Guest Residue Research Articles

Computer simulation of partitioning of ten pentapeptides Ace-WLXLL at the cyclohexane/water and phospholipid/water interfaces.

BackgroundPeptide-membrane interactions play a key role in the binding, partitioning and folding of membrane proteins, the activity of antimicrobial and fusion peptides, and a number of other processes. To gain a better understanding of the thermodynamics of such interactions, White and Wimley created an interfacial hydrophobicity scale based of the transfer free energy from water to octanol or lipid bilayers of a series of synthetic peptapeptides (Ace-WLXLL, with X being any of the twenty natural amino acids) (White and Wimley (1996) Nat. Struct. Biol. 3, 842–848). In this study, we performed molecular dynamics simulations of a representative set of ten of these peptides (X = D, K, R, N, A, T, S, I, F and W) in two membrane mimetic interfaces: water-cyclohexane (10 ns) and a fully solvated dioleoylphosphatidylcholine (DOPC) bilayer (50 ns) using both constant pressure and constant area ensembles. We focus on partitioning of the ten peptides at the cyclohexane/water and lipid/water interfaces.ResultsThe peptides rapidly equilibrate (< 2 ns) and partition at the cyclohexane/water interface. The X3 guest residue assumes average orientations that depend on the nature of the side chain. At the DOPC/water interface, dynamics is much slower and convergence is difficult to achieve on a 50 ns timescale. Nonetheless, all peptides partition to the lipid/water interface with distributions with widths of 1–2 nm. The peptides assume a broad range of side chain and backbone orientations and have only a small effect on the area of the unit cell. On average, hydrophobic guest residues partition deeper into the hydrophobic core than hydrophilic residues. In some cases the peptides penetrate sufficiently deep to somewhat affect the distribution of the C=C double bond in DOPC. The relative distribution of the X3 guest residue compared to W1 and L5 is similar in the water/cyclohexane and water/lipid simulations. Snapshots show mostly extended backbone conformations in both environments. There is little difference between simulations at a constant area of 0.66 nm2 and simulations at constant pressure that approximately yield the same average area of 0.66 nm2.ConclusionThese peptides were designed to assume extended conformations, which is confirmed by the simulations. The distribution of the X3 side chain depends on its nature, and can be determined from molecular dynamics simulations. The time scale of peptide motion at a phospholipids-water interface is too long to directly calculate the experimentally measured hydrophobicity scale to test and improve the simulation parameters. This should be possible at the water/cyclohexane interface and likely will become feasible in the future for the phospholipids/water case.

Probing the Environment of Signal−Anchor Sequences during Topogenesis in the Endoplasmic Reticulum

Signal sequences for insertion of protein into the mammalian endoplasmic reticulum orient themselves in the translocon on the basis of their flanking charges. It has recently been shown that hydrophobic N-terminal signals initially insert head-on before they invert their orientation to translocate the C-terminus. The rate of inversion is reduced with the increasing hydrophobicity of the signal due to an increased affinity for the initial bound state at the translocon. To probe the environment of the signal while its orientation is determined, different hydrophobic residues were inserted at various positions throughout a uniform oligoleucine signal sequence and the constructs were expressed in transfected COS-7 cells. The resulting topologies revealed a strikingly symmetric position dependence specifically for bulky aromatic amino acids, reflecting the structure of a lipid bilayer. Maximal N-translocation was observed when the guest residues were placed at the N- or C-terminus of the hydrophobic sequence or in the very center, corresponding to the positions of highest expected affinity of the signal sequence as a membrane-spanning helix for the bilayer. The results support the model that during topogenesis in vivo the signal sequence is exposed to the lipid membrane.

Polypeptide-Solvent Interactions Measured by Single Molecule Force Spectroscopy

AbstractStimulus-responsive biomolecules have attracted a large research interest because of their potential application in various areas such as drug delivery, actuators and sensing devices at the nanoscale. Using single-molecule force spectroscopy (SMFS) we studied elastin-like polypeptides (ELPs). These stimulus-responsive polypeptides undergo an inverse temperature transition, accompanied by a large conformational change, when the solvent quality is changed by increasing the temperature or by addition of salt. Understanding the relationship between peptide sequence and mechanisms of force generation can provide a route to engineer ELPs with desirable mechano-chemical properties. Here we studied the effect of solvent quality and type of guest residue on the mechanical properties of ELPs on the single-molecule level. We used a statistical approach to estimate polymer elasticity parameters from model fits to the data. With this approach we were able to resolve small changes in the Kuhn segment length distributions associated with different molecular architectures. We then show that these mechanical differences likely arise from differences in the hydrophobic hydration of sidegoups, in line with recent predictions from molecular dynamics simulations.

