in this context was significant cis-trans isomerism, with proline-rich peptides containing aromatic residues exhibiting 45-60% cis amide bonds, due to Pro-cis-Pro-aromatic and aromatic-cis-Pro amide bonds.     

Amphipathic alpha-helices in proteins: results from analysis of protein structures

Amphipathic alpha-helices play a crucial role in mediating the interaction of peptides and proteins with membranes. We have analyzed protein structures for the occurrence of 18-residue amphipathic helices. We find several of these alpha-helices having average hydrophobic moments and average hydrophobicities that would favor their interaction with membranes. We have analyzed the distribution of net charge, helix length, normalized frequency of occurrence, and propensities of the 20 amino acids in the delineated 18-residue helices. We have observed distinct differences in the frequencies of occurrence of polar and hydrophobic amino acids at positions 1-18 in amphipathic and nonamphipathic helices. There are also differences in propensities of the 20 amino acids to occur at positions 1-18 of amphipathic and nonamphipathic helices. Synthetic peptides corresponding to some of these surface-seeking helices do possess antibacterial and/or hemolytic activities. Knowledge of the distribution of charges in 18-residue surface-seeking amphipathic alpha-helices, as well as propensity of occurrence of amino acids at various positions, would be useful inputs in the de novo design of amphipathic peptides.     

Host-guest scale of left-handed polyproline II helix formation

Despite the clear importance of the left-handed polyproline II (PPII) helical conformation in many physiologically important processes as well as its potential significance in protein unfolded states, little is known about the physical determinants of this conformation. We present here a scale of relative PPII helix-forming propensities measured for all residues, except tyrosine and tryptophan, in a proline-based host peptide system. Proline has the highest measured propensity in this system, a result of strong steric interactions that occur between adjacent prolyl rings. The other measured propensities are consistent with backbone solvation being an important component in PPII helix formation. Side chain to backbone hydrogen bonding may also play a role in stabilizing this conformation. The PPII helix-forming propensity scale will prove useful in future studies of the conformational properties of proline-rich sequences as well as provide insights into the prevalence of PPII helices in protein unfolded states.     

Dependence of alpha-helical and beta-sheet amino acid propensities on the overall protein fold type

ABSTRACT: BACKGROUND: A large number of studies have been carried out to obtain amino acid propensities for alpha- helices and beta-sheets. The obtained propensities for alpha-helices are consistent with each other, and the pair-wise correlation coefficient is frequently high. On the other hand, the beta-sheet propensities obtained by several studies differed significantly, indicating that the context significantly affects beta-sheet propensity. RESULTS: We calculated amino acid propensities for alpha-helices and beta-sheets for 39 and 24 protein folds, respectively, and addressed whether they correlate with the fold. The propensities were also calculated for exposed and buried sites, respectively. Results showed that alpha-helix propensities do not differ significantly by fold, but beta-sheet propensities are diverse and depend on the fold. The propensities calculated for exposed sites and buried sites are similar for alpha-helix, but such is not the case for the beta-sheet propensities. We also found some fold dependence on amino acid frequency in beta-strands. Folds with a high Ser, Thr and Asn content at exposed sites in beta-strands tend to have a low Leu, Ile, Glu, Lys and Arg content (correlation coefficient = 0.90) and to have flat beta-sheets. At buried sites in beta-strands, the content of Tyr, Trp, Gln and Ser correlates negatively with the content of Val, Ile and Leu (correlation coefficient = 0.93). "All-beta" proteins tend to have a higher content of Tyr, Trp, Gln and Ser, whereas alpha/beta proteins tend to have a higher content of Val, Ile and Leu. CONCLUSIONS: The alpha-helix propensities are similar for all folds and for exposed and buried residues. However, beta-sheet propensities calculated for exposed residues differ from those for buried residues, indicating that the exposed-residue fraction is one of the major factors governing amino acid composition in beta-strands. Furthermore, the correlations we detected suggest that amino acid composition is related to folding properties such as the twist of a beta-strand or association between two beta sheets.     

