Polypeptides In Solution Research Articles

The Ribosome Alters the Folding of a Multidomain Nascent Protein

Protein folding is often studied in the context of full-length polypeptides in solution. However, the post-translational folding pathway may not recreate the pathway of folding when a protein is being synthesized by the ribosome. The effects of the ribosome and the rate of translation on folding are not well understood, partially due to the difficulty of probing the nascent chain without also affecting the ribosome. Using optical tweezers, we can look in detail at the nascent chain at the single-molecule level with minimal perturbation to the ribosome. Previous studies have shown that the ribosomal surface can act electrostatically to slow the kinetics of folding. Those studies used a linker to vary the distance from the surface and did not look at changes in sequence availability or multidomain proteins. To further understand cotranslational folding, we are studying the folding pathway of a two-domain calcium-binding protein calerythrin. Our results show that the full-length protein in solution folds robustly through a C-domain intermediate, but truncated versions of the protein can fold to an N domain or a misfolded state. During translation, the C-terminal residues required for native folding are not yet available, but the other observed intermediates could fold, in principle. By observing the folding at various sequence positions, and thus nascent chain lengths, we found that the ribosome can not only slow folding, but can also affect the unfolding rates, particularly increasing the unfolding rate of the misfolded state. The ribosome thus prevents the formation of this unproductive state via two mechanisms: decreasing the probability of folding and decreasing the stability of the folded state. This study gives further insight into the importance of the ribosome to regulate protein structure, and opens up new questions about the interplay between elongation and folding.

Ice Growth Inhibition in Antifreeze Polypeptide Solution by Short-Time Solution Preheating.

The objective of this study is to enhance the inhibition of ice growth in the aqueous solution of a polypeptide, which is inspired by winter flounder antifreeze protein. We carried out measurements on unidirectional freezing of the polypeptide solution. The thickness of the solution was 0.02 mm, and the concentration of polypeptide was varied from 0 to 2 mg/mL. We captured successive microscopic images of ice/solution interfaces, and measured the interface velocity from the locations of tips of the pectinate interface in the images. We also simultaneously measured the temperature by using a small thermocouple. The ice/solution interface temperature was defined by the temperature at the tips. It was found that the interface temperature was decreased with an increasing concentration of polypeptide. To try varying the activity of the polypeptide, we preheated the polypeptide solution and cooled it before carrying out the measurements. Preheating for 1–5 hours was found to cause a further decrease in the interface temperature. Furthermore, wider regions of solution and ice with inclined interfaces in the pectinate interface structure were observed, compared with the case where the solution was not preheated. Thus, the ice growth inhibition was enhanced by this preheating. To investigate the reason for this enhancement, we measured the conformation and aggregates of polypeptide in the solution. We also measured the local concentration of polypeptide. It was found that the polypeptide aggregates became larger as a result of preheating, although the polypeptide conformation was unchanged. These large aggregates caused both adsorption to the interface and the wide regions of supercooled solution in the pectinate interface structure.

Arrested Phase Separation of Elastin-like Polypeptide Solutions Yields Stiff, Thermoresponsive Gels

The preparation of new responsive hydrogels is crucial for the development of soft materials for various applications, including additive manufacturing and biomedical implants. Here, we report the discovery of a new mechanism for forming physical hydrogels by the arrested phase separation of a subclass of responsively hydrophobic elastin-like polypeptides (ELPs). When moderately concentrated solutions of ELPs with the pentapeptide repeat (XPAVG)n (where X is either 20% or 60% valine with the remainder isoleucine) are warmed above their inverse transition temperature, phase separation becomes arrested, and hydrogels can be formed with shear moduli on the order of 0.1-1 MPa at 20 wt % in water. The longest stress relaxation times are well beyond 10(3) s. This result is surprising because ELPs are classically known for thermoresponsive coacervation that leads to macrophase separation, and solids are typically formed in the bulk or by supplemental cross-linking strategies. This new mechanism can form gels with remarkable mechanical behavior based on simple macromolecules that can be easily engineered. Small angle scattering experiments indicate that phase separation arrests to form a network of nanoscale domains, exhibiting rheological and structural features consistent with an arrested spinodal decomposition mechanism. Gel nanostructure can be modeled as a disordered bicontinuous network with interdomain, intradomain, and curvature length scales that can be controlled by sequence design and assembly conditions. These studies introduce a new class of reversible, responsive materials based on a classic artificial biopolymer that is a versatile platform to address critical challenges in industrial and medical applications.

