Random Coil Model Research Articles

The Denatured State Ensemble Contains Significant Local and Long-Range Structure under Native Conditions: Analysis of the N-Terminal Domain of Ribosomal Protein L9

The denatured state ensemble (DSE) represents the starting state for protein folding and the reference state for protein stability studies. Residual structure in the DSE influences the kinetics of protein folding, the propensity to aggregate, and protein stability. The DSE that is most relevant for folding is the ensemble populated under native conditions, but the stability of proteins and the cooperativity of their folding normally prevent direct characterization of this ensemble. Indirect experiments have been used to infer residual structure in the DSE under nondenaturing conditions, but direct characterization is rare. The N-terminal domain of ribosomal protein L9 (NTL9) is a small mixed α-β domain that folds cooperatively on the millisecond time scale. A destabilized double mutant of NTL9, V3A/I4A-NTL9, populates the DSE in the absence of denaturant and is in slow exchange with the native state on the nuclear magnetic resonance time scale. The DSE populated in buffer was compared to the urea-induced DSE. Analysis of (1)H and (13)C chemical shifts reveals residual secondary structure in the DSE in buffer, which is stabilized by both local and long-range interactions. (15)N R2 relaxation rates deviate from random coil models, suggesting hydrophobic clustering in the DSE. Paramagnetic relaxation enhancement experiments show that there are transient long-range contacts in the DSE in buffer. In contrast, the urea-induced DSE has significantly less residual secondary structure and markedly fewer long-range contacts; however, the urea-induced DSE deviates from a random coil.

Pressure Dependence of 15N Chemical Shifts in Model Peptides Ac-Gly-Gly-X-Ala-NH2

High pressure NMR spectroscopy has developed into an important tool for studying conformational equilibria of proteins in solution. We have studied the amide proton and nitrogen chemical shifts of the 20 canonical amino acids X in the random-coil model peptide Ac-Gly-Gly-X-Ala-NH2, in a pressure range from 0.1 to 200 MPa, at a proton resonance frequency of 800 MHz. The obtained data allowed the determination of first and second order pressure coefficients with high accuracy at 283 K and pH 6.7. The mean first and second order pressure coefficients <B115N> and <B215N> for nitrogen are 2.91 ppm/GPa and −2.32 ppm/GPa2, respectively. The corresponding values <B11H> and <B21H> for the amide protons are 0.52 ppm/GPa and −0.41 ppm/GPa2. Residual dependent 1J1H15N-coupling constants are shown.

Photoinduced orientation in natural rubber

Azobenzene molecules and their derivatives have been widely investigated for their potential applications in optical and electrooptical devices. We have prepared a new guest–host system from natural rubber (NR) impregnated with azobenzene derivative Sudan Red B (SRB). The effects of stretching and immersion time on photoinduced orientation were investigated by birefringence signal measurements. We have found that the molecular orientation increase when the samples are stretched and decrease with the increase of immersion time. The first behavior was explained by using the random coil model and the latter was attributed to increase of the aggregation of SRB into NR matrix.

Conformational propensities and residual structures in unfolded peptides and proteins

Ample evidence gathered over the last ten years indicates that unfolded and naturally disordered proteins and peptides can show local order in that short segments can adopt turn or polyproline II-like conformations. These findings show that unfolded states cannot be described by the so-called random coil model which assumes that individual amino acid residues sample the entire sterically accessible parts of the Ramachandran with very similar probabilities. This article reviews the experimental evidence for the notion that amino acid residues have different propensities for polyproline II, β-strand, helical and turn conformations in water. These propensities are changed by interactions with nearest neighbours. We show that for a substantial number of residues the conformational propensities in the unfolded state correlate with values for helix propagation and the Chou-Fasman propensities for β-strands. Based on the presented results we hypothesize that the conformational distributions of a representative set of short peptides could be used for predicting structural distributions of disordered peptides and proteins in the future.

Osmolyte effects on protein stability and solubility: A balancing act between backbone and side-chains

In adaptation biology the discovery of intracellular osmolyte molecules that in some cases reach molar levels, raises questions of how they influence protein thermodynamics. We've addressed such questions using the premise that from atomic coordinates, the transfer free energy of a native protein ( ΔG tr, N ) can be predicted by summing measured water-to-osmolyte transfer free energies of the protein's solvent exposed side chain and backbone component parts. ΔG tr, D is predicted using a self avoiding random coil model for the protein, and ΔG tr, D − ΔG tr, N , predicts the m-value, a quantity that measures the osmolyte effect on the N ⇌ D transition. Using literature and newly measured m-values we show 1:1 correspondence between predicted and measured m-values covering a range of 12 kcal/mol/M in protein stability for 46 proteins and 9 different osmolytes. Osmolytes present a range of side chain and backbone effects on N and D solubility and protein stability key to their biological roles.

