Polypeptide Conformation Research Articles

The conformation of a nascent polypeptide inside the ribosome tunnel affects protein targeting and protein folding

In this report, we describe insights into the function of the ribosome tunnel that were obtained through an analysis of an unusual 25 residue N-terminal motif (EspP(1-25) ) associated with the signal peptide of the Escherichia coli EspP protein. It was previously shown that EspP(1-25) inhibits signal peptide recognition by the signal recognition particle, and we now show that fusion of EspP(1-25) to a cytoplasmic protein causes it to aggregate. We obtained two lines of evidence that both of these effects are attributable to the conformation of EspP(1-25) inside the ribosome tunnel. First, we found that mutations in EspP(1-25) that abolished its effects on protein targeting and protein folding altered the cross-linking of short nascent chains to ribosomal components. Second, we found that a mutation in L22 that distorts the tunnel mimicked the effects of the EspP(1-25) mutations on protein biogenesis. Our results provide evidence that the conformation of a polypeptide inside the ribosome tunnel can influence protein folding under physiological conditions and suggest that ribosomal mutations might increase the solubility of at least some aggregation-prone proteins produced in E. coli.

Supramolecular Assembly of Designed α‐Helical Polypeptide‐Based Nanostructures and Luminescent Conjugated Polyelectrolytes

Designed polypeptides with controllable folding properties are utilized as supramolecular templates for fabrication of ordered nanoscale molecular and fibrous assemblies of LCPs. The properties of the LCPs as well as the three dimensional conformation of the polypeptide-scaffold determine how the polymers are arranged in the supramolecular construct, which highly affects the properties of the hybrid material. The ability to control the polypeptide conformation and assembly into fibers provides a promising route for tuning the optical properties of LCPs and for fabrication of complex functional supramolecules with well defined structural properties.

Surface structural conformations of fibrinogen polypeptides for improved biocompatibility

This work reports on how incorporation of silica nanocages into poly(urethane) copolymers (PU) affects conformational orientations of adsorbed fibrinogen and how different surfaces subsequently influenced HeLa cell attachment and proliferation. Incorporation of 2 wt% silica nanocages into poly(urethane) (PU4) substantially altered the surface topography of the films and some 50% of the surface was covered with the nanocages due to their preferential exposure. AFM studies revealed the deposition of a dense protein network on the soft polymeric domains of PU4 and much reduced fibrinogen adsorption on the hard nanocage domains. As on the bare SiO 2 control surface, fibrinogen molecules adsorbed on top of the hard nanocages mainly took the dominant trinodular structures in monomeric and dimeric forms. In addition, net positively charged long α chains were prone to being hidden beneath the D domains whilst γ chains predominantly remained exposed. Dynamic interfacial adsorption as probed by spectroscopic ellipsometry revealed fast changes in interfacial conformation induced by electrostatic interactions between different segments of fibrinogen and the surface, consistent with the AFM imaging. On the PU surfaces without nanocage incorporation (PUA), however, adsorbed fibrinogen molecules formed beads-like chain networks, consistent with the structure featured on the soft PU4 domains, showing very different effects of surface chemical nature. Monoclonal antibodies specific to the α and γ chains showed reduced α but increased γ chain binding at the silicon oxide control and PU4 surfaces, whilst on the PUA, C18 and amine surfaces (organic surface controls) the opposite binding trend was detected with α chain binding dominant, showing different fibrinogen conformations. Cell attachment studies revealed differences in cell attachment and proliferation, consistent with the different polypeptide conformations on the two types of surfaces, showing a strong preference to the extent of exposure of γ chains.

