Proline periodicity modulates the self-assembly properties of elastin-like polypeptides

Elastin is a self-assembling protein of the extracellular matrix that provides tissues with elastic extensibility and recoil. The monomeric precursor, tropoelastin, is highly hydrophobic yet remains substantially disordered and flexible in solution, due in large part to a high combined threshold of proline and glycine residues within hydrophobic sequences. In fact, proline-poor elastin-like sequences are known to form amyloid-like fibrils, rich in β-structure, from solution. On this basis, it is clear that hydrophobic elastin sequences are in general optimized to avoid an amyloid fate. However, a small number of hydrophobic domains near the C terminus of tropoelastin are substantially depleted of proline residues. Here we investigated the specific contribution of proline number and spacing to the structure and self-assembly propensities of elastin-like polypeptides. Increasing the spacing between proline residues significantly decreased the ability of polypeptides to reversibly self-associate. Real-time imaging of the assembly process revealed the presence of smaller colloidal droplets that displayed enhanced propensity to cluster into dense networks. Structural characterization showed that these aggregates were enriched in β-structure but unable to bind thioflavin-T. These data strongly support a model where proline-poor regions of the elastin monomer provide a unique contribution to assembly and suggest a role for localized β-sheet in mediating self-assembly interactions.     

Rapid cross-linking of elastin-like polypeptides with (hydroxymethyl)phosphines in aqueous solution

In situ gelation of injectable polypeptide-based materials is attractive for minimally invasive in vivo implantation of biomaterials and tissue engineering scaffolds. We demonstrate that chemically cross-linked elastin-like polypeptide (ELP) hydrogels can be rapidly formed in aqueous solution by reacting lysine-containing ELPs with an organophosphorous cross-linker, beta-[tris(hydroxymethyl)phosphino]propionic acid (THPP) under physiological conditions. The mechanical properties of the cross-linked ELP hydrogels were largely modulated by the molar concentration of lysine residues in the ELP and the pH at which the cross-linking reaction was carried out. Fibroblasts embedded in ELP hydrogels survived the cross-linking process and were viable after in vitro culture for 3 days. DNA quantification of ELP hydrogels with encapsulated fibroblasts indicated that there was no significant difference in DNA content between day 0 and day 3 when ELP hydrogels were formed with an equimolar ratio of THPP and lysine residues of the ELPs. These results suggest that THPP cross-linking may be a biocompatible strategy for the in situ formation of cross-linked hydrogels.     

Cooperativity between the hydrophobic and cross-linking domains of elastin

The principal protein component of the elastic fiber found in elastic tissues is elastin, an amorphous, cross-linked biopolymer that is assembled from a high molecular weight monomer. The hydrophobic and cross-linking domains of elastin have been considered separate and independent, such that changes to one region are not thought to affect the other. However, results from these solid-state 13C NMR experiments demonstrate that cooperativity in protein folding exists between the two domain types. The sequence of the EP20-24-24 polypeptide has three hydrophobic sequences from exons 20 and 24 of the soluble monomer tropoelastin, interspersed with cross-linking domains constructed from exons 21 and 23. In the middle of each cross-linking domain is a "hinge" sequence. When this pentapeptide is replaced with alanines, as in EP20-24-24[23U], its properties are changed. In addition to the expected increase in alpha-helical content and the resulting increase in rigidity of the cross-linking domains, changes to the organization of the hydrophobic regions are also observed. Using one-dimensional CPMAS (cross-polarization with magic angle spinning) techniques, including spectral editing and relaxation measurements, evidence for a change in dynamics to both domain types is observed. Furthermore, it is likely that the methyl groups of the leucines of the hydrophobic domains are also affected by the substitution to the hinge region of the cross-linking sequences. This cooperativity between the two domain types brings new questions to the phenomenon of coacervation in elastin polypeptides and strongly suggests that functional models for the protein must include a role for the cross-linking regions.     

