Polyproline II Helix Research Articles

4,4-Difluoroproline as a Unique 19F NMR Probe of Proline Conformation.

Despite the importance of proline conformational equilibria (trans versus cis amide and exo versus endo ring pucker) on protein structure and function, there is a lack of convenient ways to probe proline conformation. 4,4-Difluoroproline (Dfp) was identified to be a sensitive 19F NMR-based probe of proline conformational biases and cis-trans isomerism. Within model compounds and disordered peptides, the diastereotopic fluorines of Dfp exhibit similar chemical shifts (ΔδFF = 0-3 ppm) when a trans X-Dfp amide bond is present. In contrast, the diastereotopic fluorines exhibit a large (ΔδFF = 5-12 ppm) difference in chemical shift in a cis X-Dfp prolyl amide bond. DFT calculations, X-ray crystallography, and solid-state NMR spectroscopy indicated that ΔδFF directly reports on the relative preference of one proline ring pucker over the other: a fluorine which is pseudo-axial (i.e., the pro-4R-F in an exo ring pucker, or the pro-4S-F in an endo ring pucker) is downfield, while a fluorine which is pseudo-equatorial (i.e., pro-4S-F when exo, or pro-4R-F when endo) is upfield. Thus, when a proline is disordered (a mixture of exo and endo ring puckers, as at trans-Pro in peptides in water), it exhibits a small Δδ. In contrast, when the Pro is ordered (i.e., when one ring pucker is strongly preferred, as in cis-Pro amide bonds, where the endo ring pucker is strongly favored), a large Δδ is observed. Dfp can be used to identify inherent induced order in peptides and to quantify proline cis-trans isomerism. Using Dfp, we discovered that the stable polyproline II helix (PPII) formed in the denatured state (8 M urea) exhibits essentially equal populations of the exo and endo proline ring puckers. In addition, the data with Dfp suggested the specific stabilization of PPII by water over other polar solvents. These data strongly support the importance of carbonyl solvation and n → π* interactions for the stabilization of PPII. Dfp was also employed to quantify proline cis-trans isomerism as a function of phosphorylation and the R406W mutation in peptides derived from the intrinsically disordered protein tau. Dfp is minimally sterically disruptive and can be incorporated in expressed proteins, suggesting its broad application in understanding proline cis-trans isomerization, protein folding, and local order in intrinsically disordered proteins.

Cis-trans isomerization of peptoid residues in the collagen triple-helix

Cis-peptide bonds are rare in proteins, and building blocks less favorable to the trans-conformer have been considered destabilizing. Although proline tolerates the cis-conformer modestly among all amino acids, for collagen, the most prevalent proline-abundant protein, all peptide bonds must be trans to form its hallmark triple-helix structure. Here, using host-guest collagen mimetic peptides (CMPs), we discover that surprisingly, even the cis-enforcing peptoid residues (N-substituted glycines) form stable triple-helices. Our interrogations establish that these peptoid residues entropically stabilize the triple-helix by pre-organizing individual peptides into a polyproline-II helix. Moreover, noting that the cis-demanding peptoid residues drastically reduce the folding rate, we design a CMP whose triple-helix formation can be controlled by peptoid cis-trans isomerization, enabling direct targeting of fibrotic remodeling in myocardial infarction in vivo. These findings elucidate the principles of peptoid cis-trans isomerization in protein folding and showcase the exploitation of cis-amide-favoring residues in building programmable and functional peptidomimetics.

Cryo-EM reveals how the mastigoneme assembles and responds to environmental signal changes.

Mastigonemes are thread-like structures adorning the flagella of protists. In Chlamydomonas reinhardtii, filamentous mastigonemes find their roots in the flagella's distal region, associated with the channel protein PKD2, implying their potential contribution to external signal sensing and flagellar motility control. Here, we present the single-particle cryo-electron microscopy structure of the mastigoneme at 3.4 Å. The filament unit, MST1, consists of nine immunoglobulin-like domains and six Sushi domains, trailed by an elastic polyproline-II helix. Our structure demonstrates that MST1 subunits are periodically assembled to form a centrosymmetric, non-polar filament. Intriguingly, numerous clustered disulfide bonds within a ladder-like spiral configuration underscore structural resilience. While defects in the mastigoneme structure did not noticeably affect general attributes of cell swimming, they did impact specific swimming properties, particularly under varied environmental conditions such as redox shifts and heightened viscosity. Our findings illuminate the potential role of mastigonemes in flagellar motility and suggest their involvement in diverse environmental responses.

