Synthetic Model Peptides Research Articles

Amplifiable protein identification via residue-resolved barcoding and composition code counting.

Ultrasensitive protein identification is of paramount importance in basic research and clinical diagnostics but remains extremely challenging. A key bottleneck in preventing single-molecule protein sequencing is that, unlike the revolutionary nucleic acid sequencing methods that rely on the polymerase chain reaction (PCR) to amplify DNA and RNA molecules, protein molecules cannot be directly amplified. Decoding the proteins via amplification of certain fingerprints rather than the intact protein sequence thus represents an appealing alternative choice to address this formidable challenge. Herein, we report a proof-of-concept method that relies on residue-resolved DNA barcoding and composition code counting for amplifiable protein fingerprinting (AmproCode). In AmproCode, selective types of residues on peptides or proteins are chemically labeled with a DNA barcode, which can be amplified and quantified via quantitative PCR. The operation generates a relative ratio as the residue-resolved 'composition code' for each target protein that can be utilized as the fingerprint to determine its identity from the proteome database. We developed a database searching algorithm and applied it to assess the coverage of the whole proteome and secretome via computational simulations, proving the theoretical feasibility of AmproCode. We then designed the residue-specific DNA barcoding and amplification workflow, and identified different synthetic model peptides found in the secretome at as low as the fmol/L level for demonstration. These results build the foundation for an unprecedented amplifiable protein fingerprinting method. We believe that, in the future, AmproCode could ultimately realize single-molecule amplifiable identification of trace complex samples without further purification, and it may open a new avenue in the development of next-generation protein sequencing techniques.

Interaction of Prion Peptides with DNA Structures.

Prion protein aggregation is known to be modulated by macromolecules including nucleic acids. To clarify the role of nucleic acids in PrP pathology, we investigated the interaction between nucleic acids and the prion peptide (PrP)—a synthetic prion protein model peptide resembling a portion of the human prion protein in structure and function spanning amino acid residues 106–126. We used synthetic DNA lattices and natural DNA duplexes extracted from salmon (sDNA) bound with PrP and studied their interaction using distinct physical measurements. The formation of DNA lattices with PrP was visualized by atomic force microscopy (AFM) to investigate the influence of the PrP. PrP inhibited the growth of the double-crossover (DX) lattices significantly compared to the control peptide (CoP). We also conducted optical measurements such as ultraviolet–visible (UV–Vis), circular dichroism (CD), and Fourier transform infrared (FTIR) spectroscopies to validate the interaction between PrP and DNA immediately (D0) and after a 30-day incubation (D30) period. UV–Vis spectra showed variation in the absorbance intensities, specific for the binding of CoP and PrP to DNA. The CD analysis revealed the presence of various secondary structures, such as α-helices and β-sheets, in PrP- and PrP-bound sDNA complexes. The PrP–sDNA interaction was confirmed using FTIR by the change and shift of the absorption peak intensity and the alteration of PrP secondary structures in the presence of DNA. The cytotoxic effects of the PrP-bound sDNA complexes were assessed by a cytotoxicity assay in human neuroblastoma cells in culture. It confirmed that PrP with sDNA was less cytotoxic than CoP. This study provides new applications for DNA molecules by investigating their effect in complex with aggregated proteins. Our study unequivocally showed the beneficial effect of the interaction between DNA and the pathological prion protein. It therefore provides valuable information to exploit this effect in the development of potential therapeutics. Moreover, our work might serve as a basis for further studies investigating the role of DNA interactions with other amyloidogenic proteins.

Peptide Sequence Influence on the Differentiation of Valine and Norvaline by Hot Electron Capture Dissociation.

