Polypeptide Structure Research Articles

Reversal of the DNA-Binding-Induced Loop L1 Conformational Switch in an Engineered Human p53 Protein

The gene encoding the p53 tumor suppressor protein, a sequence-specific DNA binding transcription factor, is the most frequently mutated gene in human cancer. Crystal structures of homo-oligomerizing p53 polypeptides with specific DNA suggest that DNA binding is associated with a conformational switch. Specifically, in the absence of DNA, loop L1 of the p53 DNA binding domain adopts an extended conformation, whereas two p53 subunits switch to a recessed loop L1 conformation when bound to DNA as a tetramer. We previously designed a p53 protein, p53FG, with amino substitutions S121F and V122G targeting loop L1. These two substitutions enhanced the affinity of p53 for specific DNA yet, counterintuitively, decreased the residency time of p53 on DNA. Here, we confirmed these DNA binding properties of p53FG using a different method. We also determined by crystallography the structure of p53FG in its free state and bound to DNA as a tetramer. In the free state, loop L1 adopted a recessed conformation, whereas upon DNA binding, two subunits switched to the extended loop L1 conformation, resulting in a final structure that was very similar to that of wild-type p53 bound to DNA. Thus, altering the apo structure of p53 changed its DNA binding properties, even though the DNA-bound structure was not altered.

Rapid Generation of Amyloid from Native Proteins <em>In vitro</em>

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Immunochemistry of adenoviruses: Limitations and new horizons of gene therapy

Adenoviruses have increasingly been recognized as significant viral pathogens causing high morbidity and mortality especially among immunocompromised individuals such as transplant recipients and AIDS patients. Through the infection process, after the adenovirus fiber and penton are bonded to cell surface receptors through special amino acid moieties, secondary messengers activate protein kinases, pro-inflammatory cytokines and chemokines. Serotype and species specific antibodies also are induced. Recombinant human adenoviruses have been pivotal in the development of gene therapy strategies and have shown a great promise for the treatment of genetic disorders and malignancies. Recent studies have enlightened their harmful immunological effects dependent on fiber and hexon polypeptide structure and receptor binding. Pre-existing antibodies or those elicited by vectors neutralize input recombinant adenovirus particles rendering them ineffective. Mediators induce serious even lethal side effects and cytotoxic reactions which extinguish transgene expression. To overcome these difficulties new strategies are required in the application of recombinant adenoviruses to redirect vector entry from the natural receptors to alternative binding sites or using rare human or animal adenovirus fiber molecules to modify the native fiber structure by altering amino acid structure and creating chimeric fibers. This requires searching for, isolating and characterizing new serotypes, mutants or variants for new generation vectors. Human adenovirus 1 feline isolate (feline adenovirus) might fulfil these criteria.

Exploring Fold Space Preferences of New-born and Ancient Protein Superfamilies

The evolution of proteins is one of the fundamental processes that has delivered the diversity and complexity of life we see around ourselves today. While we tend to define protein evolution in terms of sequence level mutations, insertions and deletions, it is hard to translate these processes to a more complete picture incorporating a polypeptide's structure and function. By considering how protein structures change over time we can gain an entirely new appreciation of their long-term evolutionary dynamics. In this work we seek to identify how populations of proteins at different stages of evolution explore their possible structure space. We use an annotation of superfamily age to this space and explore the relationship between these ages and a diverse set of properties pertaining to a superfamily's sequence, structure and function. We note several marked differences between the populations of newly evolved and ancient structures, such as in their length distributions, secondary structure content and tertiary packing arrangements. In particular, many of these differences suggest a less elaborate structure for newly evolved superfamilies when compared with their ancient counterparts. We show that the structural preferences we report are not a residual effect of a more fundamental relationship with function. Furthermore, we demonstrate the robustness of our results, using significant variation in the algorithm used to estimate the ages. We present these age estimates as a useful tool to analyse protein populations. In particularly, we apply this in a comparison of domains containing greek key or jelly roll motifs.

