Polypeptides In Solution Research Articles

Investigation of the Effectiveness of Photo Deprotection of Polypeptides in Solution and within the Core of Miniemulsion-Derived Nanoparticles.

Homopolymerization of ortho-nitrobenzyl (oNB)-protected l-cysteine and l-glutamic acid was systematically studied in different solvents and at different monomer to initiator ratios, revealing the best reaction control in dimethylformamide (DMF) across a range of degrees of polymerization. In the subsequent ultraviolet (UV)-cleavage studies, it was found that quantitative deprotection upon UV exposure at 365 nm was not achievable for either of the homopolypeptides as confirmed by 1H NMR and UV/visible (UV/vis) analyses. While the poly(oNB-l-cysteine) deprotected more readily with no effect of the polypeptide molecular weight, lower molecular weight poly(oNB-l-glutamate) reached maximum deprotection faster than high molecular weight samples. This was further confirmed by the pH changes of the solution. When incorporated into the core of miniemulsion-derived nanoparticles, both oNB-protected copolypeptides were successfully deprotected as evident from a color change and a pH change in the case of poly(oNB-l-glutamate). However, the removal of the deprotection byproduct nitrosobenzaldehyde proved unsuccessful, which indicates a diffusion barrier caused by the nanoparticle's surfactant. The study provides insights and guidelines for the UV deprotection of polypeptides and demonstrates the ability to selectively UV-deprotect polypeptides in the confined space of a nanoparticle dispersion.

Helix-coil and solvation-induced reentrant nematic-smectic A transitions in a polypeptide solution

ABSTRACT A simple model is introduced to describe re-entrant isotropic-nematic and nematic-smectic A phase transitions mediated helix-coil transition and solvation within individual liquid crystalline semiflexible polymers in a solution. We consider hydrogen bonding between the polymer and solvent molecules (solvation) as an intermolecular interaction and helix-coil transformations of the polymer as an intramolecular interaction. The helix part is rigid and can form nematic and smectic A phases. On the other hand, the solvation prevents liquid crystalline ordering of the polymer chains. At high temperatures, a polymer is in a coiled state without solvation. With decreasing temperatures, solvation and helix-coil transitions compete in the individual semiflexible polymers, and the re-entrant phase transitions occur. We also discuss the chain conformation by calculating Kuhn length.

Linear and ring polypeptides complexed with oppositely charged surfactants: the cohesion of the complexes as revealed in atomistic simulations.

The use of linear supercharged unfolded polypeptides (SUPs) and oppositely charged surfactants in aqueous solution has demonstrated impressive adhesive properties. These substances possess biocompatibility, biodegradability and other necessary properties for practical application as a biomedical glue in wound repair. The success of these substances, coupled with limited knowledge about such systems, provides hope for enhancing the performance of the final product. One potential approach involves altering the topology of the polypeptide chain. In this article, we conduct a comparative analysis to examine the behavior of the ring and linear chains of a polypeptide in aqueous solution. This analysis utilizes full-atomic computer modeling to monitor the properties of the chains. We investigate the temperature dependence of the shape and size of individual polypeptides in the solution, as well as the formation of complexes via mixing the polypeptide chains with oppositely charged sodium dodecylbenzene sulfonate (SDBS) surfactant molecules in a stoichiometric ratio. Additionally, we explore the cohesive properties of the resulting complex through power experiments involving the extraction of single polypeptide chains out of the SUP-SDBS complexes.

Characterization of Secondary Structures of Model Polypeptides in Solutions with Hyper-Raman Spectroscopy.

Characterization of the secondary structures of two model polypeptides, poly-l-lysine and poly-l-glutamic acid in aqueous solutions has been demonstrated by hyper-Raman (HR) spectroscopy for the first time. Complementary to infrared (IR) and visible Raman spectroscopy, HR spectroscopy gives the amide I, II, and III bands originating from the polypeptide backbones and the CCH3 symmetric bending mode, enabling us to distinguish different conformations. The α-helix gives the broad and weak amide III band, while the β-sheet and the random coil show similar spectral patterns with different relative intensities between the amide I and II bands. HR spectra from aqueous solutions of the α-helix and the random coil of poly-l-ornithine also possess these spectral features. The HR spectra are analogous to UV resonance Raman (UVRR) spectra, indicating the signal enhancement due to the electronic resonance effect via the π-π* transition. In contrast, the vibrational frequencies of the amide I band in the HR spectra are much higher than those in the IR, visible Raman, and UVRR spectra, suggesting the non-coincidence between HR, IR, and Raman bands. Our finding suggests that HR spectroscopy is promising to provide complementary information on the secondary structures of polypeptides in aqueous solutions as a spectral approach differing from existing vibrational spectroscopic methods.