Isomeric amino acid residues such as valine (Val) and norvaline (Nva) are common in recombinant proteins. The mis-incorporation of Nva for leucine (Leu) causes heterogeneity and in some cases even toxicity. Previous studies have shown that hot electron capture dissociation (HECD) is able to differentiate Val from Nva by producing diagnostic w ions on custom designed synthetic model peptides. To broaden the utilization of HECD in proteomic studies and to define the critical structural features, a thorough investigation was performed on representative peptides including specifically designed synthetic peptides as well as biological peptides bearing tryptic digest-like features and peptides with post-translational modifications. Experimental evidence confirmed that the formation of a w ion is directly dependent upon the presence of the corresponding z ion. The results suggested that a charge carrier residue at the C-terminus is promoting the formation of diagnostic w ions for Nva. Thus, peptides resulting from trypsin digestion, with arginine (Arg) or lysine (Lys) at the C-terminus, can be analyzed using the HECD method. Post-translational modification (PTM) such as phosphorylation did not prevent the generation of the requisite side chain fragmentation w ions. These results suggest the general applicability of HECD for unambiguous identification of Val and Nva especially in structure characterization of therapeutic proteins.

Peptides as Bio-Inspired Electronic Materials: An Electrochemical and First-Principles Perspective.

Molecular electronics is at the forefront of interdisciplinary research, offering a significant extension of the capabilities of conventional silicon-based technology as well as providing a possible stand-alone alternative. Bio-inspired molecular electronics is a particularly intriguing paradigm, as charge transfer in proteins/peptides, for example, plays a critical role in the energy storage and conversion processes for all living organisms. However, the structure and conformation of even the simplest protein is extremely complex, and therefore, synthetic model peptides comprising well-defined geometry and predetermined functionality are ideal platforms to mimic nature for the elucidation of fundamental biological processes while also enhancing the design and development of single-peptide electronic components. In this Account, we first present intramolecular electron transfer within two synthetic peptides, one with a well-defined helical conformation and the other with a random geometry, using electrochemical techniques and computational simulations. This study reveals two definitive electron transfer pathways (mechanisms), the natures of which are dependent on secondary structure. Following on from this, electron transfer within a series of well-defined helical peptides, constrained by either Huisgen cycloaddition, ring-closing metathesis, or a lactam bridge, was determined. The electrochemical results indicate that each constrained peptide, in contrast to a linear counterpart, exhibits a remarkable shift of the formal potential to the positive (>460 mV) and a significant reduction of the electron transfer rate constant (up to 15-fold), which represent two distinct electronic "on/off" states. High-level calculations demonstrate that the additional backbone rigidity provided by the side-bridge constraints leads to an increased reorganization energy barrier, which impedes the vibrational fluctuations necessary for efficient intramolecular electron transfer through the peptide backbone. Further calculations reveal a clear mechanistic transition from hopping to superexchange (tunneling) stemming from side-bridge gating. We then extended our research to fine-tuning of the electronic properties of peptides through both structural and chemical manipulation, to reveal an interplay between electron-rich side chains and backbone rigidity on electron transfer. Further to this, we explored the possibility that the side-bridge constraints present in our synthetic peptides provide an additional electronic transport pathway, which led to the discovery of two distinct forms of quantum interferometer. The effects of destructive quantum interference appear essentially through both the backbone and an alternative tunneling pathway provided by the side bridge in the constrained β-strand peptide, as evidenced by a correlation between electrochemical measurements and conductance simulations for both linear and constrained β-strand peptides. In contrast, an interplay between quantum interference effects and vibrational fluctuations is revealed in the linear and constrained 310-helical peptides. Collectively, these exciting findings augment our fundamental knowledge of charge transfer dynamics and kinetics in peptides and also open up new avenues to design and develop functional bio-inspired electronic devices, such as on/off switches and quantum interferometers, for practical applications in molecular electronics.

Observation of the side chain O-methylation of glutamic acid or aspartic acid containing model peptides by electrospray ionization-mass spectrometry.