Complex coacervation in gelatin/sodium caseinate mixtures

Gelatin is a protein widely used in food, pharmaceutical and cosmetic products as an emulsifier, peptizer, thickener, and binder. Polypeptide structure of gelatin molecule facilitates its interactions with other oppositely charged ingredients often present in such products. Sodium caseinate (NaCN) due to its excellent functional and nutritional properties, is also very often used as a hydrophilic emulsifier in a wide range of dispersed systems together with other proteins, like gelatin. Since both proteins are polyelectrolytes, different type of interactions in such systems may occur. In the present study, turbidimetric, viscosimetric, surface tension, zeta potential and rheological measurements were performed in order to investigate the interactions in the mixtures of those two proteins. All these measurements showed presence of interaction between gelatin and NaCN that resulted in insoluble complex coacervate formation at certain proteins ratio.

Determination of polymer log D distributions by micellar and microemulsion electrokinetic chromatography

The characterization of the hydrophobicity of polymer compounds in solution remains a challenging issue of importance, especially for biomedical or pharmaceutical applications. To our knowledge, there is no data of polymer hydrophobicity (log D) in the literature. In this work, for the first time, the log D distributions of cationic polymers were characterized using micellar or microemulsion electrokinetic chromatography at physiological pH. The log D distributions of the polymer samples were obtained from the electrophoretic/chromatographic retardation of the polymer derivatives in presence of neutral micelles (or neutral microemulsion), using small cationic molecules for calibration. Separating electrolytes were based on a TRIS–chloride buffer containing a neutral surfactant (polyoxyethyleneglycol dodecyl ether) for the formation of micelles (in water) or microemulsion (in water/n-pentanol mixture). The log D distributions obtained at pH 7.4 using this method were in good agreement with the chemical structures of cationic polypeptides: poly(lys, phe) 1:1>poly(lys, tyr) 1:1>poly(lys, trp) 4:1>poly(lys, ser) 3:1>poly(l-lysine), where x:y represents the molar ratio of each amino acid in the copolymer. Weight average octanol–water log D values and the dispersion of the log D distribution were also defined and determined for each polymer sample.

The ATP Costs and Time Required to Degrade Ubiquitinated Proteins by the 26 S Proteasome

The degradation of ubiquitinated proteins by 26 S proteasomes requires ATP hydrolysis. To investigate if the six proteasomal ATPases function independently or in a cyclic manner, as proposed recently, we used yeast mutants that prevent ATP binding to Rpt3, Rpt5, or Rpt6. Although proteasomes contain six ATPase subunits, each of these single mutations caused a 66% reduction in basal ATP hydrolysis, and each blocked completely the 2-3-fold stimulation of ATPase activity induced by ubiquitinated substrates. Therefore, the ATPase subunits must function in a ordered manner, in which each is required for the stimulation of ATPase activity by substrates. Although ATP is essential for multiple steps in proteasome function, when the rate of ATP hydrolysis was reduced incrementally, the degradation of Ub5-DHFR (where Ub is ubiquitin and DHFR is dihydrofolate reductase) decreased exactly in parallel. This direct proportionality implies that a specific number of ATPs is consumed in degrading a ubiquitinated protein. When the ubiquitinated DHFR was more tightly folded (upon addition of the ligand folate), the rate of ATP hydrolysis was unchanged, but the time to degrade a Ub5-DHFR molecule (∼13 s) and the energy expenditure (50-80 ATPs/Ub5-DHFR) both increased by 2-fold. With a mutation in the ATPase C terminus that reduced gate opening into the 20 S proteasome, the energy costs and time required for conjugate degradation also increased. Thus, different ubiquitin conjugates activate similarly the ATPase subunit cycle that drives proteolysis, but polypeptide structure determines the time required for degradation and thus the energy cost.

