Polyproline II Research Articles

Hydrogen bonding patterns and cooperativity in polyproline II helical bundles

Hydrogen bond cooperativity (HBC) plays an important role in stabilizing protein assemblies built by α-helices and β-sheets, the most common secondary structures. However, whether HBC exists in other types of protein secondary structures such as polyproline II (PPII) helices remains unexplored. This is intriguing, since PPII systems as assembling blocks are continuously emerging across multiple fields. Here, using a combination of computational chemistry tools and molecular modeling corroborated by experimental observables, we characterize the distinct H-bonding patterns present in PPII helical bundles and establish that HBC stabilizes intermolecular PPII helices as seen in other protein assemblies such as amyloid fibrils. In addition to cooperative interactions in canonical CO···HN H-bonds, we show that analogous interactions in non-canonical CO···HαCα H-bonds are relevant in Gly-rich PPII bundles, thus compensating for the inability of glycine residues to create hydrophobic cores. Our results provide a mechanistic explanation for the assembly of these bundles.

Structural studies of KCTD1 and its disease-causing mutant P20S provide insights into the protein function and misfunction

Members of the KCTD protein family play key roles in fundamental physio-pathological processes including cancer, neurodevelopmental/neuropsychiatric, and genetic diseases. Here, we report the crystal structure of the KCTD1 P20S mutant, which causes the scalp-ear-nipple syndrome, and molecular dynamics (MD) data on the wild-type protein. Surprisingly, the structure unravels that the N-terminal region, which precedes the BTB domain (preBTB) and bears the disease-associated mutation, adopts a folded polyproline II (PPII) state. The KCTD1 pentamer is characterized by an intricate architecture in which the different subunits mutually exchange domains to generate a closed domain swapping motif. Indeed, the BTB of each chain makes peculiar contacts with the preBTB and the C-terminal domain (CTD) of an adjacent chain. The BTB-preBTB interaction consists of a PPII-PPII recognition motif whereas the BTB-CTD contacts are mediated by an unusual (+/−) helix discontinuous association. The inspection of the protein structure, along with the data emerged from the MD simulations, provides an explanation of the pathogenicity of the P20S mutation and unravels the role of the BTB-preBTB interaction in the insurgence of the disease. Finally, the presence of potassium bound to the central cavity of the CTD pentameric assembly provides insights into the role of KCTD1 in metal homeostasis.

Beyond N-Alkylation: Synthesis, Structure, and Function of N-Amino Peptides.

The growing list of physiologically important protein-protein interactions (PPIs) has amplified the need for compounds to target topologically complex biomolecular surfaces. In contrast to small molecules, peptide and protein mimics can exhibit three-dimensional shape complementarity across a large area and thus have the potential to significantly expand the "druggable" proteome. Strategies to stabilize canonical protein secondary structures without sacrificing side-chain content are particularly useful in the design of peptide-based chemical probes and therapeutics.Substitution of the backbone amide in peptides represents a subtle chemical modification with profound effects on conformation and stability. Studies focused on N-alkylation have already led to broad-ranging applications in peptidomimetic design. Inspired by nonribosomal peptide natural products harboring amide N-oxidations, we envisioned that main-chain hydrazide and hydroxamate bonds would impose distinct conformational preferences and offer unique opportunities for backbone diversification. This Account describes our exploration of peptide N-amination as a strategy for stabilizing canonical protein folds and for the structure-based design of soluble amyloid mimics.We developed a general synthetic protocol to access N-amino peptides (NAPs) on solid support. In an effort to stabilize β-strand conformation, we designed stitched peptidomimetics featuring covalent tethering of the backbone N-amino substituent to the preceding residue side chain. Using a combination of NMR, X-ray crystallography, and molecular dynamics simulations, we discovered that backbone N-amination alone could significantly stabilize β-hairpin conformation in multiple models of folding. Our studies revealed that the amide NH2 substituent in NAPs participates in cooperative noncovalent interactions that promote β-sheet secondary structure. In contrast to Cα-substituted α-hydrazino acids, we found that N-aminoglycine and its N'-alkylated derivatives instead stabilize polyproline II (PPII) conformation. The reactivity of hydrazides also allows for late-stage peptide macrocyclization, affording novel covalent surrogates of side-chain-backbone H-bonds.The pronounced β-sheet propensity of Cα-substituted α-hydrazino acids prompted us to target amyloidogenic proteins using NAP-based β-strand mimics. Backbone N-amination was found to render aggregation-prone lead sequences soluble and resistant to proteolysis. Inhibitors of Aβ and tau identified through N-amino scanning blocked protein aggregation and the formation of mature fibrils in vitro. We further identified NAP-based single-strand and cross-β tau mimics capable of inhibiting the prion-like cellular seeding activity of recombinant and patient-derived tau fibrils.Our studies establish backbone N-amination as a valuable addition to the peptido- and proteomimetic tool kit. α-Hydrazino acids show particular promise as minimalist β-strand mimics that retain side-chain information. Late-stage derivatization of hydrazides also provides facile entry into libraries of backbone-edited peptides. We anticipate that NAPs will thus find applications in the development of optimally constrained folds and modulators of PPIs.