On the pathway of the formation of secondary structures in proteins.

Protein structures are typically made up of well-defined modules, called secondary structures. A hierarchical model of protein folding may start with the formation of five-membered non-covalently-linked ring motifs involving O⋅⋅⋅C=O and N-H···N interactions connecting two consecutive peptide groups. Some of these interactions lead to polyproline II structure, which are known to occur in the unfolded state of proteins. These interactions constitute different types of γ-turns, providing the sharpest reversal of the chain direction. Occurring transiently in the unfolded state, and in tandem, they can lead to β-turns. One of the β-turns (type I) is predisposed (from a consideration of residue usage) to form the N-terminal of an α-helix, which then propagates toward its C-terminal direction. O⋅⋅⋅C=O interactions encompass four distinct types of conformational features, and one of them has very similar backbone torsion angles as the polyproline II (PPII) conformation and can thus contribute to the formation of PPII helix. An adjustment from these angles can also drive the formation of β-strand. N-H···N interactions can also constitute capping interaction at helix termini and can link a PPII helix to an α-helix. Thus, the polypeptide backbone is endowed with all the features that can initiate the formation of secondary structural elements, and the γ-turn motifs (resulting from O⋅⋅⋅C=O and N-H···N interactions) are the basic units the protein structures are made up of.

Molecular Dynamics of Lysine Dendrigrafts in Methanol–Water Mixtures

The molecular dynamics method was used to study the structure and properties of dendrigrafts of the first and second generations in methanol-water mixtures with various volume fractions of methanol. At a small volume fraction of methanol, the size and other properties of both dendrigrafts are very similar to those in pure water. A decrease in the dielectric constant of the mixed solvent with an increase in the methanol fraction leads to the penetration of counterions into the dendrigrafts and a reduction of the effective charge. This leads to a gradual collapse of dendrigrafts: a decrease in their size, and an increase in the internal density and the number of intramolecular hydrogen bonds inside them. At the same time, the number of solvent molecules inside the dendrigraft and the number of hydrogen bonds between the dendrigraft and the solvent decrease. At small fractions of methanol in the mixture, the dominant secondary structure in both dendrigrafts is an elongated polyproline II (PPII) helix. At intermediate volume fractions of methanol, the proportion of the PPII helix decreases, while the proportion of another elongated β-sheet secondary structure gradually increases. However, at a high fraction of methanol, the proportion of compact α-helix conformations begins to increase, while the proportion of both elongated conformations decreases.

Peptoid Residues Make Diverse, Hyperstable Collagen Triple-Helices.

As the only ribosomally encoded N-substituted amino acid, proline promotes distinct secondary protein structures. The high proline content in collagen, the most abundant protein in the human body, is crucial to forming its hallmark structure: the triple-helix. For over five decades, proline has been considered compulsory for synthetic designs aimed at recapitulating collagen's structure and properties. Here we describe that N-substituted glycines (N-glys), also known as peptoid residues, exhibit a general triple-helical propensity similar to or greater than proline, enabling synthesis of stable triple-helical collagen mimetic peptides (CMPs) with unprecedented side chain diversity. Supported by atomic-resolution crystal structures as well as circular dichroism and computational characterizations spanning over 30 N-gly-containing CMPs, we discovered that N-glys stabilize the triple-helix primarily by sterically preorganizing individual chains into the polyproline-II helix. We demonstrated that N-glys with exotic side chains including a "click"-able alkyne and a photosensitive side chain enable CMPs for functional applications including the spatiotemporal control of cell adhesion and migration. The structural principles uncovered in this study open up opportunities for a new generation of collagen-mimetic therapeutics and materials.