Geometry motivated alternative view on local protein backbone structures

We present an alternative to the classical Ramachandran plot (R-plot) to display local protein backbone structure. Instead of the (φ, ψ)-backbone angles relating to the chemical architecture of polypeptides generic helical parameters are used. These are the rotation or twist angle ϑ and the helical rise parameter d. Plots with these parameters provide a different view on the nature of local protein backbone structures. It allows to display the local structures in polar (d, ϑ)-coordinates, which is not possible for an R-plot, where structural regimes connected by periodicity appear disconnected. But there are other advantages, like a clear discrimination of the handedness of a local structure, a larger spread of the different local structure domains--the latter can yield a better separation of different local secondary structure motives--and many more. Compared to the R-plot we are not aware of any major disadvantage to classify local polypeptide structures with the (d, ϑ)-plot, except that it requires some elementary computations. To facilitate usage of the new (d, ϑ)-plot for protein structures we provide a web application (http://agknapp.chemie.fu-berlin.de/secsass), which shows the (d, ϑ)-plot side-by-side with the R-plot.

Stabilities and structures of islet amyloid polypeptide (IAPP22–28) oligomers: From dimer to 16-mer

BackgroundThe formation of amyloid fibrils is associated with many age-related degenerative diseases. Nevertheless, the molecular mechanism that directs the nucleation of these fibrils is not fully understood. MethodsHere, we performed MD simulations for the NFGAILS motif of hIAPP associated with the type II diabetes to estimate the stabilities of hIAPP22–28 protofibrils with different sizes: from 2 to 16 chains. In addition, to study the initial self-assembly stage, 4 and 8 IAPP22–28 chains in explicit solvent were also simulated. ResultsOur results indicate that the ordered protofibrils with no more than 16 hIAPP22–28 chains will be structurally stable in two layers, while one-layer or three-layer models are not stable as expected. Furthermore, the oligomerization simulations show that the initial coil structures of peptides can quickly aggregate and convert to partially ordered β-sheet-rich oligomers. ConclusionsBased on the obtained results, we found that the stability of an IAPP22–28 oligomer was not only related with its size but also with its morphology. The driving forces to form and stabilize an oligomer are the hydrophobic effects and backbone H-bond interaction. Our simulations also indicate that IAPP22–28 peptides tend to form an antiparallel strand orientation within the sheet. General significanceOur finding can not only enhance the understanding about potential mechanisms of hIAPP nuclei formation and the extensive structural polymorphisms of oligomers, but also provide valuable information to develop potential β-sheet formation inhibitors against type II diabetes.

Comparison of structure and biological activity of natural polypeptide from velvet antlers of Cervus elaphus with those of synthesized polypeptide

Biochemical techniques including ion-exchange chromatography, gel filtration chromatography and reversed-phase high-performance liquid chromatography(RP-HPLC) were used to isolate and purify the natural polypeptide from velvet antler(nVAP) of Cervus elaphus (C. elaphus), which has a molecular weight of 3215.8 and the primary structure of VLSAADKSNVKAAWGKVGGNAPAFGAEALLRM. The homology of the protein sequence in nVAP with known protein sequence is less than 50%, suggesting that nVAP appears to be a new bioactive substance. At a level of 0.4–50 μg/mL, nVAP promotes mitosis in epidermal cells, chondrocytes and NIH3T3 fibroblasts primarily cultured in a significant way. Given that a yield of high-purity nVAP isolated from C. elaphus is 0.001%, nVAP is artificially synthesized to prepare synthetic velvet antler polypeptide(sVAP) according to its primary structure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) of sVAP shows a single band, and its HPLC spectrum displays a single peak. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS) was used to identify sVAP to be of a molecular weight of 3200 and the consistency between primary structures of sVAP and nVAP. Bioactivity test shows that at a dose of 5–40 μg/mL, sVAP promotes the proliferation of primarily cultured epidermal cells and NIH3T3 cell line. From the traditional Chinese medicine theory, velvet antler from Cervus nippon (C. nippon) and velvet antler from C. elaphus are considered as the same medicine, but differences between biochemical base and pharmacological effect of these two velvet antlers have been observed. We compared the total polypeptide mapping of the two velvet antlers, discovering that nVAP is active polypeptide and only exists in the velvet antler of C. elaphus. sVAP is similar to nVAP in physicochemical property and biological activity. These studies extend the possible utility of sVAP to be the promising compound to prepare velvet antler polypeptide of C. elaphus.