O-methylation of the side chains of glutamic acid (E) and aspartic acid (D) residues is generally observed modification when an acidified methanol/water (MeOH/dH2O) mixture is used as a solvent system during sample preparation for proteomic research. This chemical modification may result misidentification with endogenous protein methylation; therefore, a special care should be taken during sample handling prior to mass spectrometric analysis. In the current study, we systematically examined the extent of E/D methylation and C-terminus carboxyl group of synthetic model peptides in terms of different incubation temperatures, storage times, and added acid types as well as its percentages. To monitor these effects, C-terminus amidated and free acid forms of synthetic model peptides comprised of E or D residue(s) have been analyzed by electrospray ionization-mass spectrometry (ESI-MS). Additionally, LC-MS/MS experiments were performed to confirm the formation of methylated peptide product. The results showed that the rate of methylation was increased as the temperature increases along with prolong incubation times. Moreover, the extent of methylation was remarkably high when formic acid (FA) used as a protonation agent instead of acetic acid (AA). In addition, it was found that the degree of methylation was significantly decreased by lowering acid percentages in ESI solution. More than one acidic residue containing model peptides have been also used to explore the extent of multiple methylation reaction. Lastly, the ethanol (EtOH) and isopropanol (iPrOH) have been substituted separately with MeOH in sample preparation step to investigate the extent of esterification reaction under the same experimental conditions. However, in the positive perspective of view, this method can be used as a simple, rapid and cheap method for methylation of acidic residues under normal laboratory conditions.

Assembly of Triblock Amphiphilic Peptides into One-Dimensional Aggregates and Network Formation.

Peptide assembly plays a key role in both neurological diseases and development of novel biomaterials with well-defined nanostructures. Synthetic model peptides provide a unique platform to explore the role of intermolecular interactions in the assembly process. A triblock peptide architecture designed by the Hartgerink group is a versatile system which relies on Coulomb interactions, hydrogen bonding, and hydrophobicity to guide these peptides' assembly at three different length scales: β-sheets, double-wall ribbon-like aggregates, and finally a highly porous network structure which can support gels with ≤1% by weight peptide concentration. In this study, by using molecular dynamics simulations of a structure based implicit solvent coarse grained model, we analyzed this hierarchical assembly process. Parametrization of our CG model is based on multiple-state points from atomistic simulations, which enables this model to represent the conformational adaptability of the triblock peptide molecule based on the surrounding medium. Our results indicate that emergence of the double-wall β-sheet packing mechanism, proposed in light of the experimental evidence, strongly depends on the subtle balance of the intermolecular forces. We demonstrate that, even though backbone hydrogen bonding dominates the early nucleation stages, depending on the strength of the hydrophobic and Coulomb forces, alternative structures such as zero-dimensional aggregates with two β-sheets oriented orthogonally (which we refer to as a cross-packed structure) and β-sheets with misoriented hydrophobic side chains are also feasible. We discuss the implications of these competing structures for the three different length scales of assembly by systematically investigating the influence of density, counterion valency, and hydrophobicity.

Kinetics and thermodynamics of zinc(II) and arsenic(III) binding to XPA and PARP-1 zinc finger peptides

Inhibition of DNA repair is an established mechanism of arsenic co-carcinogenesis, and may be perpetuated by the binding of As(III) to key zinc finger (zf) DNA repair proteins. Validated molecular targets of As(III) include the first zinc finger domain of Poly (ADP-Ribose) Polymerase 1 (PARP-1), and the zinc finger domain of Xeroderma Pigmentosum Complementation Group A (XPA). In order to gain an understanding of the thermodynamic and kinetic parameters of the interaction of As(III) with these two zinc finger motifs, a fluorescence based approach was used to investigate Zn(II) and As(III) binding to synthetic model peptides corresponding to the zf motif of XPA and first zf motif of PARP-1, referred to in this paper as XPAzf and PARP-1zf-1, respectively. While XPAzf and PARP-1zf-1 display similar relative affinities for As(III), PARP-1zf-1 shows a potential kinetic advantage over XPAzf for As(III) binding, with a rate constant for the fast phase of formation of As(III)-PARP-1zf-1 approximately 4-fold higher than for As(III)-XPAzf. However, the binding of Zn(II) with either peptide proceeds at a faster rate than As(III). Notably, XPAzf demonstrates comparable affinities for binding both metals, while PARP-1zf-1 shows a slightly higher affinity for Zn(II), suggesting that the relative concentrations of Zn(II) and As(III) in a system may significantly influence which species predominates in zinc finger occupancy. These results provide insight into the mechanisms underlying interactions between zinc finger structures and As(III), and highlight the potential utility of zinc supplementation in mitigating adverse effects of As(III) on zinc finger functions in vivo.