Unraveling of the E-helices and Disruption of 4-Fold Pores Are Associated with Iron Mishandling in a Mutant Ferritin Causing Neurodegeneration

Mutations in the coding sequence of the ferritin light chain (FTL) gene cause a neurodegenerative disease known as neuroferritinopathy or hereditary ferritinopathy, which is characterized by the presence of intracellular inclusion bodies containing the mutant FTL polypeptide and by abnormal accumulation of iron in the brain. Here, we describe the x-ray crystallographic structure and report functional studies of ferritin homopolymers formed from the mutant FTL polypeptide p.Phe167SerfsX26, which has a C terminus that is altered in amino acid sequence and length. The structure was determined and refined to 2.85 A resolution and was very similar to the wild type between residues Ile-5 and Arg-154. However, instead of the E-helices normally present in wild type ferritin, the C-terminal sequences of all 24 mutant subunits showed substantial amounts of disorder, leading to multiple C-terminal polypeptide conformations and a large disruption of the normally tiny 4-fold axis pores. Functional studies underscored the importance of the mutant C-terminal sequence in iron-induced precipitation and revealed iron mishandling by soluble mutant FTL homopolymers in that only wild type incorporated iron when in direct competition in solution with mutant ferritin. Even without competition, the amount of iron incorporation over the first few minutes differed severalfold. Our data suggest that disruption at the 4-fold pores may lead to direct iron mishandling through attenuated iron incorporation by the soluble form of mutant ferritin and that the disordered C-terminal polypeptides may play a major role in iron-induced precipitation and formation of ferritin inclusion bodies in hereditary ferritinopathy.

Highly Efficient “Grafting onto” a Polypeptide Backbone Using Click Chemistry

A cell's extracellular matrix consists of macromolecules, such as glycoproteins, proteoglycans, and collagen, that control both the mechanical structure and the microenvironment.[1] These properties provide physical cues that are necessary to induce various cell functions and morphologies. An important goal of tissue engineering is to mimic the environment of the extracellular matrix on several levels, mechanically, chemically, and architecturally.[2] To accomplish this goal, new synthetic methods are necessary in order to mimic the structure of these complex macromolecules. We have developed a synthetic method to form highly functionalized grafted polypeptides that can be made to mimic complex biomacromolecules such as glycoproteins and proteoglycans. While, these new synthetic polypeptides are much simpler than natural peptides, they still possess the α-helical conformation of natural polypeptides and various chemical moieties can be attached to mimic the microenvironment of the extracellular matrix. These polymers have several features that make them very attractive for biological applications including low toxicity, biodegradability, tunable structures, and well-controlled dimensions.

New polypeptide conformations and their roles in molecular diseases of the vascular wall

New polypeptide conformations are described in which the repeating unit contains intrarepeat hydrogen bonding and in which there may or may not be hydrogen bonding between the repeating units. These conformations, called β spirals, were derived from polymers of repeat peptides of elastin, the connective tissue protein of the core of the elastic fiber. It is argued that these β spirals associate to form supramolecular fibrous constructs by intermolecular hydrophobic interactions and that these structures, which contain preferred though dynamic secondary structure, form elastomeric fibers which are not random. An appropriate understanding of these structures and their important biological properties require analysis from the disciplines of quantum chemistry, statistical mechanics, and the theory of rate processes. The manner in which these β-spirals associate, which is fundamental to the mechanism of fiber formation, provides insights into the molecular pathology of the vascular wall, specifically into the disruptive effects of decreased temperature, of lipid deposition, and of pathological calcification.

Molecular orbital calculations on the conformation of polypeptides and proteins X. The conformational energy maps of the cysteinyl and methionyl residues

Molecular orbital calculations on the conformation of polypeptides and proteins X. The conformational energy maps of the cysteinyl and methionyl residues

Nonclassical helical states and diverse biological functions of sequential polypeptides

Sequential polypeptides result in new polypeptide conformations with diverse biological roles. Collagen, a polytripeptide, is a familiar and unique structural protein: gramicidin A, an L-D polydipeptide, forms cation selective channels in biomembranes; a synthetic polytripeptide forms cation-selective voltage-dependent channels in lipid bilayers; the polypentapeptide of tropoelastin is a biological elastomer; and the pohtelra- and polyhexapeptides of tropoelastin can be ascribed specific functional roles. From these few examples one recognizes the almost limitless potential of studies on polysequential peptides. In the description of new helical conformations derived from repeating peptide sequences, the concept of cyclic conformations with linear conformational correlates is useful particularly in derivation, or even design, of single-stranded helical structures with a large number of residues per turn. The repeating peptide becomes a repeating conformational unit that can repeat on a helix axis. A common repeating conformational feature is the β turn, and helical repetitions of conformational units in which the β turn is the dominant conformational feature are designated β spirals.