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.

Combining Experiments and Simulations to Examine the Temperature-Dependent Behavior of a Disordered Protein.

Intrinsically disordered proteins are a class of proteins that lack stable folded conformations and instead adopt a range of conformations that determine their biochemical functions. The temperature-dependent behavior of such disordered proteins is complex and can vary depending on the specific protein and environment. Here, we have used molecular dynamics simulations and previously published experimental data to investigate the temperature-dependent behavior of histatin 5, a 24-residue-long polypeptide. We examined the hypothesis that histatin 5 undergoes a loss of polyproline II (PPII) structure with increasing temperature, leading to more compact conformations. We found that the conformational ensembles generated by the simulations generally agree with small-angle X-ray scattering data for histatin 5, but show some discrepancies with the hydrodynamic radius as probed by pulsed-field gradient NMR spectroscopy, and with the secondary structure information derived from circular dichroism. We attempted to reconcile these differences by reweighting the conformational ensembles against the scattering and NMR data. By doing so, we were in part able to capture the temperature-dependent behavior of histatin 5 and to link the observed decrease in hydrodynamic radius with increasing temperature to a loss of PPII structure. We were, however, unable to achieve agreement with both the scattering and NMR data within experimental errors. We discuss different possible reasons for this including inaccuracies in the force field, differences in conditions of the NMR and scattering experiments, and issues related to the calculation of the hydrodynamic radius from conformational ensembles. Our study highlights the importance of integrating multiple types of experimental data when modeling conformational ensembles of disordered proteins and how environmental factors such as the temperature influence them.

Dipole Cooperativity and Polarization Frustration Determine the Secondary Structure Distribution of Short Alanine Peptides in Water.

The physical driving forces for secondary structure preferences of hydrated alanine peptide are investigated with B3LYP-D3(BJ) and the adaptive force matching (AFM) method. The AFM fit to the DFT surface, ALA2022, provides excellent agreement with the nuclear magnetic resonance scalar coupling constants from experiments. In turn, the model is used to gain insight into the physical driving forces behind secondary structure preferences of hydrated peptides. DFT calculations with and without the Conductor-like Screening Model (COSMO) show that the α helix is stabilized by solvent polarization due to dipole cooperativity. The two adjacent amide groups in β strand form a near-planar trapezoid that is not much larger than the size of water molecules. When the finite size of a water molecule is considered, the stabilization from solvent polarization for such a trapezoid is frustrated. Water molecules cannot find orientations to properly stabilize all four polar regions close to each other with such an awkward arrangement. This leads to quite substantial reduction in polarization stabilization. Although the polyproline II (PP-II) conformation is very similar to the β strand, the small twist in the backbone angles allowed much improved polarization stabilization. The improved polarization, when combined with favorable intrapeptide interactions, leads to the PP-II to be lowest in free energy. Other factors, such as the entropic TΔS and the ϕ, ψ coupling terms, are also studied but are found to play only a minor role. The insight shown in this work helps to better understand the structure of globular and intrinsic disordered proteins and facilitate future force field development.

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.

Characterizing polyproline II conformational change of collagen superhelix unit on adsorption on gold surface.