Evidence of Self-Association and Conformational Change in Nisin Antimicrobial Polypeptide Solutions: A Combined Raman and Ultrasonic Relaxation Spectroscopic and Theoretical Study

The polypeptide Nisin is characterized by antibacterial properties, making it a compound with many applications, mainly in the food industry. As a result, a deeper understanding of its behaviour, especially after its dissolution in water, is of the utmost importance. This could be possible through the study of aqueous solutions of Nisin by combining vibrational and acoustic spectroscopic techniques. The velocity and attenuation of ultrasonic waves propagating in aqueous solutions of the polypeptide Nisin were measured as a function of concentration and temperature. The computational investigation of the molecular docking between Nisin monomeric units revealed the formation of dimeric units. The main chemical changes occurring in Nisin structure in the aqueous environment were tracked using Raman spectroscopy, and special spectral markers were used to establish the underlying structural mechanism. Spectral changes evidenced the presence of the dimerization reaction between Nisin monomeric species. The UV/Vis absorption spectra were dominated by the presence of π → π* transitions in the peptide bonds attributed to secondary structural elements such as α-helix, β-sheets and random coils. The analysis of the acoustic spectra revealed that the processes primarily responsible for the observed chemical relaxations are probably the conformational change between possible conformers of Nisin and its self-aggregation mechanism, namely, the dimerization reaction. The activation enthalpy and the enthalpy difference between the two isomeric forms were estimated to be equal to ΔH1* = 0.354 ± 0.028 kcal/mol and ΔH10 = 3.008 ± 0.367 kcal/mol, respectively. The corresponding thermodynamic parameters of the self-aggregation mechanism were found to be ΔH2* = 0.261 ± 0.004 kcal/mol and ΔH20 = 3.340 ± 0.364 kcal/mol. The effect of frequency on the excess sound absorption of Nisin solutions enabled us to estimate the rate constants of the self-aggregation mechanism and evaluate the isentropic and isothermal volume changes associated with the relaxation processes occurring in this system. The results are discussed in relation to theoretical and experimental findings.

Recent advances in the solution self‐assembly of polypeptides

AbstractPolypeptides derived from amino acids bridge the synthetic polymers and proteins, and they have attracted continuous attention on their properties and functionalities. In the past decades, the self‐assembly behavior of polypeptides has been widely studied, and diversities of assembly nanostructures have been observed. In this perspective, we focus on the recent advances in the solution self‐assembly of polypeptides from two aspects. One is new type driving forces for the polypeptide self‐assembly, including liquid crystallization of polypeptides and conformation variation of helical polypeptides. Another is novel preparation methods for the polypeptide self‐assembly, including supramolecular polymerization, substrate‐mediated self‐assembly, and polymerization‐induced self‐assembly (PISA). In the end, we give a conclusion and outlook for the trends in the polypeptide solution self‐assembly.

Ultrasound-Assisted Enzymatic Extraction and Bioactivity Analysis of Polypeptides from Cordyceps militaris