Factors affecting conformations of cyclic polypeptides in the crystalline state

Criteria for geometrical parameters, such as bond lengths, bond angles and torsional angles, are needed for building models of polypeptides based on spectral data and/or minimum energy calculations. Some assumptions that are made frequently are: that the most stable form of the peptide unit is planar and in the trans conformation; that the maximum number of hydrogen bonds will be formed: that peptides dissolved in nonpolar solvents will turn their polar groups toward the interior of the molecule (and form intramolecular hydrogen bonds), whereas those dissolved in polar solvents will turn their polar groups toward the exterior. The validity of such assumptions has been examined by crystal structure analyses of a variety of cyclic peptides crystallized under varying conditions. The cyclic backbone in telrapeptides, both natural and synthetic, has been found to assume four different conformations, cis trans cis trans with a center of inversion or with a twofold rotation axis, and trans trans trans trans with a center of inversion or with a twofold rotation axis. A pair of similar cyclic pentapeptides have been found to be rotational isomers, where the backbone conformation (all trans) remains the same, but the side chains progress by one peptide unit around the ring. A hexapeptide analogue crystallized from (CH3)2SO/(CH3)2CO and from H2O/(CH3)2CO crystallizes in different space groups. Four molecules of the hexapeptide aggregate by intermolecular hydrogen bonding to form an almost identical large cavity in each case; however, in one crystal the cavity is filled with water molecules, which make extensive hydrogen bonds to the polar groups of the surrounding peptide molecules, whereas in the other case, the cavity contains (CH3)2SO that participates in only one hydrogen bond to the peptide and otherwise presents a lipophilic surface toward the polar lining of the cavity. A similar phenomenon has been observed in the large-diameter channels formed by antamanide molecules (cyclic decapeptide) where the channel is occupied either by many water molecules in definite sites and hydrogen bound to the peptide molecules, or by nonpolar disordered n-hexane molecules. In the latter two examples the character of the solvent does not influence the conformation of the peptide.

The SAAP force field: Development of the single amino acid potentials for 20 proteinogenic amino acids and Monte Carlo molecular simulation for short peptides

Molecular simulation by using force field parameters has been widely applied in the fields of peptide and protein research for various purposes. We recently proposed a new all-atom protein force field, called the SAAP force field, which utilizes single amino acid potentials (SAAPs) as the fundamental elements. In this article, whole sets of the SAAP force field parameters in vacuo, in ether, and in water have been developed by ab initio calculation for all 20 proteinogenic amino acids and applied to Monte Carlo molecular simulation for two short peptides. The side-chain separation approximation method was employed to obtain the SAAP parameters for the amino acids with a long side chain. Monte Carlo simulation for Met-enkephalin (CHO-Tyr-Gly-Gly-Phe-Met-NH2) by using the SAAP force field revealed that the conformation in vacuo is mainly controlled by strong electrostatic interactions between the amino acid residues, while the SAAPs and the interamino acid Lennard-Jones potentials are predominant in water. In ether, the conformation would be determined by the combination of the three components. On the other hand, the SAAP simulation for chignolin (H-Gly-Tyr-Asp-Pro-Glu-Thr-Gly-Thr-Trp-Gly-OH) reasonably reproduced a native-like beta-hairpin structure in water although the C-terminal and side-chain conformations were different from the native ones. It was suggested that the SAAP force field is a useful tool for analyzing conformations of polypeptides in terms of intrinsic conformational propensities of the single amino acid units.

