Exploring hydrophobicity limits of polyproline helix with oligomeric octahydroindole‐2‐carboxylic acid

The polyproline‐II helix is the most extended naturally occurring helical structure and is widely present in polar, exposed stretches and “unstructured” denatured regions of polypeptides. Can it be hydrophobic? In this study, we address this question using oligomeric peptides formed by a hydrophobic proline analogue, (2S,3aS,7aS)‐octahydroindole‐2‐carboxylic acid (Oic). Previously, we found the molecular principles underlying the structural stability of the polyproline‐II conformation in these oligomers, whereas the hydrophobicity of the peptide constructs remains to be examined. Therefore, we investigated the octan‐1‐ol/water partitioning and inclusion in detergent micelles of the oligo‐Oic peptides. The results showed that the hydrophobicity is remarkably enhanced in longer oligomeric sequences, and the oligo‐Oic peptides with 3 to 4 residues and higher are specific towards hydrophobic environments. This contrasts significantly to the parent oligoproline peptides, which were moderately hydrophilic. With these findings, we have demonstrated that the polyproline‐II structure is compatible with nonpolar media, whereas additional manipulations of the terminal functionalities feature solubility in extremely nonpolar solvents such as hexane.     

Left-handed polyproline II helix formation is (very) locally driven

The left-handed polyproline II helix (PPII) is believed to be the preferred conformation for proline-rich regions of sequence in proteins. Such regions have been postulated to be protein-protein interaction domains. The formation of this structure is studied here using simple Monte Carlo computer simulations employing the hard sphere potential. It is found that polyproline sequences adopt only the PPII structure in the simulations. Non-proline, non-glycine residues inserted as guests into polyproline host peptides are conformationally restricted by the following proline residues and tend to be part of the PPII helix. It is found through insertion of two alanine residues into polyproline that the PPII structure is not propagated through more than one non-proline residue. This finding calls into question the hypothesis that proline-rich regions will preferentially adopt this structure since many such sequences are comprised of less than 50% proline residues. Proteins 33:218–226, 1998. © 1998 Wiley-Liss, Inc.     

NMR identification of left-handed polyproline type II helices

NMR characteristics of a model left-handed 3(1)-helical peptide are reported in this study. With temperature and sequence corrections on the predicted random coil (15)N chemical shifts, a significant (15)N chemical shift deviation is observed for the model 3(1) peptide. The (15)N chemical shift differences also correlate well with the molar ellipticities (at 220 nm) of the CD spectra at different temperatures, indicating that the (15)N chemical shift is a sensitive probe for 3(1)-helices. The average (3)J(HNalpha) and (1)J(CalphaHalpha) values of the model peptide are determined to be 6.5 and 142.6 Hz, respectively, which are consistent with the values calculated from the geometry of 3(1)-helices. With careful measurements of amide (15)N chemical shifts and incorporating temperature and sequence effect corrections, the (15)N chemical shifts can be used together with (3)J(HNalpha) and (1)J(CalphaHalpha) to differentiate 3(1)-helices from random coils with high confidence. Based on the observed NMR characteristics, a strategy is developed for probing left-handed 3(1)-helical structures from other secondary structures.     

Analysis of epidermal proteins for DNA-binding activity

A group of DNA-binding proteins from the soluble extract of newborn rat epidermis have been separated by chromatography using DNA-cellulose columns. The electrophoretogram of the DNA-binding proteins eluted from a single stranded DNA-cellulose column shows five major proteins of molecular weights ranging between 25K to 40K. Both the epidermal protein filaggrin and most keratins, except two high molecular weight keratins, do not show in vitro DNA-binding activity.     

Left-handed polyproline-II helix revisited: proteins causing proteopathies

Left-handed polyproline-II type helix is a regular conformation of polypeptide chain not only of fibrous, but also of folded and natively unfolded proteins and peptides. It is the only class of regular secondary structure substantially represented in non-fibrous proteins and peptides on a par with right-handed alpha-helix and beta-structure. In this study, we have shown that polyproline-II helix is abundant in several peptides and proteins involved in proteopathies, the amyloid-beta peptides, protein tau and prion protein. Polyproline-II helices form two interaction sites in the amyloid-beta peptides, which are pivotal for pathogenesis of Alzheimer’s disease (AD). It also with high probability is the structure of the majority of tau phosphorylation sites, important for tau hyperphosphorylation and formation of neurofibrillary tangles, a hallmark of AD. Polyproline-II helices form large parts of the structure of the folded domain of prion protein. They can undergo conversion to beta-structure as a result of relatively small change of one torsional angle of polypeptide chain. We hypothesize that in prions and amyloids, in general polyproline-II helices can serve as structural elements of the normal structure as well as dormant nuclei of structure conversion, and thus play important role in structure changes leading to the formation of fibrils.     