The change in Gibbs free energy for hydrophobic associationDerivation and evaluation by means of inverse temperature transitions

The change in Gibbs free energy resulting from changes in the temperature (Tt) and heat (ΔHt) of an inverse temperature transition (ITT) of hydrophobic association is derived and utilized to develop a hydrophobicity scale for amino acid residues within an elastic model protein, (GVGVP)n. On raising the temperature, hydrophobic residues go from water to hydrophobic association. Using Tt and ΔHt of the ITT, the change in Gibbs free energy for hydrophobic association, ΔGHA0, in kcal/mol-(GXGVP) where X is the guest amino acid residue, was calculated for the naturally occurring amino acid residues in PBS. ΔGHA0 varies by more than 10 kcal/mol-(GXGVP) from the most hydrophobic tryptophan residue to the most hydrophilic glutamate residue.

Dendrimers as Guests in Molecular Recognition Phenomena

AbstractFor Abstract see ChemInform Abstract in Full Text.

The change in Gibbs free energy for hydrophobic association: Derivation and evaluation by means of inverse temperature transitions

The change in Gibbs free energy resulting from changes in the temperature ( T t) and heat (Δ H t) of an inverse temperature transition (ITT) of hydrophobic association is derived and utilized to develop a hydrophobicity scale for amino acid residues within an elastic model protein, (GVGVP) n . On raising the temperature, hydrophobic residues go from water to hydrophobic association. Using T t and Δ H t of the ITT, the change in Gibbs free energy for hydrophobic association, Δ G HA 0 , in kcal/mol-(GXGVP) where X is the guest amino acid residue, was calculated for the naturally occurring amino acid residues in PBS. Δ G HA 0 varies by more than 10 kcal/mol-(GXGVP) from the most hydrophobic tryptophan residue to the most hydrophilic glutamate residue.

Short sequences of non-proline residues can adopt the polyproline II helical conformation.

The left-handed polyproline II (P(II)) helix is a structure that has been given a great deal of attention lately because of its role in a wide variety of physiologically important processes and potential significance in protein unfolded states. Recent work by several authors has shown that residues besides proline can adopt this structure. A scale of relative P(II)-helix-forming propensities has been generated but only for single guest residues in a proline-based host system. Here, we present multiple guest residues in a proline-based host system. Using circular dichroism spectroscopy, we have shown that not only single residues, but also short sequences of non-proline residues can adopt the P(II) conformation.

Polyproline II Helix Conformation in a Proline-Rich Environment: A Theoretical Study

Interest centers here on whether a polyproline II helix can propagate through adjacent non-proline residues, and on shedding light on recent experimental observations suggesting the presence of significant PP II structure in a short alanine-based peptide with no proline in the sequence. For this purpose, we explored the formation of polyproline II helices in proline-rich peptides with the sequences Ac-(Pro) 3-X-(Pro) 3-Gly-Tyr-NH 2, with X = Pro (PPP), Ala (PAP), Gln (PQP), Gly (PGP), and Val (PVP), and Ac-(Pro) 3-Ala-Ala-(Pro) 3-Gly-Tyr-NH 2 (PAAP), by using a theoretical approach that includes a solvent effect as well as cis ↔ trans isomerization of the peptide groups and puckering conformations of the pyrrolidine ring of the proline residues. Since 13C chemical shifts have proven to be useful for identifying secondary-structure preferences in proteins and peptides, and because values of the dihedral angles ( ϕ, ψ) are the main determinants of their magnitudes, we have, therefore, computed the Boltzmann-averaged 13C chemical shifts for the guest residues in the PXP peptide ( X = Pro, Ala, Gln, Gly, and Val) with a combination of approaches, involving molecular mechanics, statistical mechanics, and quantum mechanics. In addition, an improved procedure was used to carry out the conformational searches and to compute the solvent polarization effects faster and more accurately than in previous work. The current theoretical work and additional experimental evidence show that, in short proline-rich peptides, alanine decreases the polyproline II helix content. In particular, the theoretical evidence accumulated in this work calls into question the proposal that alanine has a strong preference to adopt conformations in the polyproline II region of the Ramachandran map.