Adsorbed α-Helical Diblock Copolypeptides: Molecular Organization, Structural Properties, and Interactions

In this work, we have developed 11-mercaptoundecanoic acid (MUA)-polypeptide "bilayer" systems by adsorbing poly(diethylene glycol-l-lysine)-poly(l-lysine) (PEGLL-PLL) diblock copolypeptide molecules of various architectures onto MUA-functionalized gold substrates. An objective of our present work is to use the PEGLL-PLL/MUA bilayer as a model system for studying the interfacial phenomena that occur when PEGLL-PLL molecules interact with carboxylic acid (COOH) moieties of nanoparticle ligands. Specifically, we have elucidated the nature of the interactions between the PEGLL-PLL and COOH moieties as well as the resulting polypeptide conformation and organization, using a combination of surface techniques-grazing-incidence IR spectroscopy, ellipsometry, and contact angle. We have also thoroughly characterized other film properties such as the packing and graft density of the polypeptide molecules as a function of the PEGLL-PLL architecture. From the IR data, the adsorption process occurs primarily by means of electrostatic interaction between the protonated PLL residues (pKa approximately 10.6) and carboxylate moieties of the MUA self-assembled monolayer (SAM) (pKa approximately 6) that is enhanced by H-bonding. The PLL block is thought to adopt a random-coil (extended) conformation, while the PEGLL block that is not interacting with the MUA molecules is found to adopt an alpha-helical conformation with an average tilt angle of -60 degrees. The PEGLL-PLL molecules have also been deduced to form a heterogeneous film and adopt liquidlike/disordered packing on the surface. The average contact angle of the MUA-polypeptide bilayer systems is -40 degrees, which implies that the diethylene glycol (EG2) side chains of the PEGLL residues may be oriented somewhat toward the surface normal. From ellipsometry measurements, it is found that PEGLLx-PLLy molecules with a longer alpha-helical block are associated with a lower graft density on the MUA surface compared to those with a shorter alpha-helical block. This observation may be attributed to the greater repulsion-steric and H-bonding effects-that is imposed by the EG2 side chains found on and projected area occupied by the longer PEGLL block. The bilayer systems have been found to be extremely stable over a 2-week period with no changes in the contact angle, thickness, polypeptide tilt angle, or conformation. Beyond that, there is a gradual decrease in the thickness and increase in the contact angle of the bilayer that could be attributed to the oxidation of the MUA SAM molecules.

Characterizing Aqueous Solution Conformations of a Peptide Backbone Using Raman Optical Activity Computations

Mounting spectroscopic evidence indicates that alanine predominantly adopts extended polyproline II (PPII) conformations in short polypeptides. Here we analyze Raman optical activity (ROA) spectra of N-acetylalanine-N′-methylamide (Ala dipeptide) in H2O and D2O using density functional theory on Monte Carlo (MC) sampled geometries to examine the propensity of Ala dipeptide to adopt compact right-handed (αR) and left-handed (αL) helical conformations. The computed ROA spectra based on MC-sampled αR and PPII peptide conformations contain all the key spectral features found in the measured spectra. However, there is no significant similarity between the measured and computed ROA spectra based on the αL- and β-conformations sampled by the MC methods. This analysis suggests that Ala dipeptide populates the αR and PPII conformations but no substantial population of αL- or β-structures, despite sampling αL- and β-structures in our MC simulations. Thus, ROA spectra combined with the theoretical analysis allow us to determine the dominant populated structures. Including explicit solute-solvent interactions in the theoretical analysis is essential for the success of this approach.