Characteristics of Human Lysozyme and Its Disease‐Related Mutants in their Unfolded States

Characteristics of Human Lysozyme and Its Disease‐Related Mutants in their Unfolded States

Toward a Physical Definition of Entanglements

To examine the role of chain entanglements on polymer melt properties, the interaction potential energy between a loop and chain at its center is evaluated. The interaction potential energy between binary contacts of two adjacent Kuhn monomers is also evaluated. The elasticity in polymer melts and the evaluation of the different contributions to flow activation energy of linear polymer chains are used to demonstrate that chain loops, or any other binary interactions between polymer chain segments, cannot justify the properties assigned to entanglements. These properties may be understood if interactions between several parallel chain segments are considered instead, which implies the assumption of the existence of short-range local ordered regions in polymer melts. Their existence is demonstrated from abundant literature experiments, molecular dynamics simulation results, and also from a detailed discussion on the interaction potential energy values. Because more than 80% of the conformational states in one chain are random sequences of chain segments, the remaining states being in short-range ordered regions, we conclude that the random coil model is not an exact model for the morphology of polymer melts, but it is still a very good description of chain conformations in the molten state.

Packing density of HS(CH2)nCOOH self-assembled monolayers

Self-assembled monolayers (SAMs) of HS(CH(2))(n)COOH, n = 5, 10, 15 deposited from ethanol solution onto gold are prepared by five approaches, and their packing densities are evaluated by X-ray photoelectron spectroscopy (XPS) measurements. The five approaches are: (1) direct deposition; (2) acetic-acid-assisted deposition; (3) butyl-amine-assisted deposition; (4) displacement of a preformed HS(CH(2))(n)CH(3) (n = 5, 10, 15) SAMs; and (5) co-deposition with HS(CH(2))(n)CH(3) (n = 5, 10, 15). Packing density metrics are calculated from measurements of SAM and substrate photoemission intensities and their attenuations by two methods. In one case the attenuated photoemissions are expressed as a ratio relative to comparable measurements on an experimental HS(CH(2))(n)CH(3) model system. In the other case a new method is introduced where a calculated attenuation based on theoretical random coil and extended chain models is used as the reference to determine a packing density fraction. Packing densities are also correlated with the S2p(Au-bonded):Au4f peak area ratios and with shifts in the C1s binding energies. SAMs prepared by the direct deposition are a partial multilayer where a second molecular layer is physisorbed onto the SAM and not removable by solvent washing. The addition of acetic acid to the deposition solution disrupts dimer associations of HS(CH(2))(n)COOH in solution and at the surface of the monolayer and yields the most ordered monolayer with the highest density of -COOH groups. The addition of butyl amine results in a labile ammonium carbonate ion pair formation but results in a lower packing density in the SAM. The displacement of the preformed HS(CH(2))(n)CH(3) SAM and the co-deposition of HS(CH(2))(n)CH(3) with HS(CH(2))(n)COOH result in SAMs with little incorporation of the -COOH component.

NMR determination of pKa values in α‐synuclein

The intrinsically unfolded protein α-synuclein has an N-terminal domain with seven imperfect KTKEGV sequence repeats and a C-terminal domain with a large proportion of acidic residues. We characterized pK(a) values for all 26 sites in the protein that ionize below pH 7 using 2D (1) H-(15) N HSQC and 3D C(CO)NH NMR experiments. The N-terminal domain shows systematically lowered pK(a) values, suggesting weak electrostatic interactions between acidic and basic residues in the KTKEGV repeats. By contrast, the C-terminal domain shows elevated pK(a) values due to electrostatic repulsion between like charges. The effects are smaller but persist at physiological salt concentrations. For α-synuclein in the membrane-like environment of sodium dodecylsulfate (SDS) micelles, we characterized the pK(a) of His50, a residue of particular interest since it is flanked within one turn of the α-helix structure by the Parkinson's disease-linked mutants E46K and A53T. The pK(a) of His50 is raised by 1.4 pH units in the micelle-bound state. Titrations of His50 in the micelle-bound states of the E46K and A53T mutants show that the pK(a) shift is primarily due to interactions between the histidine and the sulfate groups of SDS, with electrostatic interactions between His50 and Glu46 playing a much smaller role. Our results indicate that the pK(a) values of uncomplexed α-synuclein differ significantly from random coil model peptides even though the protein is intrinsically unfolded. Due to the long-range nature of electrostatic interactions, charged residues in the α-synuclein sequence may help nucleate the folding of the protein into an α-helical structure and confer protection from misfolding.