What Are the Potential Sites of Protein Arylation by N-Acetyl-p-benzoquinone Imine (NAPQI)?

Acetaminophen (paracetamol, APAP) is a safe and widely used analgesic medication when taken at therapeutic doses. However, APAP can cause potentially fatal hepatotoxicity when taken in overdose or in patients with metabolic irregularities. The production of the electrophilic and putatively toxic compound N-acetyl-p-benzoquinone imine (NAPQI), which cannot be efficiently detoxicated at high doses, is implicated in APAP toxicity. Numerous studies have identified that excess NAPQI can form covalent linkages to the thiol side chains of cysteine residues in proteins; however, the reactivity of NAPQI toward other amino acid side chains is largely unexplored. Here, we report a survey of the reactivity of NAPQI toward 11 N-acetyl amino acid methyl esters and four peptides. (1)H NMR analysis reveals that NAPQI forms covalent bonds to the side-chain functional groups of cysteine, methionine, tyrosine, and tryptophan residues. Analogous reaction products were observed when NAPQI was reacted with synthetic model peptides GAIL-X-GAILR for X = Cys, Met, Tyr, and Trp. Tandem mass spectrometry peptide sequencing showed that the NAPQI modification sites are located on the "X" residue in each case. However, when APAP and the GAIL-X-GAILR peptide were incubated with rat liver microsomes that contain many metabolic enzymes, NAPQI formed by oxidative metabolism reacted with GAIL-C-GAILR exclusively. For the peptides where X = Met, Tyr, and Trp, competing reactions between NAPQI and alternative nucleophiles precluded arylation of the target peptide by NAPQI. Although Cys residues are favorably targeted under these conditions, these data suggest that NAPQI can, in principle, also damage proteins at Met, Tyr, and Trp residues.

Influence of a Potentially Destabilizing Central Tryptophan on Transmembrane Helix Domains

Synthetic model peptides, such as GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide), provide a favorable “host” framework for investigations of the influence of chosen “guest” amino acids. For example, it is of interest to know the consequences of having a third, centrally located, tryptophan (Trp) within the hydrophobic core of a well characterized, anchored, transmembrane helix. It is crucial to note that the orientation and rotation of GWALP23 are sensitive to single-residue replacements, in part because the membrane-spanning helix exhibits only limited dynamic averaging of solid-state NMR observables such as the 2H quadrupolar splitting (Biophys. J. 101, 2939). A Trp residue was introduced in the 12th or 13th position of GWALP23, and specific deuterated alanine labels (2H-Ala) were included as probes within the core helical sequence. The 2H quadrupolar splittings from solid-state NMR spectra of GWALP23-W12 and GWALP23-W13 show that the peptide remains helical and retains a dominant preferred tilted transmembrane orientation (similar to GWALP23) in lipid bilayer membranes of DOPC, DLPC, and DMPC. Modified Gaussian and semi-static treatments of the dynamics yield similar conclusions. While a central Trp at position 12 or 13 does not alter the characteristics of bilayer-spanning GWALP23, incorporation of the peptide helix into the bilayer membrane becomes more difficult. The properties of W4, 5 GWALP23 are also being investigated, for comparison with the highly dynamic Y4, 5 and the less dynamic F4, 5 peptides. Deuterium labels at Ala3 and Ala21 will allow assessment of possible fraying of the ends of selected helices in differing lipid membrane environments.

