Effect Of Peptide Sequence Research Articles

Tn‐MUC1 Glycopeptides as Ligands: Detection of the Effect of Peptide Sequence, Site of Glycosylation, and Glycan Density on MGL Binding in H2O/D2O

ObjectiveTo apply synthetic Tn‐MUC1 glycopeptides for characterizing contributions of their structural parameters to affinity for the endogenous lectin MGL.AbstractO‐Glycosylation of mucins is assumed to serve as a versatile signal via recognition by tissue lectins. Human macrophage galactose‐type lectin (hMGL, CD301 or CLEC10A), a C‐type lectin expressed by dendritic cells and macrophages, is a receptor of Ser/Thr‐linked a‐GalNAc (T antigen nouvelle: Tn, CD175) and its a2,6‐sialylated derivative (sTn). The relative contributions of the glycan and the peptide backbone to affinity are not well understood. In this study, the chemical synthesis of the tandem‐repeat sequence of mucin MUC1, i.e. HGVTSAPDTRPAPGSTAPPA, and site‐specific Thr‐based glycopeptides facilitated calorimetric study of lectin binding. First, CD spectroscopy revealed stabilization of the polyproline II (PPII) structure with increasing density of the glycan epitope. Binding is an enthalpy‐driven process, affinity enhancement occurred for glycopeptides relative to free GalNAc. Size of enthalpic gains were associated to valency, the monoglycosylated peptides had ΔH of about −10 kcal mol−1 and the triply glycosylated ligands had ΔH −30.4 kcal mol− 1. Increases in affinity (Kd) by one order of magnitude were determined for mono‐ (5.6 μM) to triglycosylated (588 nM) MUC1 peptides. Analysis of the kinetic profiles of binding of the MUC1 glycopeptides to hMGL appears to support involvement of a “bind and jump” mechanism. The lifetimes (lower koff values) increase as the density of GalNAc increases along the MUC1 20‐mer sequence. In order to assess a role of solvent reorganization to binding to a C‐type lectin, experiments were performed in parallel in D2O. Unlike GalNAc binding, H/D exchange resulted in more favorable enthalpic contributions, for the monoglycosylated analogs accompanied by compensating higher entropic penalty. Peptide backbone presence makes a difference, as glycan density was revealed to do. In summary, our results suggest that contact building of MUC1 glycopeptides with hMGL critically depends on structural characteristics in the vicinity of the glycan and its density.Support or Funding InformationThis work was supported by the National Institutes of Health Grant CA242351 M. C.

Effect of Peptide Sequence on the LCST-Like Transition of Elastin-Like Peptides and Elastin-Like Peptide-Collagen-Like Peptide Conjugates: Simulations and Experiments.

Elastin-like polypeptides (ELPs) are thermoresponsive biopolymers that undergo an LCST-like phase transition in aqueous solutions. The temperature of this LCST-like transition, Tt , can be tuned by varying the number of repeat units in the ELP, sequence and composition of the repeat units, the solution conditions, and via conjugation to other biomacromolecules. In this study, we show how and why the choice of guest (X) residue in the VPGXG pentad repeat tunes the Tt of short ELPs, (VPGXG)4, in the free state and when conjugated to collagen-like peptides (CLPs). In experiments, the (VPGWG)4 chain (in short, WWWW) has a Tt < 278 K, while (VPGFG)4 or FFFF has a Tt > 353 K in both free ELP and ELP-CLP systems. The Tt for the FWWF ELP sequence decreases from being >353 K for free ELP to <278 K for the corresponding ELP-CLP system. The decrease in Tt upon conjugation to CLP has been shown to be due to the crowding of ELP chains that decreases the entropic loss upon ELP aggregation. Even though the net hydrophobicity of ELP has been reasoned to drive the Tt , the origins of lower Tt of WWWW compared to FFFF are unclear, as there is disagreement in hydrophobicity scales in how phenylalanine (F) compares to tryptophan (W). Motivated by these experimental observations, we use a combination of atomistic and coarse-grained (CG) molecular dynamics simulations. Atomistic simulations of free and tethered ELPs show that WWWW are more prone to acquire β-turn structures than FFFF at lower temperatures. Also, the atomistically informed CG simulations show that the increased local stiffness in W than F due to the bulkier side chain in W compared to F, alone does not cause the shift in the transition of WWWW versus FFFF. The experimentally observed lower Tt of WWWW than FFFF is achieved in CG simulations only when the CG model incorporates both the atomistically informed local stiffness and stronger effective attractions localized at the W position versus the F position. The effective interactions localized at the guest residue in the CG model is guided by our atomistically observed increased propensity for β-turn structure in WWWW versus FFFF and by past experimental work of Urry et al. quantifying hydrophobic differences through enthalpy of association for W versus F.

