Deletion of the carboxyl‐terminal residue disrupts the amino‐terminal folding,self‐association,and thermal stability of an amphipathic antimicrobial peptide

Understanding the complex relationship between amino acid sequence and protein behaviors, such as folding and self‐association, is a major goal of protein research. In the present work, we examined the effects of deleting a C‐terminal residue on the intrinsic properties of an amphapathic α‐helix of mastoparan‐B (MP‐B), an antimicrobial peptide with the sequence LKLKSIVSWAKKVL‐NH2. We used circular dichroism and nuclear magnetic resonance to demonstrate that the peptide MP‐B^[1‐13] displayed significant unwinding at the N‐terminal helix compared with the parent peptide of MP‐B, as the temperature increased when the residue at position 14 was deleted. Pulsed‐field gradient nuclear magnetic resonance data revealed that MP‐B forms a larger diffusion unit than MP‐B^[1‐13] at all experimental temperatures and continuously dissociates as the temperature increases. In contrast, the size of the diffusion unit of MP‐B^[1‐13] is almost independent of temperature. These findings suggest that deleting the flexible, hydrophobic amino acid from the C‐terminus of MP‐B is sufficient to change the intrinsic helical thermal stability and self‐association. This effect is most likely because of the modulation of enthalpic interactions and conformational freedom that are specified by this residue. Our results implicate terminal residues in the biological function of an antimicrobial peptide. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

The predominant roles of the sequence periodicity in the self‐assembly of collagen‐mimetic mini‐fibrils

Collagen fibrils represent a unique case of protein folding and self‐association. We have recently successfully developed triple‐helical peptides that can further self‐assemble into collagen‐mimetic mini‐fibrils. The 35 nm axially repeating structure of the mini‐fibrils, which is designated the d‐period, is highly reminiscent of the well‐known 67 nm D‐period of native collagens when examined using TEM and atomic force spectroscopy. We postulate that it is the pseudo‐identical repeating sequence units in the primary structure of the designed peptides that give rise to the d‐period of the quaternary structure of the mini‐fibrils. In this work, we characterize the self‐assembly of two additional designed peptides: peptide Col877 and peptide Col108rr. The triple‐helix domain of Col877 consists of three pseudo‐identical amino acid sequence units arranged in tandem, whereas that of Col108rr consists of three sequence units identical in amino acid composition but different in sequence. Both peptides form stable collagen triple helices, but only triple helices Col877 self‐associate laterally under fibril forming conditions to form mini‐fibrils having the predicted d‐period. The Co108rr triple helices, however, only form nonspecific aggregates having no identifiable structural features. These results further accentuate the critical involvement of the repeating sequence units in the self‐assembly of collagen mini‐fibrils; the actual amino acid sequence of each unit has only secondary effects. Collagen is essential for tissue development and function. This novel approach to creating collagen‐mimetic fibrils can potentially impact fundamental research and have a wide range of biomedical and industrial applications.     

High stability of self‐assembled peptide nanowires against thermal,chemical, and proteolytic attacks

Understanding the self‐assembly of peptides into ordered nanostructures is recently getting much attention since it can provide an alternative route for fabricating novel bio‐inspired materials. In order to realize the potential of the peptide‐based nanofabrication technology, however, more information is needed regarding the integrity or stability of peptide nanostructures under the process conditions encountered in their applications. In this study, we investigated the stability of self‐assembled peptide nanowires (PNWs) and nanotubes (PNTs) against thermal, chemical, proteolytic attacks, and their conformational changes upon heat treatment. PNWs and PNTs were grown by the self‐assembly of diphenylalanine (Phe–Phe), a peptide building block, on solid substrates at different chemical atmospheres and temperatures. The incubation of diphenylalanine under aniline vapor at 150°C led to the formation of PNWs, while its incubation with water vapor at 25°C produced PNTs. We analyzed the stability of peptide nanostructures using multiple tools, such as electron microscopy, thermal analysis tools, circular dichroism, and Fourier‐transform infrared spectroscopy. Our results show that PNWs are highly stable up to 200°C and remain unchanged when incubated in aqueous solutions (from pH 1 to 14) or in various chemical solvents (from polar to non‐polar). In contrast, PNTs started to disintegrate even at 100°C and underwent a conformational change at an elevated temperature. When we further studied their resistance to a proteolytic environment, we discovered that PNWs kept their initial structure while PNTs fully disintegrated. We found that the high stability of PNWs originates from their predominant β‐sheet conformation and the conformational change of diphenylalanine nanostructures. Our study suggests that self‐assembled PNWs are suitable for future nano‐scale applications requiring harsh processing conditions. Biotechnol. Bioeng. 2010; 105: 221–230. © 2009 Wiley Periodicals, Inc.     