Amino acid propensities are position-dependent throughout the length of alpha-helices

The 20 commonly occurring amino acids have been shown to have distinct position-dependent, helix-forming propensities near the ends of alpha-helices. Here, we show that the amino acids also have very strong position-dependent propensities throughout the length of a helix. Most helices are amphiphilic, and they have a strong tendency to both begin and end on the solvent-inaccessible face of the helix. These position-specific propensities should provide valuable parameters to guide de novo protein design, and should allow more precise prediction of helical topology in natural proteins.     

Molecular modeling of conformational properties of oligodepsipeptides

A depsipeptide is a chemical structure consisting of both ester and amide bonds. Quantum mechanics calculations have been performed to investigate the conformational properties of a depsidipeptide in the gas and solution phases. Similar to an alanine dipeptide, the depsidipeptide exhibits a strong preference for the polyproline II (PPII) helical conformation. Meanwhile, due to the changes in the intramolecular interaction, the propensity for beta-sheets and alpha-helices diminishes while an unusual inclination for the (phi,psi) = (-150 degrees ,0 degrees ) conformation was observed. A molecular mechanics model has been developed for polydepsipeptides based on the quantum mechanical study. Both simulated annealing and replica exchange molecular dynamics simulations have been carried out on oligodepsipeptide sequences with alternating depsi and natural residues in solution. Novel helical structures have been indicated from the simulations. When glycine is used as the alternating natural amino acid residue, the PPII conformation of a depsi residue stabilizes the peptide into a right-handed helical structure while the alpha-helical conformation of the depsi residue favors an overall left-handed helical structure. The free energy analysis indicates that both the left- and the right-handed helices are equally likely to exist. When charged lysine is introduced as the alternating natural residue, however, it is found that the depsipeptide sequence prefers an extended conformation as in PPII. Our results indicate that the depsipeptide is potentially useful in designing protein mimetics with controllable structure, function, and chemistry.     

Stereoelectronic effects on the transition barrier of polyproline conformational interconversion

There has been growing interest in polyproline type II (PPII) helices since PPII helices have been found in folded and unfolded proteins and involved in a variety of biological activities. Polyproline can also form type I helices (PPI) which are very different from PPII conformation and only exist in certain organic solvents. Recent studies have shown that stereoelectronic effects play a critical role in stabilizing a PPI or PPII helix. Here, we have synthesized a series of host–guest peptides with an electron‐withdrawing substituent at the 4R or 4S position of proline and used a kinetic approach to further explore stereoelectronic effects on the transition barrier of the interconversion between PPI and PPII conformations. Time‐dependent circular dichroism measurements revealed that the rates of PPII → PPI conversion were reduced upon incorporating the hydroxyl‐, fluoro‐, and methoxy‐groups at the 4R position while the rates would be increased if these substituents were at the 4S position. We quantified the changes in transition free energy by comparing their rate constants. (4R,2S)‐4‐Fluoroproline and (4S,2S)‐4‐fluoroproline have the largest effect on the transition energy barrier for PPII → PPI conversion. Our results provide important insights into the role of stereoelectronic effects on the PPII → PPI transition state barrier, which has not been reported in past thermodynamic studies.     

A survey of left-handed polyproline II helices

Left-handed polyproline II helices (PPII) are contiguous elements of protein secondary structure in which the phi and psi angles of constituent residues are restricted to around -75 degrees and 145 degrees, respectively. They are important in structural proteins, in unfolded states and as ligands for signaling proteins. Here, we present a survey of 274 nonhomologous polypeptide chains from proteins of known structure for regions that form these structures. Such regions are rare, but the majority of proteins contain at least one PPII helix. Most PPII helices are shorter than five residues, although the longest found contained 12 amino acids. Proline predominates in PPII, but Gln and positively charged residues are also favored. The basis of Gln's prevalence is its ability to form an i, i + 1 side-chain to main-chain hydrogen bond with the backbone carbonyl oxygen of the proceeding residue; this helps to fix the psi angle of the Gln and the phi and psi of the proceeding residue in PPII conformations and explains why Gln is favored at the first position in a PPII helix. PPII helices are highly solvent exposed, which explains why apolar amino acids are disfavored despite preferring this region of phi/psi space when in isolation. PPII helices have perfect threefold rotational symmetry and within these structures we find significant correlation between the hydrophobicity of residues at i and i + 3; thus, PPII helices in globular proteins can be considered to be amphipathic.     