Structural and Dynamic Properties of Water within the Solvation Layer around Various Conformations of the Glycine-based Polypeptide

Several conformations of the solvated glycine-based polypeptides were investigated using molecular dynamics simulations. Some properties of water in the neighboring space around these molecules were investigated. It was found that water forms a well-defined layer-the first solvation shell-around the peptide molecule, and thickness of this layer is independent of the peptide structure and is equal to approximately 0.28 nm. Within this layer, water molecules show marked orientations relative to a peptide surface. Using the two-particle contribution to entropy as a measure of structural ordering of water, we found that the first solvation shell contributes 95% or more to the total water ordering around the peptide molecule. In investigating the dynamic properties of water, diffusion coefficients and lifetime of the hydrogen bond, clear differences between solvation layer and the bulk water were observed. It was found that the translational diffusion coefficient, D(T), decreases by 30% or more compared to bulk water; also, the lifetime of the water-water hydrogen bond clearly increases. The rotational diffusion coefficient, however, decreases only slightly, no more than approximately 10%. These differences correspond to the slightly higher energy of the hydrogen bond, and to its slightly distorted geometry. Analyzing the translational dynamics of water in the vicinity of the peptide molecule, it was deduced that the structure of the first solvation shell becomes more rigid than the structure of the bulk water. Investigation of a "pure hydrophobic" form of the polypeptide shows that the structure and the properties of water within the solvation shell are predominantly determined by the hydrophobic effect. The specific interactions between water molecules and various charge groups of the peptide molecule modifies this effect only slightly.

Properties of pH-Responsive Mixed Aggregates of Polystyrene-block-poly(l-lysine) and Nonionic Surfactant in Solution and Adsorbed at a Solid Surface

Poly(styrene) 388- block-poly( l-lysine) 138 could be dispersed in water with the aid of the nonionic surfactant C 12E 6. Light scattering and direct imaging techniques show that the copolymer/surfactant aggregates are polydisperse spherical micelles. The rather broad size distribution can be attributed to the glassy state of the polystyrene core of the micelles hampering equilibration. Nevertheless, the poly( l-lysine) block remains pH sensitive in these mixed aggregates and circular dichroism measurements show that poly( l-lysine) block adopts a random coil conformation at low pH and an beta-sheet conformation at pH > or = 11 without any change in the micellar shape. Samples prepared by evaporation of drops of the solutions on graphite wafers exhibit different wetting patterns depending on the polypeptide conformation as indicated by atomic force microscopy.

Semistiff polymer model of unfolded proteins and its application to NMR residual dipolar couplings

We present a new statistical model of unfolded proteins in which the stiffness of polypeptide backbone is taken into account. We construct and solve a mean field equation which has the form of a diffusion equation and derive the distribution function for conformations of unfolded polypeptides. Accounting for the stiffness of the protein backbone results in a more accurate description of general properties of a polypeptide chain, such as its gyration radius. We then use the distribution function of a semistiff protein within a previously developed theoretical framework [J. Biomol. NMR 39, 1 (2007)] to determine the nuclear magnetic resonance (NMR) residual dipolar couplings (RDCs) in unfolded proteins. The calculated RDC profiles (dependence of the RDC value on the residue number) exhibit a more prominent bell-like shape and a better agreement with experimental data as compared to the previous results obtained with the random flights chain model.

Chain Dynamics of Nascent Polypeptides Emerging from the Ribosome

Very little is known about the conformation of polypeptides emerging from the ribosome during protein biosynthesis. Here, we explore the dynamics of ribosome-bound nascent polypeptides and proteins in Escherichia coli by dynamic fluorescence depolarization and assess the population of cotranslationally active chaperones trigger factor (TF) and DnaK. E. coli cell-free technology and fluorophore-linked E. coli Met-tRNA f Met enable selective site-specific labeling of nascent proteins at the N-terminal methionine. For the first time, direct spectroscopic evidence captures the generation of independent nascent chain motions for a single-domain protein emerging from the ribosome (apparent rotational correlation time approximately 5 ns), during the intermediate and late stages of polypeptide elongation. Such motions are detected only for a sequence encoding a globular protein and not for a natively unfolded control, suggesting that the independent nascent chain dynamics may be a signature of folding-competent sequences. In summary, we observe multicomponent, severely rotationally restricted, and strongly chain length/sequence-dependent nascent chain dynamics.