Synthesis and conformational analysis of macrocyclic peptides consisting of both α‐helix and polyproline helix segments

Macrocycles are interesting molecules because their topological features and constrained properties significantly affect their chemical, physical, biological, and self‐assembling properties. In this report, we synthesized unique macrocyclic peptides composed of both an α‐helix and a polyproline segment and analyzed their conformational properties. We found that the molecular stiffness of the rod‐like polyproline segment and the relative orientation of the two different helical segments strongly affect the efficiency of the macrocyclization reaction. Conformational analyses showed that both the α‐helix and the polyproline II helix coexisted within the macrocyclic peptides and that the polyproline segment exerts significant effect on the overall helical stability and conformation of the α‐helical segment. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 279–286, 2014.     

A method for prediction of the locations of linker regions within large multifunctional proteins,and application to a type I polyketide synthase

Multifunctional proteins often appear to result from fusion of smaller proteins and in such cases typically can be separated into their ancestral components simply by cleaving the linker regions that separate the domains. Though possibly guided by sequence alignment, structural evidence, or light proteolysis, determination of the locations of linker regions remains empirical. We have developed an algorithm, named UMA, to predict the locations of linker regions in multifunctional proteins by quantification of the conservation of several properties within protein families, and the results agree well with structurally characterized proteins. This technique has been applied to a family of fungal type I iterative polyketide synthases (PKS), allowing prediction of the locations of all of the standard PKS domains, as well as two previously unidentified domains. Using these predictions, we report the cloning of the first fragment from the PKS norsolorinic acid synthase, responsible for biosynthesis of the first isolatable intermediate in aflatoxin production. The expression, light proteolysis and catalytic abilities of this acyl carrier protein-thioesterase didomain are discussed.     

The synaptic acetylcholinesterase tetramer assembles around a polyproline II helix

Functional localization of acetylcholinesterase (AChE) in vertebrate muscle and brain depends on interaction of the tryptophan amphiphilic tetramerization (WAT) sequence, at the C-terminus of its major splice variant (T), with a proline-rich attachment domain (PRAD), of the anchoring proteins, collagenous (ColQ) and proline-rich membrane anchor. The crystal structure of the WAT/PRAD complex reveals a novel supercoil structure in which four parallel WAT chains form a left-handed superhelix around an antiparallel left-handed PRAD helix resembling polyproline II. The WAT coiled coils possess a WWW motif making repetitive hydrophobic stacking and hydrogen-bond interactions with the PRAD. The WAT chains are related by an approximately 4-fold screw axis around the PRAD. Each WAT makes similar but unique interactions, consistent with an asymmetric pattern of disulfide linkages between the AChE tetramer subunits and ColQ. The P59Q mutation in ColQ, which causes congenital endplate AChE deficiency, and is located within the PRAD, disrupts crucial WAT-WAT and WAT-PRAD interactions. A model is proposed for the synaptic AChE(T) tetramer.     

A tetrapeptide-based method for polyproline II-type secondary structure prediction

We describe a new method for polyproline II-type (PPII) secondary structure prediction based on tetrapeptide conformation properties using data obtained from all globular proteins in the Protein Data Bank (PDB). This is the first method for PPII prediction with a relatively high level of accuracy (approximately 60%). Our method uses only frequencies of different conformations among oligopeptides without any additional parameters. We also attempted to predict alpha-helices and beta-strands using the same approach. We find that the application of our method reveals interrelation between sequence and structure even for very short oligopeptides (tetrapeptides).     

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.     

The effect of the polyproline II (PPII) conformation on the denatured state entropy

Polyproline II (PPII) is reported to be a dominant conformation in the unfolded state of peptides, even when no prolines are present in the sequence. Here we use isothermal titration calorimetry (ITC) to investigate the PPII bias in the unfolded state by studying the binding of the SH3 domain of SEM-5 to variants of its putative PPII peptide ligand, Sos. The experimental system is unique in that it provides direct access to the conformational entropy change of the substituted amino acids. Results indicate that the denatured ensemble can be characterized by at least two thermodynamically distinct states, the PPII conformation and an unfolded state conforming to the previously held idea of the denatured state as a random collection of conformations determined largely by hard-sphere collision. The probability of the PPII conformation in the denatured states for Ala and Gly were found to be significant, approximately 30% and approximately 10%, respectively, resulting in a dramatic reduction in the conformational entropy of folding.     