Study on Optimal Conditions of Alcalase Enzymatic Hydrolysis of Soybean Protein Isolate

Soybean protein isolate was hydrolyzed to obtain soybean polypeptide solution using Alcalase as hydrolase. Degree of hydrolysis and the recovery rate of protein were used to characterize the soybean protein hydrolysis reaction result. Influence factors of soybean protein hydrolysis reaction including the substrate concentration, temperature, pH, enzyme concentration characterized by E/S (ratio of Enzyme and Substrate) and hydrolysis time were systematically studied with single factor and multi-level experimental. The optimal reaction conditions of soybean protein isolate hydrolyzed by Alcalase were that the substrate concentration was 8%; E/S was 3.6 AU/100 g substrate; pH was 8.0; temperature was 60°C and hydrolysis time was 2 h. The study also showed that the hydrolytic activity of the Alcalase to soybean protein isolate was stronger in the front 1 h; the hydrolytic activity of Alcalase weakened gradually with time when reaction time was longer than 2 h.

Force field-dependent solution properties of glycine oligomers.

Molecular simulations can be used to study disordered polypeptide systems and to generate hypotheses on the underlying structural and thermodynamic mechanisms that govern their function. As the number of disordered protein systems investigated with simulations increase, it is important to understand how particular force fields affect the structural properties of disordered polypeptides in solution. To this end, we performed a comparative structural analysis of Gly(3) and Gly(10) in aqueous solution from all atom, microsecond molecular dynamics (MD) simulations using the CHARMM 27 (C27), CHARMM 36 (C36), and Amber ff12SB force fields. For each force field, Gly(3) and Gly(10) were simulated for at least 300 ns and 1 μs, respectively. Simulating oligoglycines of two different lengths allows us to evaluate how force field effects depend on polypeptide length. Using a variety of structural metrics (e.g., end-to-end distance, radius of gyration, dihedral angle distributions), we characterize the distribution of oligoglycine conformers for each force field and show that each sample conformation space differently, yielding considerably different structural tendencies of the same oligoglycine model in solution. Notably, we find that C36 samples more extended oligoglycine structures than both C27 and ff12SB.

Thermal expansivities of peptides, polypeptides and proteins as measured by pressure perturbation calorimetry

The main goal of this work was to provide direct experimental evidence that the expansivity of peptides, polypeptides and proteins as measured by pressure perturbation calorimetry (PPC), can serve as a proxy to characterize relative compactness of proteins, especially the denatured state ensemble. This is very important as currently only small angle X-ray scattering (SAXS), intrinsic viscosity and, to a lesser degree, fluorescence resonance transfer (FRET) experiments are capable of reporting on the compactness of denatured state ensembles. We combined the expansivity measurements with other biophysical methods (far-UV circular dichroism spectroscopy, differential scanning calorimetry, and small angle X-ray scattering). Three case studies of the effects of conformational changes on the expansivity of polypeptides in solution are presented. We have shown that expansivity appears to be insensitive to the helix–coil transition, and appears to reflect the changes in hydration of the side-chains. We also observed that the expansivity is sensitive to the global conformation of the polypeptide chain and thus can be potentially used to probe hydration of different collapsed states of denatured or even intrinsically disordered proteins.

Correction: Phase behaviour and complex coacervation of aqueous polypeptide solutions

Correction for ‘Phase behaviour and complex coacervation of aqueous polypeptide solutions’ by Dimitrios Priftis et al., Soft Matter, 2012, 8, 9396–9405.

Extended structure of rat islet amyloid polypeptide in solution.

The process of islet amyloid polypeptide (IAPP) formation and the prefibrillar oligomers are supposed to be one of the pathogenic agents causing pancreatic β-cell dysfunction. The human IAPP (hIAPP) aggregates easily and therefore, it is difficult to characterize its structural features by standard biophysical tools. The rat version of IAPP (rIAPP) that differs by six amino acids when compared with hIAPP, is not prone to aggregation and does not form amyloid fibrils. Similar to hIAPP it also demonstrates random-coiled nature in solution. The structural propensity of rIAPP has been studied as a hIAPP mimic in recent works. However, the overall shape of it in solution still remains elusive. Using small angle X-ray scattering (SAXS) measurements combined with nuclear magnetic resonance (NMR) and molecular dynamics simulations (MD) the solution structure of rIAPP was studied. An unambiguously extended structural model with a radius of gyration of 1.83 nm was determined from SAXS data. Consistent with previous studies, an overall random-coiled feature with residual helical propensity in the N-terminus was confirmed. Combined efforts are necessary to unambiguously resolve the structural features of intrinsic disordered proteins.