MOLECULAR DYNAMICS STUDIES OF CRAMBIN AND BPTI IN TERMS OF ABEEM/MM METHOD

Recently, the ABEEM/MM method (atom-bond electronegativity equalization method fused into molecular mechanics) has successfully been applied to study polypeptide conformations. In order to further test the reliability of this method as well as the transferability of the parameters (including ABEEM and force field parameters), molecular dynamics simulation studies on Crambin and BPTI (298 K, in vacuo) have been performed in terms of ABEEM/MM fluctuating charge force field. Some of their structural properties were obtained, and relative to the X-ray structures, the root-mean-square deviations (RMSD) of bond lengths, bond angles and key dihedral angles, and the coordinate RMSDs of atoms were calculated. The ABEEM/MM results are fairly consistent with those from X-ray experiment.

Interplay among side chain sequence, backbone composition, and residue rigidification in polypeptide folding and assembly

The extent to which polypeptide conformation depends on side-chain composition and sequence has been widely studied, but less is known about the importance of maintaining an alpha-amino acid backbone. Here, we examine a series of peptides with backbones that feature different repeating patterns of alpha- and beta-amino acid residues but an invariant side-chain sequence. In the pure alpha-backbone, this sequence corresponds to the previously studied peptide GCN4-pLI, which forms a very stable four-helix bundle quaternary structure. Physical characterization in solution and crystallographic structure determination show that a variety of alpha/beta-peptide backbones can adopt sequence-encoded quaternary structures similar to that of the alpha prototype. There is a loss in helix bundle stability upon beta-residue incorporation; however, stability of the quaternary structure is not a simple function of beta-residue content. We find that cyclically constrained beta-amino acid residues can stabilize the folds of alpha/beta-peptide GCN4-pLI analogues and restore quaternary structure formation to backbones that are predominantly unfolded in the absence of cyclic residues. Our results show a surprising degree of plasticity in terms of the backbone compositions that can manifest the structural information encoded in a sequence of amino acid side chains. These findings offer a framework for the design of nonnatural oligomers that mimic the structural and functional properties of proteins.

Natively Unfolded Protein Stability as a Coil-to-Globule Transition in Charge/Hydropathy Space

In the absence of experimental assignments, the empirical charge/hydropathy correlation for the prediction of natively unfolded protein sequences (Uversky, V. N.; Gillespie, J. R.; Fink, A. L. Proteins: Struct., Funct., Genet. 2000, 41, 415-427) provides perhaps the most intuitive description of gross polypeptide conformation. The success of this correlation rests on an essential chain length independence of the boundary line between expanded and compact conformations, conversely stabilized by highly charged/weakly hydrophobic residues or weakly charged/highly hydrophobic residues, respectively. We present extensive simulation results for coarse-grained polypeptides over a wide range of sequence hydrophobicities, charges, and lengths. A coil-to-globule transition in sequence composition space analogous to the charge/hydropathy correlation is observed. A near sequence length independent stability boundary is only found when counterions for the charged peptides are explicitly included, as a result of counterion condensation stabilization of repulsive electrostatic interactions on the globule surface. The observed counterion adsorption is shown to be in quantitative agreement with theoretical condensation predictions. We argue that alternate functionalities, beyond charge and hydrophobicity, empirically known to correlate with conformational disorder can be incorporated into our minimalist polypeptide model to study the interplay between independent predictors of unfolded sequences.

CRANKITE: A fast polypeptide backbone conformation sampler.

BackgroundCRANKITE is a suite of programs for simulating backbone conformations of polypeptides and proteins. The core of the suite is an efficient Metropolis Monte Carlo sampler of backbone conformations in continuous three-dimensional space in atomic details.MethodsIn contrast to other programs relying on local Metropolis moves in the space of dihedral angles, our sampler utilizes local crankshaft rotations of rigid peptide bonds in Cartesian space.ResultsThe sampler allows fast simulation and analysis of secondary structure formation and conformational changes for proteins of average length.