In situ cross-linking of elastin-like polypeptide block copolymers for tissue repair

Rapid cross-linking of elastin-like polypeptides (ELPs) with hydroxymethylphosphines (HMPs) in aqueous solution is attractive for minimally invasive in vivo implantation of biomaterials and tissue engineering scaffolds. In order to examine the independent effect of the location and number of reactive sites on the chemical cross-linking kinetics of ELPs and the mechanical properties of the resulting hydrogels, we have designed ELP block copolymers comprised of cross-linkable, hydrophobic ELP blocks with periodic Lys residues (A block) and aliphatic, hydrophilic ELP blocks with no cross-linking sites (B block); three different block architectures, A, ABA, and BABA were synthesized in this study. All ELP block copolymers were rapidly cross-linked with HMPs within several minutes under physiological conditions. The inclusion of the un-cross-linked hydrophilic block, its length relative to the cross-linkable hydrophobic block, and the block copolymer architecture all had a significant effect on swelling ratios of the cross-linked hydrogels, their microstructure, and mechanical properties. Fibroblasts embedded in the ELP hydrogels survived the cross-linking process and remained viable for at least 3 days in vitro when the gels were formed from an equimolar ratio of HMPs and Lys residues of ELPs. DNA quantification of the embedded cells indicated that the cell viability within triblock ELP hydrogels was statistically greater than that in the monoblock gels at day 3. These results suggest that the mechanical properties of ELP hydrogels and the microenvironment that they present to cells can be tuned by the design of the block copolymer architecture.     

Structural determinants for the binding of ubiquitin-like domains to the proteasome

HHR23A, a protein implicated in nucleotide excision repair, belongs to a class of proteins containing both a ubiquitin-like (Ubl) domain and one or more ubiquitin-associated (UBA) domains, suggesting a role in the ubiquitin-proteasome pathway as well. The Ubl domain binds with high affinity to the second ubiquitin-interacting motif (UIM) of the S5a subunit of the proteasome. Here we present the solution structures of the HHR23A Ubl domain, the second UIM of S5a (UIM-2), and the Ubl:S5a-UIM-2 complex. The HHR23A Ubl domain is structurally similar to ubiquitin. The S5a UIM forms an alpha-helix with an unexpected hairpin loop that contributes to the binding interface with Ubl. The molecular determinants of the Ubl-proteasome interaction are revealed by analysis of the structures, chemical shift mapping, mutant binding studies and sequence conservation.     

影响类弹性蛋白多肽自组装成微球的因素及其作用机制

影响类弹性蛋白多肽(ELPs)自组装成微球的因素较多,目前尚缺乏系统研究。以类弹性蛋白多肽[KV8F]n为对象,利用动态光散射仪测定了不同条件下其自组装成微球的粒径。结果表明:随着分子量的增加ELPs形成的微球粒径也随之增大,粒径的均一度减小;当盐浓度低于0.4 mol/L时,盐浓度的增加,微球粒径相应增加,而盐浓度高于0.4 mol/L则呈减少的趋势,但粒径均大于1.1μm;而当ELPs末端融合木聚糖酶和1,3-丙二醇氧化还原酶后,其自组装形成的微球粒径急剧减小,约为游离ELPs的1/10,分别为151.0 nm和174.2 nm。导致这种现象的原因可能是酶分子和ELPs通过静电引力相互作用后,酶分子的空间位阻妨碍了ELPs分子的聚集。     

Structural characterization of ordered domains in a hydrophobic membrane protein

A delipidized proteolipid protein fraction was purified from organic solvent extracts of bovine cerebral cortex and studied by means of diffraction, electron microscopic, and ir techniques. Special use was made of an electron diffraction procedure which minimized the electron damage to the biological specimens. The ir spectroscopy of the apoprotein fraction indicated the presence of polypeptides in extended β-conformation, possibly in the antiparallel mode of packing. Electron microscopy of the fraction, negatively stained in organic media, made apparent the presence of both ordered and amorphous material. Only the former, characterized by repeating units of about 40–45 Å in diameter and varying length, produced diffraction patterns in the selected area mode exhibiting a highly undistorted lattice. The two-dimensional cell parameters of the protein fraction were a = 4.79 Å, b = 7.20 Å, and γ = 90°. The plane group symmetry, corresponding to the systematic absences, was p 2gg, consistent with the β-pleated sheet structure of simple polypeptides.     