Confined mobility in biomembranes modeled by early stage Brownian motion

An equation of motion, derived from the fractal analysis of the Brownian particle trajectory, makes it possible to calculate the time dependence of the mean square displacement for early times, before the Einstein formula becomes valid. The diffusion coefficient increases with the distance travelled which can be restricted by the geometrical conditions. The corresponding diffusion coefficient cannot increase further to achieve a value characteristic for unrestricted environment. Explicit formula is derived for confined diffusivity related to the unrestricted one as dependent on the maximum particle mean square displacement possible normalized by the square of its mean free path. The model describes the lipid and protein diffusion in tubular membranes with different radii, originally fitted by the modified Saffman–Delbrück equation, and the lateral mobility of synthetic model peptides for which the diffusion coefficient is inversely proportional to the radius of the diffusing object and to the thickness of the membrane.

The Cysteine-rich Region of Type VII Collagen Is a Cystine Knot with a New Topology

Collagens are a group of extracellular matrix proteins with essential functions for skin integrity. Anchoring fibrils are made of type VII collagen (Col7) and link different skin layers together: the basal lamina and the underlying connective tissue. Col7 has a central collagenous domain and two noncollagenous domains located at the N and C terminus (NC1 and NC2), respectively. A cysteine-rich region of hitherto unknown function is located at the transition of the NC1 domain to the collagenous domain. A synthetic model peptide of this region was investigated by CD and NMR spectroscopy. The peptide folds into a collagen triple helix, and the cysteine residues form disulfide bridges between the different strands. The eight cystine knot topologies that are characterized by exclusively intermolecular disulfide bridges have been analyzed by molecular modeling. Two cystine knots are energetically preferred; however, all eight disulfide bridge arrangements are essentially possible. This novel cystine knot is present in type IX collagen, too. The conserved motif of the cystine knot is CX3CP. The cystine knot is N-terminal to the collagen triple helix in both collagens and therefore probably impedes unfolding of the collagen triple helix from the N terminus.

Influence of pH and Histidine Residues on Membrane-Spanning Helical Peptides

Synthetic model peptides such as GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide)provide a useful host framework for investigations of the influence of polar amino acids, for example histidine residues, within the hydrophobic core of a transmembrane helix. Importantly, membrane-spanning GWALP23 is quite sensitive to single-residue replacements, in part because the transmembrane helix exhibits only limited dynamic averaging of solid-state NMR observables such as the 2H quadrupolar splitting (Biophys. J. 101, 2939). We inserted His residues into position 12 and/or 13 of GWALP23 (replacing either L12 or A13) and incorporated specific 2H-Ala labels within the helical core sequence. Solid-state 2H NMR spectra of GWALP23-H12 reveal a marked difference in peptide behavior between acidic and neutral pH conditions. At neutral pH, GWALP23-H12 exhibits a well-defined tilted transmembrane orientation in both DOPC and DLPC bilayer membranes. To prevent the acid catalyzed degradation of lipids, we employed ether-linked DOPC bilayers to observe the effect of low pH on the L12H mutant. Under acidic conditions GWALP23-H12 is highly dynamic and exhibits multiple states. Indeed, the multi-state behavior of GWALP23-H12, when His is charged between pH 1.5 and pH 3, resembles closely that of GWALP23-R12 at neutral pH (J. Am. Chem. Soc. 132, 5803). The dramatic change in the behavior of GWALP23-H12 indicates a pKa value less than 3 for His near the center of a lipid bilayer. Investigations are in progress to chemically exchange the C2 imidazole hydrogen of His for deuterium in the peptide, toward a goal of enabling direct observation of the His ring by solid-state 2H NMR over a range of pH and buffer conditions.