Cover Picture: On the “Tertiary Structure” of Poly-Carbenes; Self-Assembly of sp3-Carbon-Based Polymers into Liquid-Crystalline Aggregates (Chem. Eur. J. 35/2013)

The “tertiary structure” of polycarbenes has a remarkable influence on the material properties of these synthetic polymers. Although the tertiary structure of polypeptides is known to determine important functional properties of proteins, a similar influence of supramolecular aggregation of synthetic polymers on their material properties is less established. In their Full Paper on page 11577 ff., de Bruin and co-workers describe the self-assembly of syndiotactic polycarbene polymers st-PEA into van der Waals stabilized liquid-crystalline (LC) aggregates. This behaviour is due to self-aggregation of st-PEA into triple helix aggregates and is unexpected for such a flexible sp3-carbon backbone.

The preferred conformation of dipeptides in the context of biosynthesis

Globular proteins are folded polypeptide structures comprising stretches of secondary structures (helical (α- or 310 helix type), polyproline helix or β-strands) interspersed by regions of less well-ordered structure ("random coil"). Protein fold prediction is a very active field impacting inte alia on protein engineering and misfolding studies. Apart from the many studies of protein refolding from the denatured state, there has been considerable interest in studying the initial formation of peptides during biosynthesis, when there are at the outset only a few residues in the emerging polypeptide. Although there have been many studies employing quantum chemical methods of the conformation of dipeptides, these have mostly been carried out in the gas phase or simulated water. None of these conditions really apply in the interior confines of the ribosome. In the present work, we are concerned with the conformation of dipeptides in this low dielectric environment. Furthermore, only the residue types glycine and alanine have been studied by previous authors, but we extend this repertoire to include leucine and isoleucine, position isomers which have very different structural propensities.

Natural velvet antler polypeptide conformation prediction and molecular docking study with TGF-β1 complex

Based on the chain A structures of hemoglobin (PDB code: 1HDS, 1IBE, 1FAW, 3AT5), the three dimensional (3D) structure of natural velvet antler polypeptide (nVAP) was constructed by homology modeling and molecular dynamics (MD) method. The structural rationality was further checked by Profile-3D and Procheck, both of which confirmed that the 3D structure of nVAP was reasonable. The modeled structure indicates that the stable conformation of nVAP is composed of two α-helixes. The extracellular domains of transforming growth factor-β1 receptor I (TβRI-ED) and II (TβRII-ED) were docked with nVAP, respectively. The results show that both of TβR-EDs have high affinity with nVAP which locates near the active center of TβRII-ED integrating with transforming growth factor-β1 (TGF-β1). Otherwise, nVAP can also insert near the "pre-helix extension" of TβRI-ED, which is the key domain to interact on TGF-β1 and TβRII-ED. With the perturbation of nVAP, TβRI-ED can not be recruited by TGF-β1:TβRII-ED complex rigorously. The intracellular domain of TβRI (TβRI-ID) is not phosphorylated and activated by TβRII. This study shows that nVAP prefers tethering TβRI-ED which is more crucial in TGF-β1:TβRII-ED:TβRI-ED complex. Thus nVAP can disturb the TGF-β1 binding pattern by interacting on TβRs (TβRI and TβRII), further intercepting TGF-β1 pathway downstream.