A proline-rich motif in the large intracellular loop of the glycine receptor α1 subunit interacts with the Pleckstrin homology domain of collybistin

IntroductionThe inhibitory glycine receptor (GlyR), a mediator of fast synaptic inhibition, is located and held at neuronal synapses through the anchoring proteins gephyrin and collybistin. Stable localization of neurotransmitter receptors is essential for synaptic function. In case of GlyRs, only beta subunits were known until now to mediate synaptic anchoring. ObjectivesWe identified a poly-proline II helix (PPII) in position 365–373 of the intra-cellular TM3-4 loop of the human GlyRα1 subunit as a novel potential synaptic anchoring site. The potential role of the PPII helix as synaptic anchoring site was tested. MethodsGlycine receptors and collybistin variants were generated and recombinantly expressed in HEK293 cells and cultured neurons. Receptor function was assessed using patch-clamp electrophysiology, protein-protein interaction was studied using co-immuno-precipitation and pulldown experiments. ResultsRecombinantly expressed collybistin bound to isolated GlyRα1 TM3-4 loops in GST-pulldown assays. When the five proline residues P365A, P366A, P367A, P369A, P373A (GlyRα1P1-5A) located in the GlyRα1-PPII helix were replaced by alanines, the PPII secondary structure was disrupted. Recombinant GlyRα1P1-5A mutant subunits displayed normal cell surface expression and wildtype-like ion channel function, but binding to collybistin was abolished. The GlyRα1-collybistin interaction was independently confirmed by o-immunoprecipitation assays using full-length GlyRα1 subunits. Surprisingly, the interaction was not mediated by the SH3 domain of collybistin, but by its Pleckstrin homology (PH) domain. The mutation GlyRα1P366L, identified in a hyperekplexia patient, is also disrupting the PPII helix, and caused reduced collybistin binding. ConclusionOur data suggest a novel interaction between α1 GlyR subunits and collybistin, which is physiologically relevant in vitro and in vivo and may contribute to postsynaptic anchoring of glycine receptors.

Stabilization of Single Turn Polyproline II Helices via Macrocyclic Hydrocarbon Staples.

Polyproline II helix (PPII) is one of the secondary structures in proteins that play an important role in various biological processes. In this study, we have developed a new macrocyclization strategy that efficiently reinforces a model tetrapeptide into a PPII structure. We also elucidated some relationships between structural features and PPII stability in this model. This new macrocyclic stapling strategy can serve as a useful chemical tool to manipulate the PPII structure for various applications.

Enhancement of direct membrane penetration of arginine-rich peptides by polyproline II helix structure

The left-handed, extended polyproline II (PPII) helix is a unique secondary structure which potently modulates peptide/protein functions through its constraint conformation. To investigate the effect of PPII helix on the direct cell membrane penetration of arginine-rich peptides, we designed a polyproline-containing arginine-rich peptide P9R7W (PPPPPPPPPRRRRRRRW) by introducing nine proline residues into a linear R7W (RRRRRRRW) peptide. Circular dichroism spectroscopy showed that P9R7W has the PPII helix structure in solution whereas R7W is predominantly in random coil structure. Tryptophan fluorescence measurements demonstrated that P9R7W binds to negatively charged lipid vesicles with similar affinity to R7W, in which there was no significant change in the PPII helix structure. Flow cytometry and confocal laser scanning microscopy analyses showed that P9R7W has an ability to penetrate into CHO-K1 cells more efficiently compared to R7W with no cytotoxicity. Consistently, a channel current analysis unveiled that P9R7W penetrates planar lipid bilayer membranes more efficiently than R7W without significant membrane perturbation. Our results indicate that the PPII helix structure can enhance the membrane penetration efficiency of arginine-rich peptides without lipid membrane perturbation.

The ribosome-associated complex RAC serves in a relay that directs nascent chains to Ssb

The conserved ribosome-associated complex (RAC) consisting of Zuo1 (Hsp40) and Ssz1 (non-canonical Hsp70) acts together with the ribosome-bound Hsp70 chaperone Ssb in de novo protein folding at the ribosomal tunnel exit. Current models suggest that the function of Ssz1 is confined to the support of Zuo1, however, it is not known whether RAC by itself serves as a chaperone for nascent chains. Here we show that, via its rudimentary substrate binding domain (SBD), Ssz1 directly binds to emerging nascent chains prior to Ssb. Structural and biochemical analyses identify a conserved LP-motif at the Zuo1 N-terminus forming a polyproline-II helix, which binds to the Ssz1-SBD as a pseudo-substrate. The LP-motif competes with nascent chain binding to the Ssz1-SBD and modulates nascent chain transfer. The combined data indicate that Ssz1 is an active chaperone optimized for transient, low-affinity substrate binding, which ensures the flux of nascent chains through RAC/Ssb.