Model Peptides Uncover the Role of the β-Secretase Transmembrane Sequence in Metal Ion Mediated Oligomerization

The β-secretase or β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the enzyme responsible for the formation of amyloid-β peptides, which have a major role in Alzheimer pathogenesis. BACE1 has a transmembrane sequence (TMS), which makes it unique among related proteases. We noticed that the BACE1 TMS contains an uncommon sulfur-rich motif. The sequence MxxxCxxxMxxxCxMxC spans the entire TMS, resembles metal ion binding motifs, and is highly conserved among homologues. We used a synthetic 31-mer model peptide comprising the TMS to study metal ion binding and oligomerization. Applying diverse biochemical and biophysical techniques, we detected dimer and trimer formation of the TMS peptide with copper ions. Replacement of the central Cys466 by Ala essentially abolished these effects. We show that the peptide undergoes a redox reaction with copper ions resulting in a disulfide bridge involving Cys466. Further, we find peptide trimerization that depends on the presence of monovalent copper ions and the sulfhydryl group of Cys466. We identified Cys466 as a key residue for metal ion chelation and to be the core of an oligomerization motif of the BACE1-TMS peptide. Our results demonstrate a novel metal ion controlled oligomerization of the BACE1 TMS, which could have an enormous therapeutic importance against Alzheimer disease.

Characterization of Tyr‐Leu‐Gly, a novel anxiolytic‐like peptide released from bovine α S ‐casein

We found previously that dipeptide YL exhibits orally active anxiolytic activity comparable to diazepam. The YL sequence is often observed in the primary structure of natural food proteins. In the present study, we investigated whether YL and YL analogues are released from bovine αS-casein by gastrointestinal proteases. YLG, corresponding to αS1-casein (aa 91-93), was more effectively released from αS-casein than YL by pepsin-pancreatin digestion, mimicking gastrointestinal enzymatic conditions. Using the synthetic model peptide, we determined that trypsin cleaved the N terminus of YLG, and elastase and carboxypeptidase contributed to cleave the C-terminus. YLG exhibited orally active anxiolytic-like activity in the elevated plus maze and open-field tests in mice. The anxiolytic-like activity of YLG was inhibited by WAY100135, SCH23390 or bicuculline, antagonists of serotonin 5-HT1A, dopamine D1, and GABA(A) receptors, respectively; however, YLG had no affinity for these receptors. The pepsin-pancreatin digest of αS-Casein also exhibited anxiolytic-like activity. Meanwhile, anxiolytic-like activity of α-casozepine, an αS1-casein-derived decapeptide with YL sequence in the N terminus, was blocked by WAY100135, SCH23390, or bicuculline, equally to YLG and YL; however, it was not detected in the pepsin-pancreatic digest. Taken together, we found that YLG is released after pepsin-pancreatic digestion of αS-casein and exhibits potent anxiolytic-like activity via activation of serotonin, dopamine, and the GABA receptor system.

Comparison of four synthetic model peptides to understand the role of modular motifs in the self-assembly of silk fibroin

To understand the effects of the modular motifs on the silk fibroin self-assembly, regenerated silk fibroin (RSF) and synthetic SF model peptides (GS6: GAGAGS; GS10: GAGAGSGAAS; GY8: GAGAGVGY; GY6: GAGAGY) were prepared and then their self-assembly behaviors were investigated by atomic force microscopy, scanning electron microscopy, attenuated total reflectance Fourier transform infrared spectroscopy, and circular dichroism. The self-assembly behaviors were dependent on modular motifs, amino acid compositions, and concentrations. At relatively high concentrations, GS6 and GS10 self-assembled from disordered aggregates (random coil) to nanofibrils (antiparallel β-sheet), and GY8 self-assembled from disordered aggregates (random coil) to small nanolayer-like features (antiparallel β-sheet). At relatively low concentrations, GS6, GS10, and GY8 experienced a height decrease of protofilament features from 0.8 ± 0.1 nm (random coil) to 0.6 ± 0.1 nm (antiparallel β-sheet). GY6 did not self-assemble at relatively high or low concentration. The secondary structure and morphology transition times of RSF and SF model peptides were: RSF GS10 > GY8 > GY6. These results provide useful insights into the role of modular motifs in the self-assembly of silk fibroin. Furthermore, our work will be beneficial to the peptide sequence design of silk-based biomaterials with targeted properties.