Stable aqueous solutions of keratin polypeptides: their properties in solution and at interfaces

Stable aqueous solutions of keratin polypeptides: their properties in solution and at interfaces

Understanding and Describing the Liquid-Crystalline States of Polypeptide Solutions: A Coarse-Grained Model of PBLG in DMF

A perturbation theory is employed to construct a free-energy functional capable of describing the isotropic and nematic phases of attractive rod-like particles. An algebraic van der Waals–Onsager equation of state is then developed to determine the global phase behavior of prolate particles for various aspect ratios and strengths of the attractive potential. Compared with the phase diagram of athermal systems, the incorporation of an attractive potential is seen to stabilize the nematic state, leading to an increase in the degree of orientational order of the system at high densities. The most salient feature for systems with large aspect ratios is the existence of regions of nematic–nematic phase separation. A simple attractive-rod model is used to describe the phase behavior of solutions of a relatively rigid polypeptide, poly(γ-benzyl-l-glutamate) (PBLG), in dimethylformamide (DMF); this system exhibits a unique phase separation between two liquid-crystalline states. The coarse-grained representation o...

A Novel Tri-Enzyme System in Combination with Laser-Driven NMR Enables Efficient Nuclear Polarization of Biomolecules in Solution

NMR is an extremely powerful, yet insensitive technique. Many available nuclear polarization methods that address sensitivity are not directly applicable to low-concentration biomolecules in liquids and are often too invasive. Photochemically induced dynamic nuclear polarization (photo-CIDNP) is no exception. It needs high-power laser irradiation, which often leads to sample degradation, and photosensitizer reduction. Here, we introduce a novel tri-enzyme system that significantly overcomes the above challenges, rendering photo-CIDNP a practically applicable technique for NMR sensitivity enhancement in solution. The specificity of the nitrate reductase (NR) enzyme is exploited to selectively in situ reoxidize the reduced photo-CIDNP dye FMNH2. At the same time, the oxygen-scavenging ability of glucose oxidase (GO) and catalase (CAT) is synergistically employed to prevent sample photodegradation. The resulting tri-enzyme system (NR-GO-CAT) enables prolonged sensitivity-enhanced data collection in 1D and 2D heteronuclear NMR, leading to the highest photo-CIDNP sensitivity enhancement (48-fold relative to SE-HSQC) achieved to date for amino acids and polypeptides in solution. NR-GO-CAT extends the concentration limit of photo-CIDNP NMR down to the low micromolar range. In addition, sensitivity (relative to the reference SE-HSQC) is found to be inversely proportional to sample concentration, paving the way for the future analysis of even more diluted samples.

Study on Soybean Polypeptide Refining Process

Soybean polypeptide mixture was decolorized by adopting activated carbon adsorption decolourization process. Anion/cation exchange process was used in the desalination processing of soybean polypeptide. It was showed that excellent decolorizing and desalting effect to soybean peptide solution was gained in the decolourization and desalination process adopted in this experiment. Optimal dosage of activated carbon was 1.2% solution amount (w/w). After decolorizing soybean polypeptide solution color was yellow. Proportion of anion resin and cation resin of ion exchange resin was 2:3(V/V). The volume of solution processed was 5 times as the volume of anion exchange resin. After desalination processing, ash content of soybean peptide solution reduced to 2.11% (dry basis), salinity decreased by 86%, on the other hand taste of soybean polypeptide was lite.

Differential Hydration Induced Coil-Sphere Transition of Gelatin Chains in Binary Liquid Solutions

Specific hydration induced coil-globule transition in gelatin-B(GB) chain, a polypeptide, in ethanol, ethylene glycol and glycerol solutions (organo-solutions) was studied by Raman spectroscopy, viscosity and dynamic light scattering techniques to map its differential hydration behavior in mono, di and tri-ol solutions (alcohols are non-solvents for this polypeptide). The chain stiffness, determined from the hydrodynamic radius to radius of gyration ratio Rg/Rh, was estimated to be around 0.67 in water which was found to be dependent on the nature of alcohol and its concentration. This clearly attributed same random coil conformation to GB in all thesolutions at low alcohol concentration, but chain collapse to a near-globular shape (Rg/Rh=0.77) was observed when alcohol concentration was increased which was found to occur in glycerol at ≈ 60% (v/v), in ethylene glycol at ≈ 35% and in ethanol at ≈ 40% (v/v) concentrations consistence with the intrinsic viscosity data. The Huggins (KH) and Kraemer (KK) solute-solvent interaction parameter determined from the concentration dependence plot of relative viscosity revealed poorer interaction between GB and organo-solvents with increase in concentration of the organic phase. Differential chain hydration was evaluated from characteristic Raman active modes of water molecule. Three signatures Raman peaks were observed at 3200, 3310, and 3460 cm-1 specific to structured, partially structured and amorphous water respectively. The peak at 3310 cm-1 was observed to decrease with alcohol concentration while the peak at 3200 cm-1 was observed to increase indicating the increase in the water structure in all the solutions and depletion of water density near the first hydration sheath of the protein molecule which caused GB chain to collapse.