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.     

Evolutionary conservation of the polyproline II conformation surrounding intrinsically disordered phosphorylation sites

Intrinsically disordered (ID) proteins function in the absence of a unique stable structure and appear to challenge the classic structure-function paradigm. The extent to which ID proteins take advantage of subtle conformational biases to perform functions, and whether signals for such mechanism can be identified in proteome-wide studies is not well understood. Of particular interest is the polyproline II (PII) conformation, suggested to be highly populated in unfolded proteins. We experimentally determine a complete calorimetric propensity scale for the PII conformation. Projection of the scale into representative eukaryotic proteomes reveals significant PII bias in regions coding for ID proteins. Importantly, enrichment of PII in ID proteins, or protein segments, is also captured by other PII scales, indicating that this enrichment is robustly encoded and universally detectable regardless of the method of PII propensity determination. Gene ontology (GO) terms obtained using our PII scale and other scales demonstrate a consensus for molecular functions performed by high PII proteins across the proteome. Perhaps the most striking result of the GO analysis is conserved enrichment (P < 10⁻⁸) of phosphorylation sites in high PII regions found by all PII scales. Subsequent conformational analysis reveals a phosphorylation-dependent modulation of PII, suggestive of a conserved “tunability” within these regions. In summary, the application of an experimentally determined polyproline II (PII) propensity scale to proteome-wide sequence analysis and gene ontology reveals an enrichment of PII bias near disordered phosphorylation sites that is conserved throughout eukaryotes.     

A rare polyglycine type II‐like helix motif in naturally occurring proteins

Common structural elements in proteins such as α‐helices or β‐sheets are characterized by uniformly repeating, energetically favorable main chain conformations which additionally exhibit a completely saturated hydrogen‐bonding network of the main chain NH and CO groups. Although polyproline or polyglycine type II helices (PP_II or PG_II) are frequently found in proteins, they are not considered as equivalent secondary structure elements because they do not form a similar self‐contained hydrogen‐bonding network of the main chain atoms. In this context our finding of an unusual motif of glycine‐rich PG_II‐like helices in the structure of the acetophenone carboxylase core complex is of relevance. These PG_II‐like helices form hexagonal bundles which appear to fulfill the criterion of a (largely) saturated hydrogen‐bonding network of the main‐chain groups and therefore may be regarded in this sense as a new secondary structure element. It consists of a central PG_II‐like helix surrounded by six nearly parallel PG_II‐like helices in a hexagonal array, plus an additional PG_II‐like helix extending the array outwards. Very related structural elements have previously been found in synthetic polyglycine fibers. In both cases, all main chain NH and CO groups of the central PG_II‐helix are saturated by either intra‐ or intermolecular hydrogen‐bonds, resulting in a self‐contained hydrogen‐bonding network. Similar, but incomplete PG_II‐helix patterns were also previously identified in a GTP‐binding protein and an antifreeze protein.     

Transient non-specific DNA binding dominates the target search of bacterial DNA-binding proteins

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Polyproline II helix is the preferred conformation for unfolded polyalanine in water

Does aqueous solvent discriminate among peptide conformers? To address this question, we computed the solvation free energy of a blocked, 12‐residue polyalanyl‐peptide in explicit water and analyzed its solvent structure. The peptide was modeled in each of 4 conformers: α‐helix, antiparallel β‐strand, parallel β‐strand, and polyproline II helix (P_II). Monte Carlo simulations in the canonical ensemble were performed at 300 K using the CHARMM 22 forcefield with TIP3P water. The simulations indicate that the solvation free energy of P_II is favored over that of other conformers for reasons that defy conventional explanation. Specifically, in these 4 conformers, an almost perfect correlation is found between a residue's solvent‐accessible surface area and the volume of its first solvent shell, but neither quantity is correlated with the observed differences in solvation free energy. Instead, solvation free energy tracks with the interaction energy between the peptide and its first‐shell water. An additional, previously unrecognized contribution involves the conformation‐dependent perturbation of first‐shell solvent organization. Unlike P_II, β‐strands induce formation of entropically disfavored peptide:water bridges that order vicinal water in a manner reminiscent of the hydrophobic effect. The use of explicit water allows us to capture and characterize these dynamic water bridges that form and dissolve during our simulations. Proteins 2004. © 2004 Wiley‐Liss, Inc.