Structural variation manipulates the differential oxidative susceptibility and conformational stability of apolipoprotein E isoforms

A growing amount of evidence implicates the involvement of apolipoprotein E (apoE) in the development of late-onset and sporadic forms of Alzheimer's disease (AD). It is now generally believed that the epsilon4 allele is associated with AD and the oxidative stress is more pronounced in AD. However, only limited data are available on apoE isoform-specificity and its relationship to both the oxidative susceptibility and conformational stability of apoE. In this article, we use site-directed mutagenesis to investigate the structural role of amino acid residue 112, which is the only differing residue between apoE3 and E4. We examine the structural variation manipulating the oxidative susceptibility and conformational stability of apolipoprotein E isoforms. Arg112 in apoE4 was changed to Ala and Glu. Previous research has reported that apoE4 is more susceptible to free radicals than apoE3. In protein oxidation experiments, apoE4-R112A becomes more resistant to free radicals to the same extent as apoE3. In contrast, apoE4-R112E becomes the most susceptible protein to free radicals among all the apoE proteins. We also examine the conformational stability and the quaternary structural change by fluorescence spectroscopy and analytical ultracentrifugation, respectively. ApoE3 and E4 show apparent three- and two-state unfolding patterns, respectively. ApoE4-R112A, similar to apoE3, demonstrates a biphasic denaturation with an intermediate that appears. The denaturation curve for apoE4-R112E, however, also displays a biphasic profile but with a slight shoulder at approximately 1.5M GdmCl, implying that an unstable intermediate existed in the denaturation equilibrium. The size distribution of apoE isoforms display similar patterns. ApoE4-R112E, however, has a greater tendency to dissociate from high-molecular-weight species to tetramers. These experimental data suggest that the amino acid residue 112 governs the differences in salt-bridges between these two isoforms and thus has a significant impact on the free radical susceptibility and structural variation of the apoE isoforms.     

Structure and conformational stability of a tetrameric thermostable N‐succinylamino acid racemase

The N‐succinylamino acid racemases (NSAAR) belong to the enolase superfamily and they are large homooctameric/hexameric species that require a divalent metal ion for activity. We describe the structure and stability of NSAAR from Geobacillus kaustophilus (GkNSAAR) in the absence and in the presence of Co²⁺ by using hydrodynamic and spectroscopic techniques. The Co²⁺, among other assayed divalent ions, provides the maximal enzymatic activity at physiological pH. The protein seems to be a tetramer with a rather elongated shape, as shown by AU experiments; this is further supported by the modeled structure, which keeps intact the largest tetrameric oligomerization interfaces observed in other homooctameric members of the family, but it does not maintain the octameric oligomerization interfaces. The native functional structure is mainly formed by α‐helix, as suggested by FTIR and CD deconvoluted spectra, with similar percentages of structure to those observed in other protomers of the enolase superfamily. At low pH, the protein populates a molten‐globule‐like conformation. The GdmCl denaturation occurs through a monomeric intermediate, and thermal denaturation experiments indicate a high thermostability. The presence of the cofactor Co²⁺ did alter slightly the secondary structure, but it did not modify substantially the stability of the protein. Thus, GkNSAAR is one of the few members of the enolase family whose conformational propensities and stability have been extensively characterized. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 757–772, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Effect of acidic pH on the stability of α‐synuclein dimers

Environmental factors, such as acidic pH, facilitate the assembly of α‐synuclein (α‐Syn) in aggregates, but the impact of pH on the very first step of α‐Syn aggregation remains elusive. Recently, we developed a single‐molecule approach that enabled us to measure directly the stability of α‐Syn dimers. Unlabeled α‐Syn monomers were immobilized on a substrate, and fluorophore‐labeled monomers were added to the solution to allow them to form dimers with immobilized α‐Syn monomers. The dimer lifetimes were measured directly from the fluorescence bursts on the time trajectories. Herein, we applied the single‐molecule tethered approach for probing of intermolecular interaction to characterize the effect of acidic pH on the lifetimes of α‐Syn dimers. The experiments were performed at pH 5 and 7 for wild‐type α?Syn and for two mutants containing familial type mutations E46K and A53T. We demonstrate that a decrease of pH resulted in more than threefold increase in the α‐Syn dimers lifetimes with some variability between the α‐Syn species. We hypothesize that the stabilization effect is explained by neutralization of residues 96–140 of α‐Syn and this electrostatic effect facilitates the association of the two monomers. Given that dimerization is the first step of α‐Syn aggregation, we posit that the electrostatic effect thereby contributes to accelerating α‐Syn aggregation at acidic pH. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 715–724, 2016.     