The dynamic process of protein binding onto a metal surface is a frequent occurrence as gold nanoparticles are increasingly being used in biomedical applications, including wound treatment and drug transport. Collagen, as a major component of the extracellular matrix, has potentially advantageous biomedical applications, due to its excellent biocompatibility and elasticity properties. Therefore, a mechanistic comprehension of how and which species in collagen interact with gold nanoparticles is a prerequisite for collagen-gold complexes in clinical application. However, the dynamic behavior of collagen with the polyproline II (PPII) conformation on gold sheets at the molecular level is too complex to capture under current experimental conditions. Here, using molecular dynamics simulations, we investigate the adsorption process and conformational behavior of the tripeptide Gly-Pro-Hyp with the repetitive unit of the collagen superhelix on the gold surface as a function of number of repeating units from 1 to 10. The different numbers of repeating units all prefer to approach the gold surface and adsorb via charged residues at the C-terminal or N-terminal ends, tending to form arch structures on the gold surface. Compared with the various tripeptide units in solution still retaining the native PPII conformation, the presence of the gold surface affects the formation of hydrogen bonds between the protein and water molecules, thus disrupting the PPII conformation of collagen. Specifically, the interaction between the gold surface and HYP limits the rotation of the dihedral angle of collagen, resulting in a tendency for the PPII conformation of the gold surface to transform to the β-sheet conformation. The results provide an indication of how to improve the interaction between the terminal groups and the gold surface for the design of a bioavailable protein-gold material for medicinal purposes.

Influence of polymer molar mass and mixture stoichiometry on polyelectrolyte complexes of poly(l-arginine) and Poly(l-glutamic acid)

Poly(l-arginine) (PARG) and poly(l-glutamic acid) (PLGA) homopolypeptides were custom synthesized by precision N-carboxyanhydride ring-opening polymerization methods with two molar masses, matched for pairs of cationic and anionic polypeptides (degrees of polymerization n = 100 and n = 500). The conformations of the homopolypeptides were probed using circular dichroism (CD) and FTIR spectroscopy which revealed the presence of mainly polyproline II (PPII) conformation. Small-angle X-ray scattering (SAXS) showed concentration-dependent polyelectrolyte peaks and form factor with high q scaling due to the excluded volume behaviour of the wormlike chains. We then examined polyelectrolyte complexation in mixtures of pairs of PARG and PLGA polypeptides with matched molar masses. Precipitation was generally observed and the structures of precipitates, supernatant and resuspended precipitates were investigated using CD, SAXS and cryo-TEM. These revealed that, contrary to prior suggestions in the literature, the precipitates contain mostly polypeptides in a PPII-like conformation, and there is only a minimal β-sheet content (which is enhanced upon drying the sample during preparation for certain measurements). The precipitates have a fractal-like structure as revealed by cryo-TEM and SAXS. Our findings on the structure of polypeptide complex precipitates contribute to the understanding of phase separation of polyelectrolyte complexes and coacervation and may shed light on the formation of inter-cellular bodies of proteins and peptides such as Lewy and other inclusion bodies.

Insight into polyproline II helical bundle stability in an antifreeze protein denatured state

The use of polyproline II (PPII) helices in protein design is currently hindered by limitations in our understanding of their conformational stability and folding. Recent studies of the snow flea antifreeze protein (sfAFP), a useful model system composed of six PPII helices, suggested that a low denatured state entropy contributes to folding thermodynamics. Here, circular dichroism spectroscopy revealed minor populations of PPII like conformers at low temperature. To get atomic level information on the conformational ensemble and entropy of the reduced, denatured state of sfAFP, we have analyzed its chemical shifts and {1H}-15N relaxation parameters by NMR spectroscopy at four experimental conditions. No significant populations of stable secondary structure were detected. The stiffening of certain N-terminal residues at neutral versus acidic pH and shifted pKa values leads us to suggest that favorable charge-charge interactions could bias the conformational ensemble to favor the formation the C1-C28 disulfide bond during nascent folding, although no evidence for preferred contacts between these positions was detected by paramagnetic relaxation enhancement under denaturing conditions. Despite a high content of flexible glycine residues, the mobility of the sfAFP denatured ensemble is similar for denatured α/β proteins both on fast ps/ns as well as slower μs/ms timescales. These results are in line with a conformational entropy in the denatured ensemble resembling that of typical proteins and suggest that new structures based on PPII helical bundles should be amenable to protein design.