Aqueous two-phase system formation kinetics for elastin-like polypeptides of varying chain length

The kinetics of aqueous two-phase system (ATPS) formation for elastin-like polypeptides (ELP) with defined chemical composition and chain length was investigated by dark field microscopy in an on-chip format with a linear temperature gradient. Scattering intensities from peptide solutions in the presence and absence of sodium dodecyl sulfate (SDS) were recorded as a function of temperature and time, simultaneously. It was found that the formation of the ATPS for three ELPs of different molecular weights (36 075, 59 422, and 129 856 Da) in the absence of SDS followed a coalescence mechanism, and the rate constant and activation energy were independent of chain length. With the introduction of SDS into the ELP solutions, the rate constants were attenuated more strongly with increasing chain length. Moreover, the coalescence process in the presence of SDS showed non-Arrhenius kinetics as a function of temperature. For the two shorter ELPs, ATPS formation occurred via coalescence at all SDS concentrations and temperatures investigated. On the other hand, the coalescence process was greatly suppressed for the longest ELP at elevated temperatures and higher SDS concentrations. Under these circumstances, ATPS formation was forced to proceed via a mixed Ostwald ripening and coalescence mechanism.     

Swelling and mechanical behaviors of chemically cross-linked hydrogels of elastin-like polypeptides

Genetically engineered elastin-like polypeptides consisting of Val-Pro-Gly-X-Gly repeats, where X was chosen to be Lys every 7 or 17 pentapeptides (otherwise X was Val), were synthesized and expressed in E. coli, purified, and chemically cross-linked using tris-succinimidyl aminotriacetate to produce hydrogels. Swelling experiments indicate hydrogel mass decreases by 80-90% gradually over an approximate 50 degrees C temperature range. Gels ranged in stiffness from 0.24 to 3.7 kPa at 7 degrees C and from 1.6 to 15 kPa at 37 degrees C depending on protein concentration, lysine content, and molecular weight. Changes in gel stiffness and loss angle with cross-linking formulation suggest a low-temperature gel structure that is nearly completely elastic, where force is transmitted almost exclusively through fully extended polypeptide chains and chemical cross-links, and a high-temperature gel structure, where ELP chains are contracted and force is transmitted through chemical cross-links as well as frictional contact between polypeptide chains.     

类弹性蛋白多肽的从头设计、非色谱纯化及盐效应

旨在克隆、表达与纯化类弹性蛋白多肽,并测定类弹性蛋白的相变温度对不同的盐敏感程度。从头设计了类弹性蛋白多肽的序列并人工合成其编码基因片段,克隆至改造后的表达载体pET-22b中,构建重组表达载体,转化至大肠杆菌BL21(DE3) 中并诱导表达,采用可逆相变循环经高速离心对其进行纯化,并考察了盐类型及浓度对类弹性蛋白相变温度的影响。结果表明：0.4 mmol/L的Na2CO3能使25 μmol/L类弹性蛋白多肽 [KV8F-20] 相变温度降低至19 ℃,此类弹性蛋白多肽序列有望开发成一新型纯化标签,为今后     

类弹性蛋白多肽的从头设计、非色谱纯化及盐效应

近年来,因病毒侵害人类每年都要蒙受巨大的经济损失和社会损失。犬肾细胞MDCK以其具有的培养容易、增殖快、流感病毒感染效率高等特点,成为适用于流感病毒疫苗生产的重要细胞系之一。以MDCK细胞为研究对象,在自制无血清培养基中成功实现了MDCK细胞从有血清培养到无血清培养的驯化;并通过单细胞悬浮培养驯化过程实现了MDCK细胞的无血清单细胞悬浮培养,获得了适于无血清单细胞悬浮生长的ssf-MDCK细胞株,无血清单细胞悬浮批培养最大活细胞密度可达3.81×106 cells/mL,最大比生长速率可达0.056 h?     