Secondary-structure analysis of denatured proteins by vacuum-ultraviolet circular dichroism spectroscopy

To elucidate the structure of denatured proteins, we measured the vacuum-ultraviolet circular dichroism (VUVCD) spectra from 260 to 172 nm of three proteins (metmyoglobin, staphylococcal nuclease, and thioredoxin) in the native and the acid-, cold-, and heat-denatured states, using a synchrotron-radiation VUVCD spectrophotometer. The circular dichroism spectra of proteins fully unfolded by guanidine hydrochloride (GdnHCl) were also measured down to 197 nm for comparison. These denatured proteins exhibited characteristic VUVCD spectra that reflected a considerable amount of residual secondary structures. The contents of alpha-helices, beta-strands, turns, poly-L-proline type II (PPII), and unordered structures were estimated for each denatured state of the three proteins using the SELCON3 program with Protein Data Bank data and the VUVCD spectra of 31 reference proteins reported in our previous study. Based on these contents, the characteristics of the four types of denaturation were discussed for each protein. In all types of denaturation, a decrease in alpha-helices was accompanied by increases in beta-strands, PPII, and unordered structures. About 20% beta-strands were present even in the proteins fully unfolded by GdnHCl in which beta-sheets should be broken. From these results, we propose that denatured proteins constitute an ensemble of residual alpha-helices and beta-sheets, partly unfolded (or distorted) alpha-helices and beta-strands, PPII, and unordered structures.     

Stereoelectronic effects on polyproline conformation

The polyproline type II (PPII) helix is a prevalent conformation in both folded and unfolded proteins, and is known to play important roles in a wide variety of biological processes. Polyproline itself can also form a type I (PPI) helix, which has a disparate conformation. Here, we use derivatives of polyproline, (Pro)10, (Hyp)10, (Flp)10, and (flp)10, where Hyp is (2S,4R)-4-hydroxyproline, Flp is (2S,4R)-4-fluoroproline, and flp is (2S,4S)-4-fluoroproline, to probe for a stereoelectronic effect on the conformation of polyproline. Circular dichroism spectral analyses show that 4R electron-with-drawing substituents stabilize a PPII helix relative to a PPI helix, even in a solvent that favors the PPI conformation, such as n-propanol. The stereochemistry at C4 ordains the relative stability of PPI and PPII helices, as (flp)10 forms a mixture of PPI and PPII helices in water and a PPI helix in n-propanol. The conformational preferences of (Pro)10 are intermediate between those of (Hyp)10/(Flp)10 and (flp)10. Interestingly, PPI helices of (flp)10 exhibit cold denaturation in n-propanol with a value of T(s) near 70 degrees C. Together, these data show that stereoelectronic effects can have a substantial impact on polyproline conformation and provide a rational means to stabilize a PPI or PPII helix.     

Proline-induced distortions of transmembrane helices

Proline residues in the transmembrane (TM) alpha-helices of integral membrane proteins have long been suspected to play a key role for helix packing and signal transduction by inducing regions of helix distortion and/or dynamic flexibility (hinges). In this study we try to characterise the effect of proline on the geometric properties of TM alpha-helices. We have examined 199 transmembrane alpha-helices from polytopic membrane proteins of known structure. After examining the location of proline residues within the amino acid sequences of TM helices, we estimated the helix axes either side of a hinge and hence identified a hinge residue. This enabled us to calculate helix kink and swivel angles. The results of this analysis show that proline residues occur with a significant concentration in the centre of sequences of TM alpha-helices. In this location, they may induce formation of molecular hinges, located on average about four residues N-terminal to the proline residue. A superposition of proline-containing TM helices structures shows that the distortion induced is anisotropic and favours certain relative orientations (defined by helix kink and swivel angles) of the two helix segments.     

In situ conformation of spider silk proteins in the intact major ampullate gland and in solution

To understand the spinning process of dragline silk by spiders, the protein conformation before spinning has to be determined. Raman confocal spectromicroscopy has been used to study the conformation of the proteins in situ in the intact abdominal major ampullate gland of Nephila clavipes and Araneus diadematus spiders. The spectra obtained are typical of natively unfolded proteins and are very similar to that of a mixture of recombinant silk proteins. Vibrational circular dichroism reveals that the conformation is composed of random and polyproline II (PPII) segments with some alpha-helices. The alpha-helices seem to be located in the C-terminal part whereas the repetitive sequence is unfolded. The PPII structure can significantly contribute to the efficiency of the spinning process in nature.     