Viroporin activity and membrane topology of classic swine fever virus p7 protein

Viroporins are a group of viral proteins that participate in viral replication cycles, including modification of membrane permeability and promotion of viral release. Although biological data have been accumulated on viroporion-like proteins of other viruses belonging to family Flaviviridae, the viroporin activity and membrane topology of p7 protein from classical swine fever virus (CSFV), a member of the genus Pestivirus of the family Flaviviridae, are largely unknown. In this study, sequence analysis of the primary structure of p7 polypeptide demonstrates that p7 contains two putative transmembrane regions connected by a short hydrophilic segment. Expression of p7 protein in Escherichia coli leads to the permeabilization of bacterial cells to small molecules. The p7 protein also enhances the permeability of mammalian cells, increasing the intracellular Ca2+ concentration and the permeability of cells to the translation inhibitor Hygromycin B. This protein is an integral membrane protein and can form homo-oligomers. It mainly localizes to the ER at the early stage of the expression and can be transferred to the plasma membrane at the late stage of the expression. Detergent permeabilization assays confirmed that the p7 protein is a 2-pass transmembrane protein and its N and C termini are exposed to the ER lumen. Deletion analysis showed that amino acid residues 41–63 may be essential for the viroporin activity of the protein. Our studies demonstrate that CSFV p7 possesses properties commonly associated with viroporins, which could be a potential target for the development of a therapeutic intervention for classic swine fever virus infection.

An albumin-derived peptide scaffold capable of binding and catalysis.

We have identified a 101-amino-acid polypeptide derived from the sequence of the IIA binding site of human albumin. The polypeptide contains residues that make contact with IIA ligands in the parent protein, and eight cysteine residues to form disulfide bridges, that stabilize the polypeptide structure. Seventy-four amino acids are located in six α-helical regions, while the remaining thirty-seven amino acids form six connecting coil/loop regions. A soluble GST fusion protein was expressed in E. coli in yields as high as 4 mg/l. This protein retains the IIA fragment’s capacity to bind typical ligands such as warfarin and efavirenz and other albumin’s functional properties such as aldolase activity and the ability to direct the stereochemical outcome of a diketone reduction. This newly cloned polypeptide thus represents a valuable starting point for the construction of libraries of binders and catalysts with improved proficiency.

Stimuli‐responsive polypeptide materials prepared by ring‐opening polymerization of α‐amino acid N‐carboxyanhydrides

AbstractDesign and synthesis of biodegradable stimuli‐responsive polypeptides are important areas considering their promising applications in biomedical fields. This article summarizes the most recent progresses in the development of stimuli‐responsive polypeptide materials prepared via ring‐opening polymerization of α‐amino acid N‐carboxyanhydrides. We discuss the design, synthesis and structure‐property correlation of emerging materials including thermo‐responsive, redox‐responsive, photo‐responsive and biomolecule responsive polypeptides. Considering the unique structural features of amino acids, we try to emphasize that the thermo‐responsive properties not only depend on the amino acid structure but also rely on the secondary structures of polypeptides. © 2013 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2013

Ion cooperativity and the effect of salts on polypeptide structure – a molecular dynamics study of BBA5 in salt solutions

Using molecular dynamics simulations, we investigated effects of inorganic salts on the structure and dynamics of a short alpha,beta-polypeptide, BBA5. The simulations showed that three model salts, NaI, NaF, and Na2SO3, have very different effects on the structure of the polypeptide. The addition of NaI to the aqueous solution caused denaturation and significantly weakened hydrogen bonds of the polypeptide. Na2SO3 strengthened the hydrophobic interactions and increased hydrogen bonding of the polypeptide. Preferred binding of Na+ to the backbone carbonyl groups of BBA5 occurred in the NaI solution, consistent with the weakened protein backbone hydrogen bonds, whereas Na+ is excluded more from the vicinity of the protein backbone in the Na2SO3 solution. This difference in Na+ binding correlates well with the different propensities of the counter ions approaching the protein surface: SO3(2-) is much more strongly expelled from the protein apolar surface than I-, and demonstrates the importance of cation-anion cooperativity in affecting protein structures. The binding of the two salts to and their effects on the hydration of the protein surface depends strongly on the polarity of the latter. However, both salts reduce the flexibility of the polypeptide and the fluctuation of its hydration layer. These simulations showed that the chaotropic NaI affects protein structure mainly through a direct binding of Na+ to the backbone and I- to the protein surface. The main effect of Na2SO3 manifests in strengthening the hydrophobic interaction and consequently the hydrogen bonding of the protein, more likely through an "indirect" mechanism. In addition, the simulations showed that NaF has a similar effect as Na2SO3 (but weaker than the latter, consistent with their positions in the Hofmeister series).