Cordyceps militaris is rich in protein, polysaccharide, cordycepin, and other active components, with anticancer and antioxidation functions. In order to improve the economic value of C. militaris, the protein was extracted from its fruiting body by alkali-soluble acid precipitation process, and the extraction technology was optimized by orthogonal test. The polypeptide was obtained by digesting those proteins with a complex enzyme. And the antimicrobial and anticancer activities of those polypeptides were evaluated by measuring inhibitory zone and cytotoxicity. The results showed that the optimal extraction conditions of protein were as follows: pH of 8.5, material-to-water ratio of 1 : 28, extraction time of 3.5 h, extraction three times, and the highest protein yield was 45.06%. The optimum enzymatic hydrolysis process of C. militaris polypeptide solution was as follows: the ratio of alkaline protease to papain was 4 : 3, the optimum temperature was 55°C, pH was 7.2, the enzyme dosage was 7000 U/mL, the enzymolysis time was 3.5 h, and the highest yield of peptide was 16.73%. Under those conditions, the polypeptides prepared from C. militaris (<3000 Da) showed good antibacterial activity against Escherichia coli, Bacillus subtilis, and Staphylococcus aureus, with inhibitory zones of (12.08 ± 0.22), (6.67 ± 0.12), and (10.32 ± 0.23) mm, respectively. The results showed that the SAO-S (IC50 = 0.49 mg/L) and T24 (IC50 = 0.23 mg/L) were significantly inhibited by C. militaris polypeptide. Results from this study suggest that polypeptides can be utilized as a new approach for bioactive compounds production from C. militaris.

A polypeptide coating for preventing biofilm on implants by inhibiting antibiotic resistance genes

Aging population has been boosting the need for orthopedic implants. However, biofilm has been a major obstacle for orthopedic implants due to its insensitivity to antibiotics and tendency to drive antimicrobial resistance. Herein, an antibacterial polypeptide coating with excellent in vivo adhesive capacity was prepared to prevent implants from forming biofilms and inducing acquired antibiotic resistance. A peptide-based copolymer, poly[phenylalanine10-stat-lysine12]-block-3,4-dihydroxy-l-phenylalanine [Poly(Phe10-stat-Lys12)-DOPA] was modularly designed, where poly(Phe10-stat-Lys12) is antibacterial polypeptide with high antibacterial activity, and DOPA provides strong adhesion in both wet and dry microenvironments. Meanwhile, compared to traditional “graft-onto” methods, this antibacterial coating can be facilely achieved by immersing Titanium substrates into antibacterial polypeptide solution for 5 min at room temperature. The poly(Phe10-stat-Lys12)-DOPA polymer showed good antibacterial activity with minimum inhibitory concentrations against S. aureus and E. coli of 32 and 400 μg/mL, respectively. Compared to obvious antimicrobial resistance of S. aureus after continuous treatment with vancomycin, this antibacterial coating doesn't drive antimicrobial resistance upon long-term utilization. Transcriptome sequencing and qPCR tests further confirmed that the antibacterial coating was able to inhibit the expression of multiple peptide resistance factor (mprF) and lipoteichoic acid modification D-alanylation genes (dltB and dltC) that can increase the net positive charge of bacterial cell wall to induce the resistance to cationic antimicrobial peptides. In vivo experiments confirmed that this poly(Phe10-stat-Lys12)-DOPA coating can both effectively prevent biofilm formation through surface contact sterilization and avoid local and systemic infections. Overall, we proposed a facile method for preparing antibacterial orthopedic implants with longer indwelling time and without inducing antimicrobial resistance by coating a polypeptide-based polymer on the implants.

Tagging Peptides with a Redox Responsive Fluorescent Probe Enabled by Photoredox Difunctionalization of Phenylacetylenes with Sulfinates and Disulfides.

Herein, we describe a photoredox three-component atom-transfer radical addition (ATRA) reaction of aryl alkynes directly with dialkyl disulfides and alkylsulfinates, circumventing the utilization of chemically unstable and synthetically challenging S-alkyl alkylthiosulfonates as viable addition partners. A vast array of (E)-β-alkylsulfonylvinyl alkylsulfides was prepared with great regio- and stereoselectivity. Moreover, this powerful tactic could be employed to tag cysteine residues of complex polypeptides in solution or on resin merging with solid phase peptide synthesis (SPPS) techniques. A sulfonyl-derived redox responsive fluorescent probe could be conveniently introduced on the peptide, which displays green fluorescence in cells while showing blue fluorescence in medium. The photophysical investigations reveal that the red shift of the emission fluorescence is attested to reduction of carbonyl group to the corresponding hydroxyl moiety. Interestingly, the fluorescence change of tagged peptide could be reverted in cells by treatment of H2O2, arising from the reoxidation of hydroxyl group back to ketone by the elevated level of reactive oxygen species (ROS).