The effect of salt and temperature on the conformational changes of P1LEA-22, a repeat unit of plant Late Embryogenesis Abundant proteins.

The effect of choline chloride on the conformational dynamics of the 11-mer repeat unit P1LEA-22 of group 3 Late Embryogenesis Abundant (G3LEA) proteins was studied. Circular dichroism data of aqueous solutions of P1LEA-22 revealed that the peptide favors a polyproline II (PPII) helix structure at low temperature, with increasing temperature promoting a gain of unstructured conformations. Furthermore, increases in sample FeCl3 or choline chloride concentrations causes a gain in PPII helical structure at low temperature. The potential role of PPII structure in intrinsically disordered and G3LEA proteins is discussed, including its ability to easily access other secondary structural conformations such as α-helix and β-sheet, which have been observed for dehydrated G3LEA proteins. The observed effect of FeCl3 and choline chloride salts on P1LEA-22 suggests favorable cation interactions with the PPII helix, supporting ion sequestration as a G3LEA protein function. As choline chloride is suggested to improve salt tolerance and protect cell membrane in plants at low temperature, our results support adoption of the PPII structure as a possible damage-preventing measure of Late Embryogenesis Abundant proteins.

Prediction of polyproline II secondary structure propensity in proteins.

Background: The polyproline II helix (PPIIH) is an extended protein left-handed secondary structure that usually but not necessarily involves prolines. Short PPIIHs are frequently, but not exclusively, found in disordered protein regions, where they may interact with peptide-binding domains. However, no readily usable software is available to predict this state. Results: We developed PPIIPRED to predict polyproline II helix secondary structure from protein sequences, using bidirectional recurrent neural networks trained on known three-dimensional structures with dihedral angle filtering. The performance of the method was evaluated in an external validation set. In addition to proline, PPIIPRED favours amino acids whose side chains extend from the backbone (Leu, Met, Lys, Arg, Glu, Gln), as well as Ala and Val. Utility for individual residue predictions is restricted by the rarity of the PPIIH feature compared to structurally common features. Conclusion: The software, available at http://bioware.ucd.ie/PPIIPRED, is useful in large-scale studies, such as evolutionary analyses of PPIIH, or computationally reducing large datasets of candidate binding peptides for further experimental validation.

Impacts of the Terminal Charged Residues on Polyproline Conformation.

Cis-trans isomerization of proline is involved in various biological processes, such as protein folding, cell signaling, and ion-channel gating. Polyproline is a useful system for better understanding proline isomerization because it exists predominantly as two forms, all-cis polyproline I (PPI) and all-trans polyproline II (PPII) helices. The stability of PPI and PPII can be modulated by various effects, including aromatic-proline interactions, terminal charges, and stereoelectronic effects. Here, we used a series of oligoproline peptides in which positively charged or negatively charged amino acids were incorporated into the termini to investigate their effects on polyproline conformation. Circular dichroism measurements show that a cationic residue at the C-terminus or an anionic residue at the N-terminus increases the stability of a PPII helix; in particular, the C-terminal cationic residues impose an enormous impact on PPII stability. The electrostatic attractions between a cationic sidechain and the C-terminal carboxylate exhibit a greater effect than those between an anionic sidechain and the N-terminal ammonium on the conversion of PPII to PPI, suggesting that the stabilization effect of electrostatic interactions on PPII is directional. In contrast, incorporating a cationic residue seems more favorable than adding an anionic residue into the N-terminus because the cationic residue can stabilize PPI. Moreover, the predicted dipole moments from optimized oligopeptide models reveal that the macrodipole of the peptides with a cationic residue at the C-terminus exhibits the opposite direction to that of other peptides in the PPI conformation, suggesting that such a dipole distortion may cause these peptides to disfavor PPI helices. Together, we have found that the introduction of terminal electrostatic interactions can have a significant effect on PPII stability, providing useful information to the design of polyproline-based scaffolds for biomedical applications.