Protein amyloids develop an intrinsic fluorescence signature during aggregation

We report observations of an intrinsic fluorescence in the visible range, which develops during the aggregation of a range of polypeptides, including the disease-related human peptides amyloid-β(1-40) and (1-42), lysozyme and tau. Characteristic fluorescence properties such as the emission lifetime and spectra were determined experimentally. This intrinsic fluorescence is independent of the presence of aromatic side-chain residues within the polypeptide structure. Rather, it appears to result from electronic levels that become available when the polypeptide chain folds into a cross-β sheet scaffold similar to what has been reported to take place in crystals. We use these findings to quantify protein aggregation in vitro by fluorescence imaging in a label-free manner.

Thermo-responsive “hairy-rod” polypeptides for smart antitumor drug delivery

A series of thermo-responsive “hairy-rod” polypeptides was efficiently synthesized by grafting of azide-terminated (co)polymers of 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA) or 2-(2-(2-methoxyethoxy)ethoxy)ethyl methacrylate (MEO3MA) (i.e., N3-PMEOiMA) onto poly(γ-propargyl-L-glutamate) (PPLG) through a “click” reaction. The thermo-responsiveness and secondary structure of the resultant polypeptides (i.e. PLG-g-PMEOiMA) were shown to be dependent on the MEO2MA:MEO3MA ratio in PMEOiMA side chains. PLG40-g-P(MEO2MA18-co-MEO3MA9) (P2) and PLG40-g-P(MEO2MA7-co-MEO3MA18) (P4) with lower critical solution temperatures at 25.6 and 34.8 °C, and cloud points at 26.9 and 35.6 °C in physiological saline, respectively, were selected to self-assemble into micelles for doxorubicin (DOX) loading and release. In vitro DOX release from DOX-loaded micelles could be accelerated by a decrease of temperature and pH. Confocal laser scanning microscopy and flow cytometry confirmed the efficient internalization and intracellular DOX release of DOX-loaded micelles towards HeLa cells (a human cervical carcinoma cell line). In vitro methyl thiazolyl tetrazolium assays revealed that the polypeptides were cytocompatible, and DOX-loaded micelles showed efficient cellular proliferation inhibition. Hemolysis tests indicated that micelles were hemocompatible, and the encapsulation with polypeptides significantly reduced the hemolysis ratio of DOX. Therefore, the thermo-responsive polypeptide micelles, which are stable in physiological condition while releasing payloads in the acidic intracellular microenvironment, are promising for smart drug delivery.

Synthesis of Poly-L-lysine and Its Structural Response to Environmental pH

Using L-lysine as raw material, Polylysine (PLL) was synthesized through four steps. The structures of intermediums and the final product were confirmed by IR and 1 H NMR. Furthermore, some key synthetic steps were optimized in the process. On the basis, response ability to environmental pH of PLL structures was investigated by SEM and circular dichroism spectra. Experimental results indicated that polypeptide structure of PLL has good response ability to pH of environment. Under different pH conditions, distinct morphology was formed. Under acidic conditions, PLL presented a specific coil conformation, whereas orderly pine needle was observed under alkaline conditions. Keywords polylysine; N-carboxy-(N e -benzyloxycarbonyl)-L-lysine anhydride (NCA); synthesis; pH; response