溶液中の高分子に対するrandom coil modelについて

溶液中の高分子に対するrandom coil modelについて

Alpha‐helix formation studied with pressure perturbation calorimetry

Pressure perturbation calorimetry (PPC) is a relatively unexplored technique that quantifies the temperature dependence of a solute's thermal expansion coefficient. PPC data provides information about solute‐solvent interactions. To investigate the formation of an alpha‐helix in solution, PPC was used to characterize helix‐solvent and random coil‐solvent interactions. Helix and random coil models were obtained by using two peptides, Ac‐DKDGYISAAE‐NH2 (P12) and Ac‐DKDGDGYISAAEAAAQ‐NH2 (P16) which contain the same sequence for the first twelve amino acids. Circular dichroism spectropolarimetry was used to determine the helix content. In the presence of La3+ the last four residues of P16 form an alpha‐helix. When La3+ is absent, both peptides are random coils, with a small amount of residual helix, which decreases with increasing temperature. The temperature dependence of the corrected thermal expansion coefficient suggests that the helix‐solvent interactions are more hydrophobic, while random coil‐solvent interactions are more hydrophilic. Therefore, alpha‐helix formation is marked by a change from hydrophilic to hydrophobic solvent interaction. This result is consistent with the accepted view that helix formation results in a net loss of hydrophilic surface area.

Quantifying the Surface Coverage of Conjugate Molecules on Functionalized Nanoparticles

Here we present a method to determine the surface coverage or surface density of biological molecules conjugated to nanoparticle surfaces. Electrospray-differential mobility analysis (ES-DMA) is used to determine a coating thickness by measuring the change in the size of gold nanoparticles before and after modification with thiol-derivatized single-stranded DNA. The DNA surface coverage is then obtained from the coating thickness through the use of a simple random coil model. The method requires neither fluorescent tagging nor calibration curves. We believe ES-DMA to be a broadly applicable nanometrology tool for the characterization of biologically conjugated nanoparticles.

Variation of Linker Length in Ratiometric Fluorescent Sensor Proteins Allows Rational Tuning of Zn(II) Affinity in the Picomolar to Femtomolar Range

Ratiometric fluorescent sensor proteins with a very high and tunable affinity for Zn(II) were created by connecting two fluorescently labeled metal binding domains, CFP−Atox1 and WD4−YFP, using a series of flexible peptide linkers. A simple random-coil model describing the conformational distribution of the linker allowed a quantitative understanding of the effect of the linker length on both the change in emission ratio and the Zn(II) affinity.

Exopolysaccharides of Two Cyanobacterial Strains from Roman Hypogea

The cytomorphology and the exopolysaccharides of two heterocystous cyanobacteria, Scytonema ocellatum CP8-2 and Fischerella maior NAV 10 bis, isolated from biofilms collected in two Roman hypogea, were investigated from a cytochemical, ultrastructural and biochemical point of view. Cytochemical stains and observation using light and transmission electron microscopy showed a bi-layered sheath in both cyanobacteria, with a different composition in sulphated and carboxylic groups. RP-HPLC of the extracted polysaccharides forming the mucous matrix showed the neutral sugars glucose and arabinose and the charged galacturonic acid as the main monosaccharides. Circular dichroism spectra recorded at varying pH and temperature showed that these polymers followed a random coil model and did not present a structured organised conformation at ambient temperature. The outcomes of this study increase the understanding of colonization and biodeterioration of important archaeological and cultural sites by cyanobacterial biofilms and the role of the exopolymeric substances involved.

Salmon Calcitonin and Amyloid β: Two Peptides with Amyloidogenic Capacity Adopt Different Conformational Manifolds in Their Unfolded States

The molecular conformations of salmon calcitonin in aqueous solution have been investigated by exploiting the different influences of excitonic coupling on the amide I band profile in the isotropic and anisotropic Raman, FTIR, and vibrational circular dichroism spectra of a polypeptide. The N-terminal loop, caused by a disulfide bridge between cysteines at positions 1 and 7, was modeled by performing a conformational search by molecular mechanics calculations. The remaining part of the peptide chain was modeled as a mixture of three sequences containing different fractions of residues adopting poly-l-proline II (PPII), extended beta-strand, and alpha-helix-like conformations. This yielded an excellent reproduction of the experimentally observed amide I' band profiles. A comparison with recent data on the beta-amyloid fragment Abeta(1)(-)(28) revealed a lower PPII content and more conformational heterogeneity for calcitonin. Thus, our results underscore the notion that individual structural propensities of amino acid residues give rise to structural differences between the unfolded states of even long peptide chains, at variance with expectations based on a random or statistical coil model.