Conformational dynamics in the disordered region of human CPEB3 linked to memory consolidation

BackgroundCurrent understanding of the molecular basis of memory consolidation points to an important function of amyloid formation by neuronal-specific isoforms of the cytoplasmic polyadenylation element binding (CPEB) protein family. In particular, CPEB is thought to promote memory persistence through formation of self-sustaining prion-like amyloid assemblies at synapses, mediated by its intrinsically disordered region (IDR) and leading to permanent physical alterations at the basis of memory persistence. Although the molecular mechanisms by which amyloid formation takes place in CPEB have been described in invertebrates, the way amyloid formation occurs in the human homolog CPEB3 (hCPEB3) remains unclear. Here, we characterize by NMR spectroscopy the atomic level conformation and ps-ms dynamics of the 426-residue IDR of hCPEB3, which has been associated with episodic memory in humans.ResultsWe show that the 426-residue N-terminal region of hCPEB3 is a dynamic, intrinsically disordered region (IDR) which lacks stable folded structures. The first 29 residues, M1QDDLLMDKSKTQPQPQQQQRQQQQPQP29, adopt a helical + disordered motif, and residues 86–93: P83QQPPPP93, and 166–175: P166PPPAPAPQP175 form polyproline II (PPII) helices. The (VG)5 repeat motif is completely disordered, and residues 200–250 adopt three partially populated α-helices. Residues 345–355, which comprise the nuclear localization signal (NLS), form a modestly populated α-helix which may mediate STAT5B binding. These findings allow us to suggest a model for nascent hCPEB3 structural transitions at single residue resolution, advancing that amyloid breaker residues, like proline, are a key difference between functional versus pathological amyloids.ConclusionOur NMR spectroscopic analysis of hCPEB3 provides insights into the first structural transitions involved in protein–protein and protein-mRNA interactions. The atomic level understanding of these structural transitions involved in hCPEB3 aggregation is a key first step toward understanding memory persistence in humans, as well as sequence features that differentiate beneficial amyloids from pathological ones.AreasBiophysics, Structural Biology, Biochemistry & Neurosciences.

Local interactions and transient secondary structures govern backbone dynamics of intrinsically disordered proteins

The lack of well-defined structures in intrinsically disordered proteins (IDPs) calls for a fundamental reassessment how their amino-acid sequences codes for functions. Some attention has been paid to nascent structures, but a missing link is sequence-dependent backbone dynamics. In our previous study, we showed that sequence-dependent backbone dynamics in the N-terminal disordered region of ChiZ arises from the formation of correlated segments stabilized by polyproline II (PPII) propensities and salt bridges.

Combination of epigenetic regulation with gene therapy-mediated immune checkpoint blockade induces anti-tumour effects and immune response in vivo

Immunotherapy has become a powerful cancer treatment, but only a small fraction of patients have achieved durable benefits due to the immune escape mechanism. In this study, epigenetic regulation is combined with gene therapy-mediated immune checkpoint blockade to relieve this immune escape mechanism. PPD (i.e., mPEG-b-PLG/PEI-RT3/DNA) is developed to mediate plasmid-encoding shPD-L1 delivery by introducing multiple interactions (i.e., electrostatic, hydrogen bonding, and hydrophobic interactions) and polyproline II (PPII)-helix conformation, which downregulates PD-L1 expression on tumour cells to relieve the immunosuppression of T cells. Zebularine (abbreviated as Zeb), a DNA methyltransferase inhibitor (DNMTi), is used for the epigenetic regulation of the tumour immune microenvironment, thus inducing DC maturation and MHC I molecule expression to enhance antigen presentation. PPD plus Zeb combination therapy initiates a systemic anti-tumour immune response and effectively prevents tumour relapse and metastasis by generating durable immune memory. This strategy provides a scheme for tumour treatment and the inhibition of relapse and metastasis.