DPD Simulation of Protein Conformations: From α-Helices to β-Structures.

We suggest a coarse-grained model for DPD simulations of polypeptides in solutions. The model mimics hydrogen bonding that stabilizes α-helical and β-structures using dissociable Morse bonds between quasiparticles representing the peptide groups amenable to hydrogen bonding. We demonstrate the capabilities of the model by simulating transitions between coil-like, globular, α-helical, and β-hairpin configurations of model peptides, varying Morse potential parameters, the hydrophobicities of residue side chains, and pH, which determines the charges of residue side chains. We construct a model triblock polypeptide mimicking the sequence of residues α-synuclein at two different pHs. The conformations of this model polypeptide depend on pH similarly to the behavior observed experimentally. The suggested approach to accounting for hydrogen bond formation within the general DPD framework may make the DPD method a competitive alternative to CGMD for modeling equilibrium and dynamic properties of proteins and polypeptides, especially during their transport in confined environments.

Physical Gelation of Polypeptide–Polyelectrolyte–Polypeptide (ABA) Copolymer in Solution

The gelation behavior of polypeptide–polyelectrolyte–polypeptide in solution is investigated by Monte Carlo techniques. It is found that different hydrogen bonding styles, parallel and antiparallel, provide a major influence on the sol–gel transition and the chain conformation. The antiparallel hydrogen bonding style favors the bridge conformation, while loop conformation has less structural limitation with parallel bonding. However, in the parallel style, polyelectrolyte blocks are aligned in the same side of β-sheets, which induces more steric constrains and the electrostatic repulsion. Consequently, compared with the parallel bonding style, the sol–gel transition of antiparallel style occurs at lower hydrogen bonding energy and the dihedral angle θ of antiparallel β-sheets is a little higher. The increase of charging fraction of polyelectrolyte blocks prompts the sol–gel transition of both bonding styles but causes an impact on the number of hydrogen bond and the dihedral angle, indicating a decrease in the structure integrity.

Rheological properties of cysteine-containing elastin-like polypeptide solutions and hydrogels.

The rheological properties of cysteine-containing elastin-like polypeptide (Cys-ELP) solutions and Cys-ELP hydrogels are reported. The Cys-ELP solutions exhibit a surprisingly high apparent viscosity at low shear rate. The high viscosity is attributed to the formation of an interfacial cross-linked "skin" at the sample surface, rather than the bulk of the Cys-ELP solution. At higher shear rate, the interfacial cross-linked film breaks, and its influence on the viscosity of the Cys-ELP solution can be ignored. Cys-ELP hydrogels are formed by mixing Cys-ELP and hydrogen peroxide (H(2)O(2)). At fixed concentration of Cys-ELP, the gelation time can be tuned by the concentration of H(2)O(2). Cys-ELP hydrogels have the typical characteristics of covalent cross-linked networks, as the storage moduli are larger than the loss moduli and are independent of frequency in dynamic oscillatory frequency sweep experiments. The plateau moduli obtained from linear frequency sweep experiments are much lower than those estimated from the number of thiol groups along the Cys-ELP chain, indicating that only a small fraction of thiols form elastically active cross-links. From the small value of the fraction of elastically active cross-links, the Cys-ELP hydrogel is concluded to be an inhomogenous network. Under steady shear, a 2.5 wt % Cys-ELP hydrogel shear thickens at shear rates lower than that necessary for fracture.

Test of a method for sampling the internal degrees of freedom of a flexible solute molecule based on adiabatic decoupling and temperature or force scaling