Dissection of the Structural Features of a Fungicidal Antibody-Derived Peptide.

The synthetic peptide T11F (TCRVDHRGLTF), derived from the constant region of human IgM antibodies, proved to exert a significant activity in vitro against yeast strains, including multidrug resistant isolates. Alanine substitution of positively charged residues led to a decrease in candidacidal activity. A more dramatic reduction in activity resulted from cysteine replacement. Here, we investigated the conformational properties of T11F and its alanine-substituted derivatives by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Peptide interaction with Candida albicans cells was studied by confocal and scanning electron microscopy. T11F and most of its derivatives exhibited CD spectra with a negative band around 200 nm and a weaker positive band around 218 nm suggesting, together with NMR coupling constants, the presence of a polyproline II (PPII) helix, a conformational motif involved in a number of biological functions. Analysis of CD spectra revealed a critical role for phenylalanine in preserving the PPII helix. In fact, only the F11A derivative presented a random coil conformation. Interestingly, the loss of secondary structure influenced the rate of killing, which turned out to be significantly reduced. Overall, the obtained results suggest that the PPII conformation contributes in characterising the cell penetrating and fungicidal properties of the investigated peptides.

Solvent Mediation of Peptide Conformations: Polyproline Structures in Water, Methanol, Ethanol, and 1-Propanol as Determined by Ion Mobility Spectrometry-Mass Spectrometry.

Ion mobility spectrometry and circular dichroism spectroscopy are used to examine the populations of the small model peptide, polyproline-13 in water, methanol, ethanol, and 1-propanol over a range of solution temperatures (from 288 to 318K). At low temperatures, the less-polar solvents (1-propanol and ethanol) favor the all-cis polyproline I helix (PPI); as the temperature is increased, the trans-configured polyproline II helix (PPII) is formed. In polar solvents (methanol and water), PPII is favored at all temperatures. From the experimental data, we determine the relative stabilities of the eight structures in methanol, ethanol, and 1-propanol, as well as four in water, all with respect to PPII. Although these conformers show relatively small differences in free energies, substantial variability is observed in the enthalpies and entropies across the structures and solvents. This requires that enthalpies and entropies be highly correlated: in 1-propanol, cis-configured PPI conformations are energetically favorable but entropically disfavored. In more polar solvents, PPI is enthalpically less favorable and entropy favors trans-configured forms. While either ΔH0 or ΔS0 can favor different structures, no conformation in any solvent is simultaneously energetically and entropically stabilized. These data present a rare opportunity to examine the origin of conformational stability. Graphical Abstract ᅟ.

Influence of resveratrol on interactions between negatively charged DPPC/DPPG membranes and positively charged poly-l-lysine

Resveratrol (Res), a natural polyphenol present in different plants and vegetables, exhibits potential therapeutic activity with cardioprotective, antineurodegenerative, antioxidant, and antitumor action. In this study, the effect of Res on the mutual interactions between positively charged poly-l-lysine (PLL) and negatively charged dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol (DPPC/DPPG) membranes was studied using Fourier-transform infrared (FTIR) spectroscopy supported by principal component analysis (PCA). The interactions between PLL and DPPC/DPPG membranes were strongly affected by the presence of Res molecules. Depending on the Res concentration and method of its supply (through the water or lipid phase) to the studied peptide–membrane systems, the membrane-induced transition of PLL from an α-helix to an extended left-handed polyproline II helix (PPII) occurred at different temperatures, with different cooperativity, or was even completely suppressed. The influence of PLL on the conformational (trans/gauche) state of the hydrocarbon chain region of the lipid membranes and the hydration state of the polar/apolar membrane interface was also modulated by Res, depending on the membrane phase state.