Supramolecular assembly of collagen triblock peptides.

The relationship between primary sequence and collagen triple-helix formation is relatively well characterized, while higher levels of structural assembly from these sequences is poorly understood. To address this gap, a new collagen-like triblock peptide design was used to study the relationship between amino acid sequence and supramolecular assembly. Four collagen-like peptides with the sequence (Glu)(5)(Gly-Xaa-Hyp-Gly-Pro-Hyp)(6)(Glu)(5) and corresponding to Xaa = alanine, proline, serine, or valine, and an analogous peptide without the glutamic acid end blocks, were solubilized in water at high concentrations (20-150 mg/mL) and analyzed in optical polarizing microscopy and transmission electron microscopy. Some of the peptides self-assembled into supramolecular structures, the nature of which was determined by the core collagen-like sequence. The globular end blocks appeared necessary for these short triple-helix-forming peptides to spontaneously organize into supramolecular structures in solution and also provided enhanced thermal stability based on CD analysis. The results indicate a strong dependence of the peptide triblock assembly behavior on the identity of the guest residue Xaa; nematic order when Xaa was valine, no organization when Xaa was serine, and banded spherulites displaying a cholesteric-like twist when Xaa was proline or alanine. According to these results, the identity of the amino acid in position Xaa of the triplet Gly-Xaa-Yaa dramatically determined the type of supramolecular assembly formed by short triple helices based on collagen-triblock like sequences. Moreover, the structural organization observed for these collagen-triblock peptides was analogous to some assemblies observed for native collagen in vivo and in vitro. The amino acid sequence in the native collagen proteins may therefore be a direct determinant of the different supramolecular architectures found in connective tissues.

Modulating Protein Folding Rates in Vivo and in Vitro by Side-chain Interactions between the Parallel β Strands of Green Fluorescent Protein

We have identified pairs of residues across the two parallel beta strands of green fluorescent protein that facilitate native strand register of the surface-exposed beta barrel. After constructing a suitable host environment around two guest residues, minimizing interactions of the guest residues with surrounding side-chains yet maintaining the wild-type protein structure and the chromophore environment, we introduced a library of cross-strand pairings by cassette mutagenesis. Colonies of Escherichia coli transformed with the library differ in intracellular fluorescence. Most of the fluorescent pairs have predominantly charged and polar guest site residues. The magnitude and the rate of fluorescence acquisition in vivo from transformed E. coli cells varies among the mutants despite comparable levels of protein expression. Spectroscopic measurements of purified mutants show that the native protein structure is maintained. Kinetic studies using purified protein with fully matured chromophores demonstrate that the mutants span a 10-fold range in folding rates with undetectable differences in unfolding rates. Thus, green fluorescent protein provides an ideal system for monitoring determinants of in vivo protein folding. Cross-strand pairings affect both protein stability and folding kinetics by favoring the formation of native strand register preferentially to non-native strand alignments.

Effect of chiral guest residue on the conformation of short peptide containing the GLY-DPG-GLY segment: a cd investigation

Abstract: The Gly-Dpg-Gly segment (Dpg = a ,a-Dipropylglycine) has already been shown to adopt a fully extended conformation in solution as well as in solid state. In this paper we investigate the effect of chiral residues and the terminal aromatic groups on extended conformation in solution with a Gly-Dpg-Gly backbone. CD studies on these peptides indicate that a chiral L-Leu residue at the C-terminus leads to a type I'/11' P-tum structure while a L-Leu residue at the N-terminus leads to a type,I/11P-turn. In addition we demonstrate the role of aromatic residues in the enhancement of CD intensities in the far UV region.

Structure-based conformational preferences of amino acids.