Simulation of the folding equilibrium of a polypeptide in solution is a computational challenge. Standard molecular dynamics (MD) simulations of such systems cover hundreds of nanoseconds, which is barely sufficient to obtain converged ensemble averages for properties that depend both on folded and unfolded peptide conformations. If one is not interested in dynamical properties of the solute, techniques to enhance the conformational sampling can be used to obtain the equilibrium properties more efficiently. Here the effect on particular equilibrium properties at 298 K of adiabatically decoupling the motion a β-hepta-peptide from the motion of the solvent and subsequently up-scaling its temperature or down-scaling the forces acting on it is investigated. The ensemble averages and rate of convergence are compared to those for standard MD simulations at two different temperatures and a simulation in which the temperature of the solute is increased to 340 K while keeping the solvent at 298 K. Adiabatic decoupling with a solute mass scaling factor s m = 100 and a temperature scaling factor of s T = 1.1 seems to slightly increase the convergence of several properties such as enthalpy of folding, NMR NOE atom–atom distances and 3J-couplings compared to a standard MD simulation at 298 K. Convergence is still slower than that observed at 340 K. The system with a temperature of 340 K for the solute and 298 K for the solvent without scaling of the mass converges fastest. Using a force scaling factor s V = 0.909 perturbs the system too much and leads to a destabilization of the folded structure. The sampling efficiency and possible distortive effects on the configurational distribution of the solute degrees of freedom due to adiabatic decoupling and temperature or force scaling are also analysed for a simpler model, a dichloroethane molecule in water. It appears that an up-scaling of the mass of the solute reduces the sampling more than the subsequent up-scaling of the temperature or down-scaling of the force enhances it. This means that adiabatic decoupling the solute degrees of freedom from the solvent ones followed by an up-scaling of temperature of down-scaling of the forces does not lead to significantly enhanced sampling of the folding equilibrium.

Phase behaviour and complex coacervation of aqueous polypeptide solutions

Mixing of oppositely charged polyelectrolytes in aqueous solutions may result in the formation of polyelectrolyte complexes (PEC). Phase separation and complex coacervation of polypeptides are investigated in this study. Polypeptides have identical backbones and differ only in their charged side groups, making them attractive model systems for this work. All experiments are conducted using equal chain lengths of polycation and polyanion in order to isolate and highlight effects of the interactions of the charged groups during complexation. Complex coacervation is strongly affected by the polypeptide mixing ratio (stoichiometry), ionic strength (salt concentration), total polymer concentration, pH, and temperature. To examine the effect of these parameters on complex formation we use sample turbidity as an indicator, and optical microscopy to discriminate between coacervate and precipitate. We establish phase diagrams as a function of polybase content and salt concentration using the critical salt concentrations required to reach the coacervate to solution boundary. Additionally, we examine the effect of molecular weight on the complex formation for the P(L-Lysine) (PLys)/P(L-Glutamic acid) (PGlu) system and establish a phase diagram. By determining the water content of the coacervate phase under various conditions we find that the salt content and stoichiometry of the mixed polyelectrolytes have a significant effect on the coacervate composition.

Analysis of NAD 2D-NMR Spectra of Saturated Fatty Acids in Polypeptide Aligning Media by Experimental and Modeling Approaches

The overall and detailed elucidation (including the stereochemical aspects) of enzymatic mechanisms requires the access to all reliable information related to the natural isotopic fractionation of both precursors and products. Natural abundance deuterium (NAD) 2D-NMR experiments in polypeptide liquid-crystalline solutions are a new, suitable tool for analyzing site-specific deuterium isotopic distribution profiles. Here this method is utilized for analyzing saturated C14 to C18 fatty acid methyl esters (FAMEs), which are challenging because of the crowding of signals in a narrow spectral region. Experiments in achiral and chiral oriented solutions were performed. The spectral analysis is supplemented by the theoretical prediction of quadrupolar splittings as a function of the geometry and flexibility of FAMEs, based on a novel computational methodology. This allows us to confirm the spectral assignments, while providing insights into the mechanism of solute ordering in liquid-crystalline polypeptide solutions. This is found to be dominated by steric repulsions between FAMEs and polypeptides.

Molecular Architecture Influences the Thermally Induced Aggregation Behavior of Elastin-like Polypeptides

Elastin-like polypeptides are thermally responsive polymers that exhibit phase separation above a transition temperature. The effect of molecular architecture on the temperature responsive behavior of elastin-like polypeptide solutions was investigated by characterization of solutions of three-armed star polypeptides, linear polypeptides, and their mixtures. These biosynthesized polypeptides have precise lengths and amino acid sequences. Transition temperatures were measured as a function of molecular weight and solution concentration and compared to their linear counterparts. Like their linear counterparts, the transition temperature is linearly related to log concentration. A mathematical relationship was used to fit the transition temperature data of different polypeptide lengths to a volume-based concentration using the polymer coil volume. The results of this model suggest that the linear ELP is in a random coil conformation at the transition temperature while the three-armed ELP is in a compact extended coil conformation, consistent with different pathways for aggregation. Solutions containing both trimer and linear constructs have two transition temperatures, further supporting differing aggregation behaviors.