Insight of Transmembrane Processes of Self-Assembling Nanotubes Based on a Cyclic Peptide Using Coarse Grained Molecular Dynamics Simulation.

Transmembrane self-assembling cyclic peptide (SCP) nanotubes are promising candidates for delivering specific molecules through cell membranes. The detailed mechanisms behind the transmembrane processes, as well as stabilization factors of transmembrane structures, are difficult to elucidate through experiments. In this study, the effects of peptide sequence and oligomeric state on the transmembrane capabilities of SCP nanotubes and the perturbation of embedded SCP nanotubes acting on the membrane were investigated based on coarse grained molecular dynamics simulation. The simulation results reveal that hydrophilic SCP oligomers result in the elevation of the energy barrier while the oligomerization of hydrophobic SCPs causes the reduction of the energy barrier, further leading to membrane insertion. Once SCP nanotubes are embedded, membrane properties such as density, thickness, ordering state and lateral mobility are adjusted along the radial direction. This study provides insight into the transmembrane strategy of SCP nanotubes and sheds light on designing novel transport systems.

Peptide-mediated synthesis of gold nanoparticles: effects of peptide sequence and nature of binding on physicochemical properties

Biomimetic nanotechnologies that use peptides to guide the growth and assembly of nanostructures offer new avenues for the creation of functional nanomaterials and manipulation of their physicochemical properties. However, the impacts of peptide sequence and binding motif upon the surface characteristics and physicochemical properties of nanoparticles remain poorly understood. The configurations of the biomolecules are expected to be extremely important for directing the synthesis and achieving desired material functionality, and these binding motifs will vary with the peptide sequence. Here, we have prepared a series of Au nanoparticles capped with a variety of materials-directing peptides with known affinity for metal surfaces. These nanomaterials were characterized by UV-vis and circular dichroism spectroscopies, transmission electron microscopy, and ζ-potential measurement. Then their catalytic activity for 4-nitrophenol reduction was analyzed. The results indicate that substantially different Au-peptide interfaces are generated using different peptide sequences, even when these sequences have similar binding affinity. This is consistent with recent work showing that Au-peptide binding affinity can have varying entropic and enthalpic contributions, with enthalpically- and entropically-driven binders exhibiting quite different ensembles of configurations on the Au surface. The catalytic activity, as reflected by the measured activation energy, did not correlate with the particle size or with the binding affinity of the peptides, suggesting that the reactivity of these materials is governed by the more subtle details of the conformation of the bound peptide and on the nanoparticle surface reconstruction as dictated by the peptide structure. Such variations in both nanoparticle surface reconstruction and peptide configuration could potentially be used to program specific functionality into the peptide-capped nanomaterials.

Carboxyl-Catalyzed Prototropic Rearrangements in Histidine Peptide Radicals upon Electron Transfer: Effects of Peptide Sequence and Conformation

We report an unusual prototropic rearrangement in gas-phase radicals formed by collisional electron transfer from cesium atoms to protonated peptides HAL, AHL, and ALH at 50 keV. The rearrangement depends on the peptide amino acid sequence and presence or steric accessibility of a free carboxyl group. Upon electron transfer, protonated HAL and ALH rearrange to tautomers that are detected as nondissociated anions in charge-reversal mass spectra. The isomerization is minor in protonated ALH and virtually absent in HAL amide. Electron structure calculations indicate that the gas-phase ions are preferentially protonated in the His imidazole ring and consist of multiple conformers that differ in their hydrogen bonding patterns. Electron transfer to protonated HAL and AHL triggers an exothermic and dynamically barrierless transfer of the carboxyl proton onto the C-2' position of the His ring that occurs on a 120-240 ns time scale. The kinetics of this isomerization are controlled by internal rotations in the radicals to assume conformations favoring the proton transfer. The radical conformations also affect subsequent proton migrations in zwitterionic His imidazoline intermediates that reform the COOH group and result in His ring isomerization. This autocatalytic prototropic rearrangement in gas-phase peptide radicals is analogous to enzyme catalytic reactions involving His and acidic amino acid residues. In contrast to HAL and AHL, the C-2' position is sterically inaccessible in ALH radicals. These radicals undergo proton migrations to the His ring C-5' positions that have moderate energy barriers and are less efficient. RRKM calculations on the combined B3LYP and PMP2/6-311++G(2d,p) potential energy surface of the ground doublet electronic state of the peptide radicals provided rate constants that were quantitatively consistent with the dissociations observed in the gas phase. The formation of minor sequence z(1) and z(2) fragments from AHL was interpreted as occurring in the first excited state of the radical.