Proteins can be very tolerant to amino acid substitution, even within their core. Understanding the factors responsible for this behavior is of critical importance for protein engineering and design. Mutations in proteins have been quantified in terms of the changes in stability they induce. For example, guest residues in specific secondary structures have been used as probes of conformational preferences of amino acids, yielding propensity scales. Predicting these amino acid propensities would be a good test of any new potential energy functions used to mimic protein stability. We have recently developed a protein design procedure that optimizes whole sequences for a given target conformation based on the knowledge of the template backbone and on a semiempirical potential energy function. This energy function is purely physical, including steric interactions based on a Lennard-Jones potential, electrostatics based on a Coulomb potential, and hydrophobicity in the form of an environment free energy based on accessible surface area and interatomic contact areas. Sequences designed by this procedure for 10 different proteins were analyzed to extract conformational preferences for amino acids. The resulting structure-based propensity scales show significant agreements with experimental propensity scale values, both for alpha-helices and beta-sheets. These results indicate that amino acid conformational preferences are a natural consequence of the potential energy we use. This confirms the accuracy of our potential and indicates that such preferences should not be added as a design criterion.

The Measure of Interior Disorder in a Folded Protein and Its Contribution to Stability

To investigate whether any parameter other than packing density determines the quality of packing in a folded protein, the contribution of van der Waals interactions between hydrophobic core residues to protein stability at fixed packing density is investigated experimentally in this study. To this end, we employed a novel sequence variation scheme called “residue shuffling”, defined as permutation of guest residues at equivalent host sites in a symmetric sequence frame. By comparing the stability of the analogues generated by permutation of hydrophobic core residues in a synthetic two-stranded α-helical coiled-coil scaffold, we conclude that the number of permissible rotamers, based on the avoidance of steric clashes, is another packing parameter. Rotamer number measures the degree of disorder in the hydrophobic core of a folded protein and complements packing density in evaluating packing free energy by gauging packing entropy, the dynamical aspect of packing.

Helicity of hydrophobic peptides in polar vs. non-polar environments

The helical contents of a series of designed Ala-based host–guest hydrophobic peptides (where the guest residues include the 20 commonly occurring amino acids) were determined in both polar (aqueous solution) and non-polar (n-butanol) environments using circular dichroism spectroscopy. While the relative helicities of these peptides in water (Ppolar) agree well with the helical propensity predicted by Chou and Fasman (Annu. Rev. Biochem., 1978, 47, 251) for globular proteins, their helicities in n-butanol (Pnon-polar) correlate loosely with the relative hydrophobicities of the peptides as determined by HPLC retention times. To determine the relevance of helical propensity demonstrated in these model peptides to membrane proteins, we analyzed a database of 5444 transmembrane (TM) segments and 4196 non-TM helices. The amino acid frequency of the non-TM segments is correlated (71%) with Ppolar, while the amino acid frequency of the TM segments is correlated (85%) with Pnon-polar. Further, when mean residue helicity is computed for TM and non-TM helices of comparable length using Ppolar and Pnon-polar, we found that the TM segments have 64% overlap with the non-TM helices in terms of polar helicity, but only 17% overlap in terms of non-polar helicity. The results suggest that despite the necessity to fulfill the requirement of hydrophobicity by using bulky side chains and aromatic residues, the TM segment nevertheless must maintain a relatively high level of helicity in order to assume a stable helical conformation in the membrane.

The role of PII conformations in the calculation of peptide fractional helix content.

Changes in the temperature, pH, ionic strength, or denaturant concentration of aqueous solutions of the monomeric non-alpha-helical peptide acetylYEAAAKEAPAKEAAAKAamide generate changes in its dichroic spectrum characteristic for a conformational transition. This transition has the characteristic features of a residue PII/unstructured conformational equilibrium in which PII denotes an extended left-handed helical conformation and unstructured denotes all the remaining conformations in a random coil ensemble. Replacement of the proline residue facilitates population of residues in an alpha-helical conformation. However, the ellipticity values for these non-proline peptides merge with the ellipticity of the proline peptide as the population of residues in the alpha-helix conformation is diminished. This convergence suggests that all residues in a host/guest peptide series of the same length share a common PII/unstructured conformational equilibrium in a given solvent. We propose that the fractional helix content of peptides within such a series may be estimated by using a two-state calculation in which the ellipticity for the non-alpha-helix conformations is provided by a peptide having a central proline guest residue.

Inversion of 3(10)-helix screw sense in a (D-alpha Me)Leu homo-tetrapeptide induced by a guest D-(alpha Me)Val residue.