Conformational and physicochemical characteristics of bovine skim milk obtained from cows with different genetic variants of β-casein

This study highlights differences in conformational and physicochemical characteristics of bovine skim milk and micellar casein from cows of different β-casein phenotypes. These genetic variants have been one of the predominant topics among dairy researchers due to their differences in β-casein structure, and thus their potential effects on dairy processing and human health. For characterising differences in milk protein structure, Fourier Transform Infrared (FTIR) and Nuclear Magnetic Resonance (1H NMR) spectroscopies combined with chemometric analysis were used. Additionally, physiochemical properties such as mineral content, particle size, and electrostatic charge in skim milk and micellar casein samples were analysed at 4 and 20 ᵒC. Results showed variation in the secondary structure of all three genetic variants independent of temperature. Moreover, the main differences involved a higher level of β-turn and α-helical structures in A1/A1 β-casein milk, while intermolecular β-sheets were more numerous in A1/A2 β-casein milk, whereas random or polyproline II (PPII) structures were more common in A2/A2 β-casein milk. Temperature slightly affected these differences, which was due to the dissociation of β-casein from the micelle at low temperature. In addition, A2/A2 β-casein milk and its micellar casein had a larger average particle size, which resulted in a lower negative ζ-potential. The A2/A2 β-casein samples contained greater amounts of phosphorus and less calcium compared to the other genetic variants of milk and their micellar caseins. The results also indicated that a combination of FTIR and 1H NMR spectroscopies could be used to establish conformational differences in milk and micellar caseins of different genetic variants.

Do polyproline II helix associations modulate biomolecular condensates?

Biomolecular condensates are microdroplets that form inside cells and serve to selectively concentrate proteins, RNAs and other molecules for a variety of physiological functions, but can contribute to cancer, neurodegenerative diseases and viral infections. The formation of these condensates is driven by weak, transient interactions between molecules. These weak associations can operate at the level of whole protein domains, elements of secondary structure or even moieties composed of just a few atoms. Different types of condensates do not generally combine to form larger microdroplets, suggesting that each uses a distinct class of attractive interactions. Here, we address whether polyproline II (PPII) helices mediate condensate formation. By combining with PPII‐binding elements such as GYF, WW, profilin, SH3 or OCRE domains, PPII helices help form lipid rafts, nuclear speckles, P‐body‐like neuronal granules, enhancer complexes and other condensates. The number of PPII helical tracts or tandem PPII‐binding domains can strongly influence condensate stability. Many PPII helices have a low content of proline residues, which hinders their identification. Recently, we characterized the NMR spectral properties of a Gly‐rich, Pro‐poor protein composed of six PPII helices. Based on those results, we predicted that many Gly‐rich segments may form PPII helices and interact with PPII‐binding domains. This prediction is being tested and could join the palette of verified interactions contributing to biomolecular condensate formation.

Intrinsic Conformational Preferences of the Charged Amino Acids in Disordered Proteins

Conformational equilibria in the unfolded states of proteins have many important biological roles, from regulating how some proteins fold and maintain structural stability, to describing the macromolecular details of intrinsically disordered proteins (IDPs), a protein class that does not adopt physiologically stable structures and instead utilizes unfolded states to facilitate their functions. Recently, to better understand the structural properties of IDPs, we analyzed both the sequence dependence to the mean hydrodynamic size of IDPs in water, as well as the impact of heat on the coil dimensions, showing that the sequence dependence and thermodynamic energies associated with intrinsic biases for the polyproline II (PPII) backbone conformation could be obtained. This result is important because PPII is used biologically for protein–protein and protein–nucleic acid interactions and thus has a role in molecular recognition. Experiments that evaluate how the hydrodynamic size changes with compositional changes in the IDP revealed amino acid-specific preferences for PPII that were in excellent quantitative agreement with calorimetry-measured values from small unfolded peptides and also those inferred by a survey of the protein coil library. Here, a set of charge-substitution experiments were used to measure the intrinsic preferences of the charged amino acids (i.e., lysine, arginine, glutamic acid, and aspartic acid) for the PPII backbone conformation, the results of which provide insight into how long-range charge-charge interactions and short-range effects, such as hydration and sterics, can modulate the structural and dynamical properties of IDPs, an increasingly important protein class in cell function.

Structural and Energetic Characterization of the Denatured State from the Perspectives of Peptides, the Coil Library, and Intrinsically Disordered Proteins.