Mean-square hydrophobic moment for partially helical polypeptides

A mean-square helical hydrophobic moment, 〈h²〉, is defined for polypeptides in analogy to the mean-square dipole moment, 〈μ²〉, for polymer chains. For a freely jointed polymer chain, 〈μ²〉 is given by Σm, where m_i denotes the dipole moment associated with bond i. In the absence of any correlations in the hydrophobic moments of individual amino acid residues in the helix, 〈h²〉 is specified by ΣH, where H_i denotes the hydrophobicity of residue i. The tendency for correlations in orientations of residue hydrophobic moments in helices therefore dictates the size of 〈h²〉/〈H²〉, where 〈H²〉 denotes the average value of ΣH for all helices. The value of 〈h²〉/〈H²〉 will be greater than one in amphiphilic helices. A necessary prerequisite for this diagnostic usage of 〈h²〉/〈H²〉 is that the residue hydrophobic moment be oriented prependicular to the principal axis of the helix. Matrix-generation schemes are formulated that permit rapid evaluation of 〈h²〉 and 〈H²〉. The behavior of 〈h²〉/〈H²〉 is illustrated by calculations performed for model sequential copolypeptides.     

An infrared spectroscopic study of the conformational transition of elastin-like polypeptides

The infrared spectroscopy of elastin-like polypeptides and the relation to the inverse thermal transition are discussed. To correlate the spectroscopic observations with structure a density function theory model was created that captures the essential hydrogen bonding and packing of the beta-spiral structure proposed for elastin and elastin-like polypeptides. The infrared spectrum was calculated using periodic boundary conditions and a method for estimating the difference dipole moment permits both frequencies and intensities to be obtained for the modeling of spectra. The two observed amide I bands at 1615 cm(-1) and 1656 cm(-1) are shown to arise from the beta-spiral structure. The increase in intensity of these bands with increasing salt concentration and temperature is assigned to the closer association of strands of the beta-spiral. The sharp inverse temperature transition is observed within 1 degrees C and involves a change in secondary structure that involves formation of interstrand beta-sheets for approximately 25% of the amino acids. This conclusion is consistent with available data and simulations that have been reported to date.     

Improved non-chromatographic purification of a recombinant protein by cationic elastin-like polypeptides

This paper reports an improvement in the purification of thioredoxin (Trx) expressed from E. coli by inverse transition cycling (ITC) using cationic elastin-like polypeptides (ELPs). Two ELP libraries having 2% and 5% lysine residues and molecular weights ranging from 4 to 61.1 kDa showed greater salt sensitivity in their inverse transition behavior than purely aliphatic ELPs. Expression yield of Trx-ELP fusions was an unpredictable function of guest residue composition, but reducing the molecular weight of the ELP tag generally increased Trx yield. A cationic 4.3 kDa ELP is the shortest ELP used to purify any protein by ITC to date. A 15.9 kDa ELP with a guest residue composition of K:V:F of 1:7:1 was found to be the optimal cationic tag to purify Trx, as it provided 50% greater Trx yield and only required one-fifth the added NaCl for purification of Trx as compared to previously used aliphatic ELP tags.     

Secretion of elastin-like polypeptides with different transition temperatures by Pichia pastoris

Like natural tropoelastin, polypeptides based on an elastin-like VPGXG repeat have a characteristic inverse temperature response, which leads to coacervate formation above a certain transition temperature and which could be useful for a variety of applications. The key advantage of elastin-like polypeptides (ELPs) over (tropo)elastin is a full control over this temperature response by adjustment of either the amino acid composition or the chain length, according to insights provided by extensive research. Future application of ELPs will require efficient ELP production systems, and in a previous article, we described the successful use of Pichia pastoris for secreted production of an ELP, with an overall yield of ≈ 200 mg L(-1). In this study, we investigated the influence of changed amino acid composition and chain length on the yield of secreted ELP. We have found that both parameters have a distinct impact on the overall yield, with higher yield for shorter and more hydrophilic ELPs. Because yield and transition temperature (Tt) thus appear to be positively correlated, we hypothesize that good solubility of ELP below the Tt promotes the secreted production and coacervate formation above Tt decreases it.     

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.     

Comparison of thermal behavior of two recombinantly expressed human elastin-like polypeptides for cell culture applications

Two synthetic genes that code for artificial proteins have been constructed that were modeled on the most regularly repeated hydrophobic domain of human tropoelastin. We compare the physicochemical properties of the recombinant products that differ in their primary structure; the alanine/lysine-rich cross-linking domains, which are highly conserved in mammalian tropoelastin, were either present or absent in the recombinant products. Both biopolymers showed thermoresponsive properties, and variations were observed that were dependent on solution conditions. Cell compatibility was assayed using the biopolymers as coating agents in culture experiments with a neuroblastoma cell line; cell adhesion and proliferation effects were evaluated. The cells were found to retain their neural differentiation potential. The data presented in our work support the usefulness of these versatile biopolymers for a variety of applications related to biotechnology and biomedicine.     