The terminally blocked tetrapeptide pBrBz-[D-(alpha Me)Leu]2-D-(alpha Me)Val-D-(alpha Me)Leu-OfBu is folded in the crystal state in a left-handed 3(10)-helical structure stabilized by two consecutive 1<--4 C = O...H-N intramolecular H-bonds, as determined by X-ray diffraction analysis. A CD study strongly supports the view that this conformation is also that largely prevailing in MeOH solution. A comparison with the published conformation of pBrBz-[D-(alpha Me)Leu]4-OfBu indicates that incorporation of a single internal beta-branched (alpha Me)Val guest residue into the host homo-tetrapeptide from the gamma-branched (alpha Me)Leu residue is responsible for a dramatic structural perturbation, i.e. an inversion of the 3(10) screw sense from right to left-handed.

Defect peptide chemistry: Perturbations in the structure of a homopentapeptide induced by a guest residue interrupting side‐chain regularity

The fully blocked pentapeptide Tfa-(Deg)2-L-Abu-(Deg)2-OtBu (Tfa: triflouroacetyl; Deg: C alpha, alpha-diethylglycine; Otbu: tert-butoxy) adopts in the crystal state a regular, right-handed 3(10)-helical structure stabilized by three N--H...O=C intramolecular 1 < 4 (or C10) H bonds, as determined by an x-ray diffraction analysis. However, a Fourier transform ir absorption and 1H-nmr study strongly supports the view that in deuterochloroform solution the four Deg residues at both termini of the peptide main chain are involved in successive, fully extended C5 forms. A comparison with the stable, fully developed, multiple C5 conformation of Tfa-(Deg)5-OtBu indicates that incorporation of an Abu guest residue, interrupting the side-chain uniformity of the host (Deg)5 homopeptide, while altering only marginally the conformation in a solvent of low polarity, is responsible for a dramatic perturbation of the crystal-state structure.

Modulation of the helical stability of a model peptide by ionic residues.

The mean residue ellipticity of the helical host peptide, acetyl-YEAAAKEAXAKEAAAKA-amide containing guest residues at position X, was measured as a function of pH and ionic strength at 0 degree C. Changes in ellipticity at 222 nm were interpreted in terms of a two-state helix/coil transition of a monomeric peptide. Variable pH measurements in low concentrations of KCl defined changes in helix stability resulting from the ionization of each guest residue. Variable [KCl] measurements at fixed pH generated ellipticity values for the neutral and ionic forms of each guest residue free of electrostatic and lyotropic contributions. These ellipticity values were used to calculate a helix propagation parameter for each form of a guest residue using the Lifson-Roig algorithm and assuming a universal nucleation parameter. In all cases, the propagation parameter of a residue is either unaffected or decreased by ionization of its side chain.

Residue helix parameters obtained from dichroic analysis of peptides of defined sequence.

Circular dichroic measurements of the host peptide acetyl-Y(EAAAK)3A-amide were obtained in solutions of increasing ionic strength at pH 7.0 and 0 degree C. The changes observed in the dichroic spectra are characteristic for a two-state helix/coil transition. The mean residue ellipticity at 222 nm exhibits a curvilinear dependence on ionic strength which becomes linear at ionic strengths greater than 1 M. The slope of the linear portion is assumed to represent the lyotropic character of the salt, and its extrapolated intercept is assumed to represent the mean residue ellipticity of the peptide solution freed from both electrostatic and lyotropic contributions which affect the helical stability of the host peptide. An extrapolated mean residue ellipticity value was obtained for each host peptide having a different amino acid guest residue at position 9 in the peptide sequence. These values were used to calculate a propagation parameter, s, for each residue using the Lifson-Roig algorithm for peptide helical content and assuming a common nucleation parameter of 0.003. The ability of these minimally determined residue parameters to predict the helical content of a variety of peptides is encouraging. Estimates were also made of the delta G values for the electrostatic interactions within the host peptide and for the additional interactions generated by ionic guest residues.

Comparison of the effect of five guest residues on the .beta.-sheet conformation of host (L-val)n oligopeptides

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTComparison of the effect of five guest residues on the .beta.-sheet conformation of host (L-val)n oligopeptidesV. Moretto, M. Crisma, G. M. Bonora, C. Toniolo, Hemalatha Balaram, and P. BalaramCite this: Macromolecules 1989, 22, 7, 2939–2944Publication Date (Print):July 1, 1989Publication History Published online1 May 2002Published inissue 1 July 1989https://doi.org/10.1021/ma00197a010RIGHTS & PERMISSIONSArticle Views254Altmetric-Citations142LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (709 KB) Get e-Alerts Get e-Alerts