Assignment of PolyProline II conformation and analysis of sequence--structure relationship

Background

Secondary structures are elements of great importance in structural biology, biochemistry and bioinformatics. They are broadly composed of two repetitive structures namely α-helices and β-sheets, apart from turns, and the rest is associated to coil. These repetitive secondary structures have specific and conserved biophysical and geometric properties. PolyProline II (PPII) helix is yet another interesting repetitive structure which is less frequent and not usually associated with stabilizing interactions. Recent studies have shown that PPII frequency is higher than expected, and they could have an important role in protein – protein interactions.

Methodology/Principal Findings

A major factor that limits the study of PPII is that its assignment cannot be carried out with the most commonly used secondary structure assignment methods (SSAMs). The purpose of this work is to propose a PPII assignment methodology that can be defined in the frame of DSSP secondary structure assignment. Considering the ambiguity in PPII assignments by different methods, a consensus assignment strategy was utilized. To define the most consensual rule of PPII assignment, three SSAMs that can assign PPII, were compared and analyzed. The assignment rule was defined to have a maximum coverage of all assignments made by these SSAMs. Not many constraints were added to the assignment and only PPII helices of at least 2 residues length are defined.

Conclusions/Significance

The simple rules designed in this study for characterizing PPII conformation, lead to the assignment of 5% of all amino as PPII. Sequence – structure relationships associated with PPII, defined by the different SSAMs, underline few striking differences. A specific study of amino acid preferences in their N and C-cap regions was carried out as their solvent accessibility and contact patterns. Thus the assignment of PPII can be coupled with DSSP and thus opens a simple way for further analysis in this field.     

General architecture of the alpha-helical globule

A model is presented for the arrangement of alpha-helices in globular proteins. In the model, helices are placed on certain ribs of "quasi-spherical" polyhedra. The polyhedra are chosen so as to allow the close packing of helices around a hydrophobic core and to stress the collective interactions of the individual helices. The model predicts a small set of stable architectures for alpha-helices in globular proteins and describes the geometries of the helix packings. Some of the predicted helix arrangements have already been observed in known protein structures; others are new. An analysis of the three-dimensional structures of all proteins for which co-ordinates are available shows that the model closely approximates the arrangements and packing of helices actually observed. The average deviations of the real helix axes from those in the model polyhedra is +/- 20 degrees in orientation and +/- 2 A in position (1 A = 0.1 nm). We also show that for proteins that are not homologous, but whose helix arrangements are described by the same polyhedron, the root-mean-square difference in the position of the C alpha atoms in the helices is 1.6 to 3.0 A.     

Polyproline II helix is a key structural motif of the elastic PEVK segment of titin

Titin is a family of giant elastic proteins that constitute an elastic sarcomere matrix in striated muscle. In the I-band region of the sarcomere, where titin extends and develops passive force upon stretch, titin is composed of tandem repeats of approximately 100 residue immunoglobin domains and approximately 28-residue PEVK modules. We have performed 2D NMR and circular dichroism (CD) studies of the conformations of one representative 28-mer PEVK module from human fetal titin (PEPPKEVVPEKKAPVAPPKKPEVPPVKV). NMR data of synthetic peptides of this module as well as three constituent peptides of 9 to 12 residues in aqueous solutions reveal distinguishing features for left-handed three-residue per turn PPII helices: the lack of NOE NN(i, i+1), very large NOE alphaN(i, i+1)/NN(i, i+1), no medium range NOE alphaN(i, i+2), and dihedral angles phi and psi values of -78 and 146, respectively. Structural determinations indicate the presence of three short stretches of PPII helices of 4, 5, and 6 residues that are interposed with an unordered, and presumably flexible, spacer region to give one "polyproline II helix-coil" or "PhC" motif for roughly every 10 residues. These peptides also display the characteristic PPII CD spectra: positive peak or negative shoulder band at 223 nm, negative CD band near 200 nm, and biphasic thermal titration curves that reflect varied stability of these PPII helices. We propose that this PhC motif is a fundamental feature and that the number, length, stability, and distribution of PPII is important in the understanding of the elasticity and protein interactions of the PEVK region of titin.     