Heterologous expression and optimized one-step separation of levansucrase via elastin-like polypeptides tagging system

Elastin-like polypeptides (ELPs) undergo a reversible inverse phase transition upon a change in temperature. This thermally triggered phase transition allows for a simple and rapid means of purifying a fusion protein. Recovery of ELPs-tagged fusion protein was easily achieved by aggregation, triggered either by raising temperature or by adding salt. In this study, levansucrase has been used as a model enzyme in the development of a simple one-step purification method using ELPs. The levansucrase gene cloned from Pseudomonas aurantiaca S-4380 was tagged with various sizes of ELPs to functionally express and optimize the purification of levansucrase. One of two ELPs, ELP[V-20] or ELP[V-40], was fused at the C-terminus of the levansucrase gene. A levansucrase-ELP fusion protein was expressed in Escherichia coli DH5alpha at 37 degrees C for 18 h. The molecular masses of levansucrase-ELP[V-20] and levansucrase-ELP[V-40] were determined as 56 kDa and 65 kDa, respectively. The phase transition of levansucrase-ELP[V-20] occurred at 20 degrees C in 50 mM Tris-Cl (pH 8) buffer with 3 M NaCl added, whereas the phase transition temperature (Tt) of levansucrase-ELP[V-40] was 17 degrees C with 2 M NaCl. Levansucrase was successfully purified using the phase transition characteristics of ELPs, with a recovery yield of higher than 80%, as verified by SDS-PAGE. The specific activity was measured spectrophotometrically to be 173 U/mg and 171 U/mg for levansucrase-ELP[V-20] and levansucrase-ELP[V-40], respectively, implying that the ELP-tagging system provides an efficient one-step separation method for protein purification.     

Human elastin-like polypeptides as a versatile platform for exploitation of ultrasensitive bilirubin detection by UnaG

A new, bifunctional recombinant protein was expressed as the fusion product of human elastin-like polypeptide (HELP) and the bilirubin-binding protein UnaG. The engineered product displays both the HELP-specific property of forming a functional hydrogel matrix and the UnaG-specific capacity of emitting green fluorescence upon ligand binding. The new fusion protein has been proven to be effective at detecting bilirubin in complex environments with high background noise. A cell culture model of the stress response, consisting of bilirubin released in the cell culture medium, was set up to assess the bilirubin-sensing properties of the functional matrix obtained by cross-linking the HELP moiety. Our engineered protein allowed us to monitor cell induction by the release of bilirubin in the culture medium on a nanomolar scale. This study shows that elastin-like protein fusion represents a versatile platform for the development of novel and commercially viable analytical and biosensing devices.     

Structural domains of the poliovirus polyprotein are major determinants for proteolytic cleavage at Gln-Gly pairs

The processing of poliovirus precursor polypeptides provides a valuable system in which to study the recognition and interaction of a proteolytic enzyme with its substrates. Processing of the poliovirus polyprotein includes cleavage between 9 of 13 available glutamineglycine (Q-G) pairs by the activity of a virally encoded proteinase, 3C. In this study, we assess the importance of primary, secondary, and tertiary structural determinants in the cleavage at two Q-G pairs in the capsid protein precursor, P1. Employing site-directed mutagenesis of cDNA copies of poliovirus RNA, we have made specific alterations in regions of the P1 capsid precursor and have assayed the effect of these alterations on proteinase cleavage at the two Q-G pairs. We have also introduced additional Q-G pairs into P1 and demonstrated that the proteinase can recognize some of the inserted Q-G pairs as cleavage sites. By correlating the predicted three-dimensional structures and the processing phenotypes of several altered P1 precursors, we are able to rank the importance of determinants required for P1 processing. While a Q-G pair appears to be the primary determinant in proteinase recognition, the tertiary location of a Q-G pair in the precursor either allows or prevents processing at that pair. Our results also suggest that the proper folding of at least two of the three P1 beta-barrel structures is required for efficient proteinase cleavage at Q-G pairs.