Effects of porous media on the phase behaviour of polypeptide solutions

The generalized van der Waals equation for anisotropic fluids in porous media, proposed by the authors in previous works, is used to describe the effect of porous media on the phase behavior of polypeptide solutions. By introducing the temperature dependence for the depth of the potential well and the geometric parameters of the spherocylinder, the main features of phase behavior of the polypeptide poly (γ-benzyl-L-glutamate) (PBLG) in a solution of dimethylformamide, including the existence of two nematic phases, is reproduced. It is shown that the presence of a porous medium shifts the phase diagram to the region of lower densities and lower temperatures.

BeStSel: From Secondary Structure Analysis to Protein Fold Prediction by Circular Dichroism Spectroscopy.

Far-UV circular dichroism (CD) spectroscopy is a classical method for the study of the secondary structure of polypeptides in solution. It has been the general view that the α-helix content can be estimated accurately from the CD spectra. However, the technique was less reliable to estimate the β-sheet contents as a consequence of the structural variety of the β-sheets, which is reflected in a large spectral diversity of the CD spectra of proteins containing this secondary structure component. By taking into account the parallel or antiparallel orientation and the twist of the β-sheets, the Beta Structure Selection (BeStSel) method provides an improved β-structure determination and its performance is more accurate for any of the secondary structure types compared to previous CD spectrum analysis algorithms. Moreover, BeStSel provides extra information on the orientation and twist of the β-sheets which is sufficient for the prediction of the protein fold.The advantage of CD spectroscopy is that it is a fast and inexpensive technique with easy data processing which can be used in a wide protein concentration range and under various buffer conditions. It is especially useful when the atomic resolution structure is not available, such as the case of protein aggregates, membrane proteins or natively disordered chains, for studying conformational transitions, testing the effect of the environmental conditions on the protein structure, for verifying the correct fold of recombinant proteins in every scientific fields working on proteins from basic protein science to biotechnology and pharmaceutical industry. Here, we provide a brief step-by-step guide to record the CD spectra of proteins and their analysis with the BeStSel method.

In situ formation of silver nanoparticles-contained gelatin-PEG-dopamine hydrogels via enzymatic cross-linking reaction for improved antibacterial activities

Hydrogels containing silver nanoparticles (AgNPs) were recently found to exhibit excellent antibacterial properties against both gram-negative/positive bacteria and fungi. In this study, we reported the synthesis of AgNPs-contained gelatin-polyethylene glycol-dopamine (AgNP-GPD) hydrogels via the in situ formation of AgNPs in GPD polypeptide solution, followed by an enzymatic cross-linking reaction to form hydrogels. The experimental results showed that the reducing reaction exerted by GPD polypeptides under physiological conditions can afford the formation of AgNPs in situ in the polypeptide solution without the need for additional reducing agents and/or processes such as UV or thermal treatments and then the hydrogelation of GPD polypeptide solution containing AgNPs were preceeded via enzymatic cross-linking reaction. It was found that the gelation time, hydrogel mechanical property, degree of swelling and degree of enzymatic degradation for both GPD and AgNP-GPD hydrogels can be tuned by varying enzyme/oxidative agent concentration, catechol content, and reducing reaction conditions such as reaction time and silver ion concentration. Importantly, AgNP-GPD hydrogels exhibit excellent antibacterial properties against gram-negative and gram-positive bacteria. This type of hydrogel is a promising biomaterial for biomedical applications including wound healing and tissue engineering.

Alternative Way of Increasing Stability of Polypeptides in Solution (in Memory of the Teacher)

This survey summarizes the published and new results of research conducted in leading scientific institutions and clinics in our country. It was shown that the oral administration of dilute solutions of insulin does not cause the destruction of the hormone in the stomach and the degree of its hydrolysis caused by intestinal enzymes significantly decreases. In this case, the hormone enters the bloodstream, practically realizing the natural route of insulin in the bloodstream through the liver.