Super Secondary Structure Consisting of a Polyproline II Helix and a \u03b2-Turn in Leucine Rich Repeats in Bacterial Type III Secretion System Effectors

Leucine rich repeats (LRRs) are present in over 100,000 proteins from viruses to eukaryotes. The LRRs are 20–30 residues long and occur in tandem. LRRs form parallel stacks of short β-strands and then assume a super helical arrangement called a solenoid structure. Individual LRRs are separated into highly conserved segment (HCS) with the consensus of LxxLxLxxNxL and variable segment (VS). Eight classes have been recognized. Bacterial LRRs are short and characterized by two prolines in the VS; the consensus is xxLPxLPxx with Nine residues (N-subtype) and xxLPxxLPxx with Ten residues (T-subtype). Bacterial LRRs are contained in type III secretion system effectors such as YopM, IpaH3/9.8, SspH1/2, and SlrP from bacteria. Some LRRs in decorin, fribromodulin, TLR8/9, and FLRT2/3 from vertebrate also contain the motifs. In order to understand structural features of bacterial LRRs, we performed both secondary structures assignments using four programs—DSSP-PPII, PROSS, SEGNO, and XTLSSTR—and HELFIT analyses (calculating helix axis, pitch, radius, residues per turn, and handedness), based on the atomic coordinates of their crystal structures. The N-subtype VS adopts a left handed polyproline II helix (PPII) with four, five or six residues and a type I β-turn at the C-terminal side. Thus, the N-subtype is characterized by a super secondary structure consisting of a PPII and a β-turn. In contrast, the T-subtype VS prefers two separate PPIIs with two or three and two residues. The HELFIT analysis indicates that the type I β-turn is a right handed helix. The HELFIT analysis determines three unit vectors of the helix axes of PPII (P), β-turn (B), and LRR domain (A). Three structural parameters using these three helix axes are suggested to characterize the super secondary structure and the LRR domain.

Transmembrane Polyproline Helix.

The third most abundant polypeptide conformation in nature, the polyproline-II helix, is a polar, extended secondary structure with a local organization stabilized by intercarbonyl interactions within the peptide chain. Here we design a hydrophobic polyproline-II helical peptide based on an oligomeric octahydroindole-2-carboxylic acid scaffold and demonstrate its transmembrane alignment in model lipid bilayers by means of solid-state 19F NMR. As result, we provide a first example of a purely artificial transmembrane peptide with a structural organization that is not based on hydrogen-bonding.

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.

Disrupting the intramolecular interaction between proto-oncogene c-Src SH3 domain and its self-binding peptide PPII with rationally designed peptide ligands

Proto-oncogene non-receptor tyrosine protein kinase c-Src has been involved in the development, progression and metastasis of a variety of human cancers. This protein contains two self-binding peptide (SBP) sites separately between the SH3 domain and polyproline-II (PPII) helix and between the SH2 domain and C-terminal phosphorylatable tail (CTPT), which are potential targets of anticancer drugs to regulate the kinase activity. Here, we described an integrated protocol to systematically investigate the structural basis, energetic property and dynamics behaviour of PPII binding to SH3, and to rationally design potent peptide ligands to target the SBP site of SH3–PPII interaction. Our study found that the PPII peptide is a non-typical binder that can only interact effectively with its cognate SH3 domain when it is integrated into the full-length c-Src kinase protein; stripping the peptide from the protein would considerably impair SH3 affinity by increasing entropy penalty upon the domain–peptide binding, suggesting that the protein context plays an essential role in the SBP’s biological function. Next, we identified that the PPII peptide binds to SH3 domain in a class II manner and, on this basis, we derived a series of modified versions of the wild-type PPII peptide using a structure-based rational strategy. These modified peptide mutants have been structurally optimized with respect to their molecular flexibility and interaction potency with SH3 domain, in order to minimize indirect entropy penalty and to maximize direct binding enthalpy simultaneously. Consequently, several rationally designed peptides were obtained, including PPIIm2 (TSKPQTPGRA), PPIIm5 (KPPTPPRA), PPIIm6 (FPPPPPRA) and PPIIm7 (YPPLPPRA), which exhibit a moderately or considerably increased affinity (Kd = 72, 34, 15 and 5.7 μM, respectively) relative to the wild-type PPII (TSKPQTQGLA) (Kd = 160 μM). These peptides can be used as lead molecular entities to further develop new anticancer therapeutics to regulate c-Src kinase activity by targeting the SBP site of SH3–PPII interaction.