Molecular basis for the action of the collagen-specific chaperone Hsp47/SERPINH1 and its structure-specific client recognition

Collagen is the most abundant protein in animals and is a major component of the extracellular matrix in tissues such as skin and bone. A distinctive structural feature of all collagen types is a unique triple-helical structure formed by tandem repeats of the consensus sequence Xaa-Yaa-Gly, in which Xaa and Yaa frequently are proline and hydroxyproline, respectively. Hsp47/SERPINH1 is a procollagen-specific molecular chaperone that, unlike other chaperones, specifically recognizes the folded conformation of its client. Reduced functional levels of Hsp47 were reported in severe recessive forms of osteogenesis imperfecta, and homozygous knockout is lethal in mice. Here we present crystal structures of Hsp47 in its free form and in complex with homotrimeric synthetic collagen model peptides, each comprising one Hsp47-binding site represented by an arginine at the Yaa-position of a Xaa-Yaa-Gly triplet. Two of these three binding sites in the triple helix are occupied by Hsp47 molecules, which bind in a head-to-head fashion, thus making extensive contacts with the leading and trailing strands of the collagen triple helix. The important arginine residue within the Xaa-Arg-Gly triplet is recognized by a conserved aspartic acid. The structures explain the stabilization of the triple helix as well as the inhibition of collagen-bundle formation by Hsp47. In addition, we propose a pH-dependent substrate release mechanism based on a cluster of histidine residues.

Tyrosine Replacing Tryptophan as an Anchor in GWALP Peptides

Synthetic model peptides have proven useful for examining fundamental peptide-lipid interactions. A frequently employed peptide design consists of a hydrophobic core of Leu-Ala residues with polar or aromatic amino acids flanking each side at the interfacial positions, which serve to "anchor" a specific transmembrane orientation. For example, WALP family peptides (acetyl-GWW(LA)(n)LWWA-[ethanol]amide), anchored by four Trp residues, have received particular attention in both experimental and theoretical studies. A recent modification proved successful in reducing the number of Trp anchors to only one near each end of the peptide. The resulting GWALP23 (acetyl-GGALW(5)(LA)(6)LW(19)LAGA-[ethanol]amide) displays reduced dynamics and greater sensitivity to lipid-peptide hydrophobic mismatch than traditional WALP peptides. We have further modified GWALP23 to incorporate a single tyrosine, replacing W(5) with Y(5). The resulting peptide, Y(5)GWALP23 (acetyl-GGALY(5)(LA)(6)LW(19)LAGA-amide), has a single Trp residue that is sensitive to fluorescence experiments. By incorporating specific (2)H and (15)N labels in the core sequence of Y(5)GWALP23, we were able to use solid-state NMR spectroscopy to examine the peptide orientation in hydrated lipid bilayer membranes. The peptide orients well in membranes and gives well-defined (2)H quadrupolar splittings and (15)N/(1)H dipolar couplings throughout the core helical sequence between the aromatic residues. The substitution of Y(5) for W(5) has remarkably little influence on the tilt or dynamics of GWALP23 in bilayer membranes of the phospholipids DOPC, DMPC, or DLPC. A second analogue of the peptide with one Trp and two Tyr anchors, Y(4,5)GWALP23, is generally less responsive to the bilayer thickness and exhibits lower apparent tilt angles with evidence of more extensive dynamics. In general, the peptide behavior with multiple Tyr anchors appears to be quite similar to the situation when multiple Trp anchors are present, as in the original WALP series of model peptides.