A Study to Evaluate the Efficacy and Safety of Brain Polypeptide Solution in Mild Alzheimer's Disease

A Study to Evaluate the Efficacy and Safety of Brain Polypeptide Solution in Mild Alzheimer's Disease

Mapping the amide-I vibrations of model dipeptides with secondary structure sensitivity and amino acid residue specificity, and its application to amyloid β-peptide in aqueous solution.

Vibrational spectroscopy has been known as particularly well-suited for deciphering the polypeptide's structure. To decode structural information encoded in IR spectra, we developed amide-I frequency maps on the basis of model dipeptides to correlate the amide-I frequency of interest to the combination of the calculated secondary structure dependent amide-I frequency by using DFT method and the electrostatic potentials that projected onto the amide unit from the micro-environment within molecular mechanics force field. The constructed maps were applied to model dipeptides and amyloid β-peptide fragment (Aβ25-35). The dipeptide specified map (DS map) and the hybrid map (HYB map) predicted amide-I bands of Aβ25-35 in solution satisfactorily reproduce experimental observation, and indicate the preference of forming β-sheet and random coil structure for Aβ25-35 in D2O just as the results of cluster analysis suggested. These maps with secondary structural sensitivity and amino acid residue specificity open up a way for the interpretation of amide-I vibrations and show their potentials in the understanding of molecular structure of polypeptides in solution.

Effects of H102 on the memory recognition ability and AMPK-mTOR autophagy-related pathway in AD mice

To study the effect of β-sheet blocking peptide H102 on the expression of AMPK-mTOR autophagy pathway-related protein in APP/PS1 double transgenic AD mice. Thirty male APP/PS1 transgenic male AD mice of 6 months old were randomly divided into AD group and H102 intervention group, and C57BL/6J male mice of the same age were used as control group (n=15). The mice in the HF group were administered with 5 μl (5.8 mg/kg) of H102 polypeptide solution through the nasal cavity at the same time period, and the mice in the control group and the AD group were given the same amount of blank adjuvant solution daily. The memory recognition ability was tested by a new object recognition experiment 30 days after continuous administration. Immunohistochemistry and Western blot were used to detect the expressions of phosphorylated AMP-activated protein kinase(P-AMPK),phosphorylated mammalian target of rapamycin (P-mTOR) and ratio of LC32to LC31(LC3II/I )in brain tissue. Compared with the control group, the new object recognition index (RI) of the AD group was significantly lower (P＜0.05), and the P-AMPK and LC3II/I ratios in the brain of the mice were significantly lower (P＜0.05). The expression of P-mTOR protein was increased significantly (P＜0.05). Compared with the AD group, the RI of the H102 intervention group was increased significantly (P＜0.05), and the P-AMPK and LC3II/I ratios in the brain tissue of the mice were increased significantly (P＜0.05). The expression of P-mTOR protein was decreased significantly (P＜0.05). H102 can improve the recognition and memory ability of AD mice by activating the AMPK-mTOR autophagy-related pathway.

Leinal polypeptide promotes NK cells to suppress PCa survival in vitro.

As an important component in the innate immune system, natural killer (NK) cells have been demonstrated to be clinically associated with prostate cancer (PCa) progression and castration resistance. Therefore, the development of novel agents that may enhance the cytotoxicity of NK cells possesses promising therapeutic applications. In the present study, leinal polypeptide (LP) solution was supplemented into a co-culture system of NK and PCa cells, as it was previously demonstrated that LP are able to activate NK cells, which kill PCa cells based on an MTT cell viability assay. Mechanistic dissection demonstrated that LP enhanced androgen receptor degradation, which resulted in an upregulation of MHC class I polypeptide-related sequence A (MICA) and MICB. In turn, the induced expression of MICA and MICB was able to further trigger NK cell activation, forming a positive loop between NK cells and PCa cells in the presence of LP solution.