Folding and aggregation are selectively influenced by the conformational preferences of the alpha-helices of muscle acylphosphatase

The native state of human muscle acylphosphatase (AcP) presents two alpha-helices. In this study we have investigated folding and aggregation of a number of protein variants having mutations aimed at changing the propensity of these helical regions. Equilibrium and kinetic measurements of folding indicate that only helix-2, spanning residues 55-67, is largely stabilized in the transition state for folding therefore playing a relevant role in this process. On the contrary, the aggregation rate appears to vary only for the variants in which the propensity of the region corresponding to helix-1, spanning residues 22-32, is changed. Mutations that stabilize the first helix slow down the aggregation process while those that destabilize it increase the aggregation rate. AcP variants with the first helix destabilized aggregate with rates increased to different extents depending on whether the introduced mutations also alter the propensity to form beta-sheet structure. The fact that the first alpha-helix is important for aggregation and the second helix is important for folding indicates that these processes are highly specific. This partitioning does not reflect the difference in intrinsic alpha-helical propensities of the two helices, because helix-1 is the one presenting the highest propensity. Both processes of folding and aggregation do not therefore initiate from regions that have simply secondary structure propensities favorable for such processes. The identification of the regions involved in aggregation and the understanding of the factors that promote such a process are of fundamental importance to elucidate the principles by which proteins have evolved and for successful protein design.     

Differences in the amino acid distributions of 3(10)-helices and alpha-helices.

Local determinants of 3(10)-helix stabilization have been ascertained from the analysis of the crystal structure data base. We have clustered all 5-length substructures from 51 nonhomologous proteins into classes based on the conformational similarity of their backbone dihedral angles. Several clusters, derived from 3(10)-helices and multiple-turn conformations, had strong amino acid sequence patterns not evident among alpha-helices. Aspartate occurred over twice as frequently in the N-cap position of 3(10)-helices as in the N-cap position of alpha-helices. Unlike alpha-helices, 3(10)-helices had few C-termini ending in a left-handed alpha conformation; most 3(10) C-caps adopted an extended conformation. Differences in the distribution of hydrophobic residues among 3(10)- and alpha-helices were also apparent, producing amphipathic 3(10)-helices. Local interactions that stabilize 3(10)-helices can be inferred both from the strong amino acid preferences found for these short helices, as well as from the existence of substructures in which tertiary interactions replace consensus local interactions. Because the folding and unfolding of alpha-helices have been postulated to proceed through reverse-turn and 3(10)-helix intermediates, sequence differences between 3(10)- and alpha-helices can also lend insight into factors influencing alpha-helix initiation and propagation.     

Conservation of polyproline II helices in homologous proteins: implications for structure prediction by model building.

Left-handed polyproline II (PPII) helices commonly occur in globular proteins in segments of 4-8 residues. This paper analyzes the structural conservation of PPII-helices in 3 protein families: serine proteinases, aspartic proteinases, and immunoglobulin constant domains. Calculations of the number of conserved segments based on structural alignment of homologous molecules yielded similar results for the PPII-helices, the alpha-helices, and the beta-strands. The PPII-helices are consistently conserved at the level of 100-80% in the proteins with sequence identity above 20% and RMS deviation of structure alignments below 3.0 A. The most structurally important PPII segments are conserved below this level of sequence identity. These results suggest that the PPII-helices, in addition to the other 2 secondary structure classes, should be identified as part of structurally conserved regions in proteins. This is supported by similar values for the local RMS deviations of the aligned segments for the structural classes of PPII-helices, alpha-helices, and beta-strands. The PPII-helices are shown to participate in supersecondary elements such as PPII-helix/alpha-helix. The conservation of PPII-helices depends on the conservation of a supersecondary element as a whole. PPII-helices also form links, possibly flexible, in the interdomain regions. The role of the PPII-helices in model building by homology is 2-fold; they serve as additional conserved elements in the structure allowing improvement of the accuracy of a model and provide correct chain geometry for modeling of the segments equivalenced to them in a target sequence. The improvement in model building is demonstrated in 2 test studies.