The α and polyproline II (PPII) basins are the two most populated regions of the Ramachandran map when constructed from the protein coil library, a widely used denatured state model built from the segments of irregular structure found in the Protein Data Bank. This indicates the α and PPII conformations are dominant components of the ensembles of denatured structures that exist in solution for biological proteins, an observation supported in part by structural studies of short, and thus unfolded, peptides. Although intrinsic conformational propensities have been determined experimentally for the common amino acids in short peptides, and estimated from surveys of the protein coil library, the ability of these intrinsic conformational propensities to quantitatively reproduce structural behavior in intrinsically disordered proteins (IDPs), an increasingly important class of proteins in cell function, has thus far proven elusive to establish. Recently, we demonstrated that the sequence dependence of the mean hydrodynamic size of IDPs in water and the impact of heat on the coil dimensions, provide access to both the sequence dependence and thermodynamic energies that are associated with biases for the α and PPII backbone conformations. Here, we compare results from peptide-based studies of intrinsic conformational propensities and surveys of the protein coil library to those of the sequence-based analysis of heat effects on IDP hydrodynamic size, showing that a common structural and thermodynamic description of the protein denatured state is obtained.

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.

Sequence-Dependent Correlated Segments in the Intrinsically Disordered Region of ChiZ.

How sequences of intrinsically disordered proteins (IDPs) code for their conformational dynamics is poorly understood. Here, we combined NMR spectroscopy, small-angle X-ray scattering (SAXS), and molecular dynamics (MD) simulations to characterize the conformations and dynamics of ChiZ1-64. MD simulations, first validated by SAXS and secondary chemical shift data, found scant α-helices or β-strands but a considerable propensity for polyproline II (PPII) torsion angles. Importantly, several blocks of residues (e.g., 11–29) emerge as “correlated segments”, identified by their frequent formation of PPII stretches, salt bridges, cation-π interactions, and sidechain-backbone hydrogen bonds. NMR relaxation experiments showed non-uniform transverse relaxation rates (R2s) and nuclear Overhauser enhancements (NOEs) along the sequence (e.g., high R2s and NOEs for residues 11–14 and 23–28). MD simulations further revealed that the extent of segmental correlation is sequence-dependent; segments where internal interactions are more prevalent manifest elevated “collective” motions on the 5–10 ns timescale and suppressed local motions on the sub-ns timescale. Amide proton exchange rates provides corroboration, with residues in the most correlated segment exhibiting the highest protection factors. We propose the correlated segment as a defining feature for the conformations and dynamics of IDPs.

Bacterial Aggregation Triggered by Fibril Forming Tryptophan-Rich Sequences: Effects of Peptide Side Chain and Membrane Phospholipids.

The influence of side chain residue and phospholipid characteristics of the cytoplasmic membrane upon the fibrillation and bacterial aggregation of arginine (Arg) and tryptophan (Trp) rich antimicrobial peptides (AMPs) has not been well described to date. Here, we utilized the structural advantages of HHC-10 and 4HarHHC-10 (Har, l-homoarginine) that are highly active Trp-rich AMPs and investigated their fibril formation and activity behavior against bacteria. The peptides revealed time-dependent self-assembly of polyproline II (PPII) α-helices, but by comparison, 4HarHHC-10 tended to form higher ordered fibrils due to relatively strong cation-π stacking of Trp with Har residue. Both peptides rapidly killed S. aureus and E. coli at their MICs and caused aggregation of bacteria at higher concentrations. This bacterial aggregation was accompanied by the formation of morphologically distinct electron-dense nanostructures, likely including but not limited to peptides alone. Both HHC-10-derived peptides caused blebs and buds in the E. coli membrane that are rich in POPE phospholipid that promotes negative curvature. However, the main population of S. aureus cells retained their cocci structure upon treatment with HHC peptides even at concentration higher than the MICs. In contrast, the cell aggregation was not induced by HHC fibrils that were most likely stabilized through intra-/intermolecular cation-π stacking. It is proposed that masking of these interactions might have resulted in diminished membrane association/insertion of the HHC nanostructures. The peptides caused aggregation of POPC/POPG (1/3) and POPE/POPG (3/1) liposomes. Nonetheless, disaggregation of the former vesicles was observed at ratios of lipid to peptide of greater than 6 and 24 for HHC-10 and 4HarHHC-10, respectively. Collectively, our results revealed dose-dependent bacterial aggregation mediated by Trp-rich AMPs that was profoundly influenced by the degree of peptide's self-association and the composition and intrinsic curvature of the cytoplasmic membrane lipids.