CaCO3 crystallization in 2,3-epoxypropyltrimethylammonium chloride modified gelatin solutions

Gelatin was modified by 2,3-epoxypropyltrimethyl ammonium chloride (EPTAC), and the EPTAC modified gelatin (EPTAC-m-gelatin) was used to modulate CaCO3 crystallization by a slow gas diffusion method. The influences of concentrations of EPTAC-m-gelatin and Ca2+, reaction temperature and aging time on the crystallization of CaCO3 were investigated. The CaCO3 particles were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), powder X-ray diffraction (XRD), and thermogravimetric analysis (TG) methods. We found that the electrostatic interaction between EPTAC-m-gelatin and Ca2+, the intra- and inter-molecular interactions between EPTAC-m-gelatin molecules, and the adsorption of EPTAC-m-gelatin molecules on CaCO3 particles played a key role in the crystallization of CaCO3 with different morphologies and polymorphs. Concentrations of EPTAC-m-gelatin and Ca2+, temperature and aging time showed different influences on CaCO3 crystallization and growth. Based on the analysis of the CaCO3 particles, a mechanism of CaCO3 crystallization in EPTAC-m-gelatin solution was proposed. The work would provide new insight into the CaCO3 crystallization process in the presence of polypeptide solutions.

Revisiting the Helical Cooperativity of Synthetic Polypeptides in Solution.

Using synthetic polypeptides as a model system, the theories of helix-coil transition were developed into one of the most beautiful and fruitful subjects in macromolecular science. The classic models proposed by Schellman and Zimm-Bragg more than 50 years ago, differ in the assumption on whether the configuration of multiple helical sequences separated by random coil sections is allowed in a longer polypeptide chain. Zimm also calculated the critical chain lengths that facilitate such interrupted helices in different solvent conditions. The experimental validation of Zimm's prediction, however, was not carefully examined at that time. Herein, we synthesize a series of homopolypeptide samples with different lengths, to systematically examine their helix-coil transition and folding cooperativity in solution. We find that for longer chains, polypeptides do exist as interrupted helices with scattered coil sections even in helicogenic solvent conditions, as predicted in the Zimm-Bragg model. The critical chain lengths that facilitate such interrupted helices, however, are substantially smaller than Zimm's original estimation. The inaccuracy is in part due to an approximation that Zimm made in simplifying the calculation. But more importantly, we find there exist intramolecular interactions between different structural segments in the longer polypeptides, which are not considered in the classic helix-coil theories. As such, even the Zimm-Bragg model in its exact form cannot fully describe the transition behavior and folding cooperativity of longer polypeptides. The results suggest that long "all-helix" chains may be much less prevalent in solution than previously imagined, and a revised theory is required to accurately account for the helix-coil transition of the longer chains with potential "non-local" intramolecular interactions.

Seed-Induced Heterogeneous Cross-Seeding Self-Assembly of Human and Rat Islet Polypeptides.

Amyloid peptides can misfold and aggregate into amyloid oligomers and fibrils containing conformationally similar β-sheet structures, which are linked to the pathological hallmark of many neurodegenerative diseases. These β-sheet-rich amyloid aggregates provide common structural motifs to accelerate amyloid formation by acting as seeds. However, little is known about how one amyloid peptide aggregation will affect another one (namely, cross-seeding). In this work, we studied the cross-seeding possibility and efficiency between rat islet amyloid polypeptide (rIAPP) and human islet amyloid polypeptide (hIAPP) solution with preformed aggregates at different aggregation phases, using a combination of different biophysical techniques. hIAPP is a well-known peptide hormone that forms amyloid fibrils and induces cytotoxicity to β-cells in type 2 diabetes, whereas rIAPP is a nonaggregating and nontoxic peptide. Experimental results showed that all different preformed hIAPP aggregates can cross-seed rIAPP to promote the final fibril formation but exhibit different cross-seeding efficiencies. Evidently, hIAPP seeds preformed at a growth phase show the strongest cross-seeding potential to rIAPP, which accelerates the conformational transition from random structures to β-sheet and the aggregation process at the fibrillization stage. Homoseeding of hIAPP is more efficient in initiating and promoting aggregation than cross-seeding of hIAPP and rIAPP. Moreover, the cross-seeding of rIAPP with hIAPP at the lag phase also reduced cell viability, probably because of the formation of more toxic hybrid oligomers at the prolonged lag phase. The cross-seeding effects in this work may add new insights into the mechanistic understanding of the aggregation and coaggregation of amyloid peptides linked to different neurodegenerative diseases.