Effect of Peptide Sequence on Surface Properties and Self-Assembly of an Amphiphilic pH-Responsive Peptide

Peptides that undergo a morphological change when exposed to a stimulus have been investigated for their surface and self-assembly properties. Two 15-residue sequences were designed and synthesized for the purpose of determining the role of sequence on surface properties and peptide self-assembly. The KhK (KKKFLIVIGSIIKKK) and Alternating Kh (KFLKKIVKIGKKSII) sequences were synthesized via microwave peptide synthesis according to the automated base-labile Fmoc strategy. Despite having the same amino acid content, KhK solutions exhibited an increase in contact angle with increasing pH, whereas Alternating Kh solutions demonstrated a decrease in contact angle with increasing pH. Further analysis by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) showed marked differences in the peptide solution and peptide particle morphology. Circular dichroism (CD) spectroscopy indicated that KhK consisted of primarily beta-sheet conformations at acidic and neutral pH. In Alternating Kh CD spectra, random coil conformations were predominant at acidic and neutral pH.

Nanotubules Formed by Highly Hydrophobic Amphiphilic α-Helical Peptides and Natural Phospholipids

We previously reported that the 18-mer amphiphilic α-helical peptide, Hel 13-5, consisting of 13 hydrophobic residues and five hydrophilic amino acid residues, can induce neutral liposomes (egg yolk phosphatidylcholine) to adopt long nanotubular structures and that the interaction of specific peptides with specific phospholipid mixtures induces the formation of membrane structures resembling cellular organelles such as the Golgi apparatus. In the present study we focused our attention on the effects of peptide sequence and chain length on the nanotubule formation occurring in mixture systems of Hel 13-5 and various neutral and acidic lipid species by means of turbidity measurements, dynamic light scattering measurements, and electron microscopy. We designed and synthesized two sets of Hel 13-5 related peptides: 1) Five peptides to examine the role of hydrophobic or hydrophilic residues in amphiphilic α-helical structures, and 2) Six peptides to examine the role of peptide length, having even number residues from 12 to 24. Conformational, solution, and morphological studies showed that the amphiphilic α-helical structure and the peptide chain length (especially 18 amino acid residues) are critical determinants of very long tubular structures. A mixture of α-helix and β-structures determines the tubular shapes and assemblies. However, we found that the charged Lys residues comprising the hydrophilic regions of amphiphilic structures can be replaced by Arg or Glu residues without a loss of tubular structures. This suggests that the mechanism of microtubule formation does not involve the charge interaction. The immersion of the hydrophobic part of the amphiphilic peptides into liposomes initially forms elliptic-like structures due to the fusion of small liposomes, which is followed by a transformation into tubular structures of various sizes and shapes.

T-cell activation by peptide antigen: effect of peptide sequence and method of antigen presentation.

A series of synthetic peptide analogues of a determinant recognized by the ovalbumin-specific, I-Ad-restricted, T-cell hybridoma 3DO-54.8 were synthesized. The resulting peptides were tested for activation of 3DO-54.8 cells by using glutaraldehyde-fixed cells as well as reconstituted membranes as antigen-presenting surfaces. The results show that the minimum epitope for activation of this T cell is between 7 and 11 amino acids in length. This region includes two important histidine residues. The order of preference of the various peptide analogues was the same regardless of the method of antigen presentation. However, the amount of peptide required for T-cell activation was considerably higher when reconstituted membranes, rather than fixed cells, were used as antigen-presenting surfaces.