Importance of Aromatic Anchor Residue Identity and Location for the Tilt and Dynamics of Transmembrane Peptides

Aromatic or polar amino acid residues often flank each side of a membrane-spanning alpha helix, whether in an integral membrane protein or a model peptide. The aromatic residues Trp, Tyr and to a lesser extent Phe tend to partition to the membrane-water interface and act as “anchors” to stabilize the transmembrane orientation. The synthetic model peptide, GWALP23 (acetyl-GGALW5(LA)6LW19LAGA-[ethanol]amide), has proven valuable for experimentation, with only one Trp “anchor” near each end of the transmembrane sequence. Indeed, with relatively minimal complications from the peptide dynamics, the average tilt of GWALP23 has been shown to vary systematically in lipid bilayer membranes of different thickness (see J. Biol. Chem. 285, 31723). We have employed 2H-alanines and solid-state NMR spectroscopy to investigate the consequences of moving or replacing W5 or W19 in GWALP23 with selected Tyr, Phe or Trp residues at the same or nearby locations. We find that GWALP23 peptides having Y5, F5 or W5 exhibit essentially the same average tilt in bilayer membranes of DOPC, DMPC or DLPC; with somewhat increased dynamics for the F5 peptide. When “double” anchors are present in Y4,5GWALP23 or F4,5GWALP23, the peptides appear less responsive to the bilayer thickness, as the dynamics become dramatically more extensive. Moving W19 to position 18, a 100° radial change, alters the direction of the helix tilt, as expected. We conclude that, in the absence of other functional groups, the aromatic residues determine the preferred orientations and dynamics of transmembrane peptides. Increased dynamics are observed when the ring hydrogen bonding is removed (Phe), or when two aromatic anchors are present on one side of the core transmembrane sequence.

Exploring Peptide-Membrane Interactions with Coarse-Grained MD Simulations

The interaction of α-helical peptides with lipid bilayers is central to our understanding of the physicochemical principles of biological membrane organization and stability. Mutations that alter the position or orientation of an α-helix within a membrane, or that change the probability that the α-helix will insert into the membrane, can alter a range of membrane protein functions. We describe a comparative coarse-grained molecular dynamics simulation methodology, based on self-assembly of a lipid bilayer in the presence of an α-helical peptide, which allows us to model membrane transmembrane helix insertion. We validate this methodology against available experimental data for synthetic model peptides (WALP23 and LS3). Simulation-based estimates of apparent free energies of insertion into a bilayer of cystic fibrosis transmembrane regulator-derived helices correlate well with published data for translocon-mediated insertion. Comparison of values of the apparent free energy of insertion from self-assembly simulations with those from coarse-grained molecular dynamics potentials of mean force for model peptides, and with translocon-mediated insertion of cystic fibrosis transmembrane regulator-derived peptides suggests a nonequilibrium model of helix insertion into bilayers.

Model peptides provide new insights into the role of histidine residues as potential ligands in human cellular copper acquisition via Ctr1.

Cellular acquisition of copper in eukaryotes is primarily accomplished through the Ctr family of copper transport proteins. In both humans and yeast, methionine-rich "Mets" motifs in the amino-terminal extracellular domain of Ctr1 are thought to be responsible for recruitment of copper at the cell surface. Unlike yeast, mammalian Ctr1 also contains extracellular histidine-rich motifs, although a role for these regions in copper uptake has not been explored in detail. Herein, synthetic model peptides containing the first 14 residues of the extracellular domain of human Ctr1 (MDHSHHMGMSYMDS) have been prepared and evaluated for their apparent binding affinity to both Cu(I) and Cu(II). These studies reveal a high affinity Cu(II) binding site (log K = 11.0 ± 0.3 at pH 7.4) at the amino-terminus of the peptide as well as a high affinity Cu(I) site (log K = 10.2 ± 0.2 at pH 7.4) that utilizes adjacent HH residues along with an additional His or Met ligand. These model studies suggest that the histidine domains may play a direct role in copper acquisition from serum copper-binding proteins and in facilitating the reduction of Cu(II) to the active Ctr1 substrate, Cu(I). We tested this hypothesis by expressing a Ctr1 mutant lacking only extracellular histidine residues in Ctr1-knockout mouse embryonic fibroblasts. Results from live cell studies support the hypothesis that extracellular amino-terminal His residues directly participate in the copper transport function of Ctr1.