PII structure in the model peptides for unfolded proteins: Studies on ubiquitin fragments and several alanine‐rich peptides containing QQQ, SSS, FFF, and VVV

A great deal of attention has been paid lately to the structures in unfolded proteins due to the recent discovery of many biologically functional but natively unfolded proteins and the far-reaching implications of order in unfolded states for protein folding. Recently, studies on oligo-Ala, oligo-Lys, oligo-Asp, and oligo-Glu, as well as oligo-Pro, have indicated that the left-handed polyproline II (PII) is the major local structure in these short peptides. Here, we show by NMR and CD studies that ubiquitin fragments, model unfolded peptides composed of nonrepeating amino acids, and four alanine-rich peptides containing QQQ, SSS, FFF, and VVV sequences are all present in aqueous solution predominantly in the extended PII or beta conformation. The results from this and related studies indicate that PII might be a major backbone conformation in unfolded proteins. The presence of defined local backbone structure in unfolded proteins is inconsistent with predictions from random coil models.

Polyproline II propensities from GGXGG peptides reveal an anticorrelation with β-sheet scales

There is growing appreciation of the functional relevance of unfolded proteins in biology. However, unfolded states of proteins have proven inaccessible to the usual techniques for high-resolution structural and energetic characterization. Unfolded states are still generally conceived of as statistical coils, based on the pioneering work of Flory [(1969) Statistical Mechanics of Chain Molecules (Wiley, New York)] and Tanford [(1968) Adv. Protein Chem. 23, 121-282]. Recently, several lines of independent evidence have raised doubts about the random coil model and offer support for alternative views. Here, we show that polyproline II conformation is dominant in a host-guest peptide model AcGGXGGNH(2) (X not equal glycine), in equilibrium predominantly with beta-structure. This result is inconsistent with a random coil model and the general view that these peptides are unstructured. By calculating a set of apparent DeltaG values from the measured coupling constants of the backbone amides, we can construct a polyproline II scale that correlates negatively with beta-sheet scales.

Sterics and Solvation Winnow Accessible Conformational Space for Unfolded Proteins

The magnitude of protein conformational space is over-estimated by the traditional random-coil model, in which local steric restrictions arise exclusively from interactions between adjacent chain neighbors. Using a five-state model, we assessed the extent to which steric hindrance and hydrogen bond satisfaction, energetically significant factors, impose additional conformational restrictions on polypeptide chains, beyond adjacent residues. Steric hindrance is repulsive: the distance of closest approach between any two atoms cannot be less than the sum of their van der Waals radii. Hydrogen bond satisfaction is attractive: polar backbone atoms must form hydrogen bonds, either intramolecularly or to solvent water. To gauge the impact of these two factors on the magnitude of conformational space, we systematically enumerated and classified the disfavored conformations that restrict short polyalanyl backbone chains. Applying such restrictions to longer chains, we derived a scaling law to estimate conformational restriction as a function of chain length. Disfavored conformations predicted by the model were tested against experimentally determined structures in the coil library, a non-helix, non-strand subset of the PDB. These disfavored conformations are usually absent from the coil library, and exceptions can be uniformly rationalized.

Nanoscale Morphology of Polyanhydride Copolymers

The microphase separation in polyanhydride random copolymers composed of 1,6-bis(p-carboxyphenoxy)hexane and sebacic acid is described. Though the copolymers are random, the monomers are sufficiently long and the segment−segment interaction parameter is sufficiently high to promote microphase separation when the copolymer is rich in one component. Solid-state NMR spin diffusion experiments and synchrotron small-angle X-ray scattering are used to discern the length scales of the microphase separation. Both techniques reveal a nanostructure with domain sizes less than 25 Å. This nanostructure is compared to approximate calculations of chain dimensions based on a random coil model and discussed in the context of the rational design of these materials for drug delivery applications.

Aggregation dynamics of laminin-1 in a physiological solution: A time-resolved static light scattering study

The aggregation of laminin-1 in a physiological solution was observed using time-resolved static light scattering. In a solution containing 150 mM of NaCl and 1 mM of CaCl 2, with a pH of 7.2 buffered by 50 mM Tris, and maintained at 25 °C, the weight-averaged mass (molecular weight) M w, and radius of gyration R g, of the aggregate were measured at 10 s intervals. The aggregation kinetics changed from reaction-limited cluster aggregation (RLCA) to diffusion-limited cluster aggregation (DLCA) over time. The fractal dimension estimated in the DLCA regime was 1.71 from the M w ∼ R g d f relationship, which is consistent with the d f of 1.75 obtained from an R g ∼ t 1 / d f plot. Direct calculation of d f using the scattering intensity and scattering vector revealed that d f gradually increased over time in the DLCA regime, suggesting that spontaneous restructuring of the aggregate had occurred. This restructuring would have been caused by hydrophobic contact in the aggregate. The form factor of the aggregate was well fitted by a linear random coil model and not by a simple spherical model.