Nonspecific shielding of unfavorable electrostatic intramolecular interactions in the erythropoietin native-state increase conformational stability and limit non-native aggregation

Previous equilibrium and kinetic folding studies of the glycoprotein erythropoietin indicate that sodium chloride increases the conformational stability of this therapeutically important cytokine, ostensibly by stabilizing the native-state [Banks DD, (2011) The Effect of Glycosylation on the Folding Kinetics of Erythropoietin. J Mol Biol 412:536–550]. The focus of the current report is to determine the underlying cause of the salt dependent increase in erythropoietin conformational stability and to understand if it has any impact on aggregation, an instability that remains a challenge to the biotech industry in maintaining the efficacy and shelf-life of protein therapeutics. Isothermal urea denaturation experiments conducted at numerous temperatures in the absence and presence of sodium chloride indicated that salt stabilizes erythropoietin primarily by increasing the difference in enthalpy between the native and unfolded sates. This result, and the finding that the salt induced increases in erythropoietin melting temperatures were independent of the identity of the salt cation and anion, indicates that salt likely increases the conformational stability of erythropoietin at neutral pH by nonspecific shielding of unfavorable electrostatic interaction(s) in the native-state. The addition of salt (even low concentrations of the strong chaotrope salt guanidinium hydrochloride) also exponentially decreased the initial rate of soluble erythropoietin non-native aggregation at 37 °C storage.     

Modeling the competition between aggregation and self‐assembly during virus‐like particle processing

Understanding and controlling aggregation is an essential aspect in the development of pharmaceutical proteins to improve product yield, potency and quality consistency. Even a minute quantity of aggregates may be reactogenic and can render the final product unusable. Self‐assembly processing of virus‐like particles (VLPs) is an efficient method to quicken the delivery of safe and efficacious vaccines to the market at low cost. VLP production, as with the manufacture of many biotherapeutics, is susceptible to aggregation, which may be minimized through the use of accurate and practical mathematical models. However, existing models for virus assembly are idealized, and do not predict the non‐native aggregation behavior of self‐assembling viral subunits in a tractable nor useful way. Here we present a mechanistic mathematical model describing VLP self‐assembly that accounts for partitioning of reactive subunits between the correct and aggregation pathways. Our results show that unproductive aggregation causes up to 38% product loss by competing favorably with the productive nucleation of self‐assembling subunits, therefore limiting the availability of nuclei for subsequent capsid growth. The protein subunit aggregation reaction exhibits an apparent second‐order concentration dependence, suggesting a dimerization‐controlled agglomeration pathway. Despite the plethora of possible assembly intermediates and aggregation pathways, protein aggregation behavior may be predicted by a relatively simple yet realistic model. More importantly, we have shown that our bioengineering model is amenable to different reactor formats, thus opening the way to rational scale‐up strategies for products that comprise biomolecular assemblies. Biotechnol. Bioeng. 2010;107: 550–560. © 2010 Wiley Periodicals, Inc.     

The effect of end‐group substitution on surface self‐assembly of peptides

Biofouling, the undesirable accumulation of organisms onto surfaces, affects many areas including health, water, and energy. We previously designed a tripeptide that self‐assembles into a coating that prevents biofouling. The peptide comprises three amino acids: DOPA, which allows its adhesion to the surface, and two fluorinated phenylalanine residues that direct its self‐assembly into a coating and acquire it with antifouling properties. This short peptide has an ester group at its C‐terminus. To examine the importance of this end group for the self‐assembly and antifouling properties of the peptide, we synthesized and characterized tripeptides with different end groups (ester, amide, or carboxylic group). Our results indicate that different groups at the C‐terminus of the peptide can lead to a change in the peptide assembly on the surface and its adsorption process. However, this change only affects the antifouling properties of the coating toward Gram‐positive bacteria (Staphylococcus epidermidis), whereas Gram‐negative bacteria (Escherichia coli) are not affected.     

Self‐association of TPR domains: Lessons learned from a designed,consensus‐based TPR oligomer

The tetratricopeptide repeat (TPR) motif is a protein–protein interaction module that acts as an organizing centre for complexes regulating a multitude of biological processes. Despite accumulating evidence for the formation of TPR oligomers as an additional level of regulation there is a lack of structural and solution data explaining TPR self‐association. In the present work we characterize the trimeric TPR‐containing protein YbgF, which is linked to the Tol system in Gram‐negative bacteria. By subtracting previously identified TPR consensus residues required for stability of the fold from residues conserved across YbgF homologs, we identified residues involved in oligomerization of the C‐terminal YbgF TPR domain. Crafting these residues, which are located in loop regions between TPR motifs, onto the monomeric consensus TPR protein CTPR3 induced the formation of oligomers. The crystal structure of this engineered oligomer shows an asymmetric trimer where stacking interactions between the introduced tyrosines and displacement of the C‐terminal hydrophilic capping helix, present in most TPR domains, are key to oligomerization. Asymmetric trimerization of the YbgF TPR domain and CTPR3Y3 leads to the formation of higher order oligomers both in the crystal and in solution. However, such open‐ended self‐association does not occur in full‐length YbgF suggesting that the protein's N‐terminal coiled‐coil domain restricts further oligomerization. This interpretation is borne out in experiments where the coiled‐coil domain of YbgF was engineered onto the N‐terminus of CTPR3Y3 and shown to block self‐association beyond trimerization. Our study lays the foundations for understanding the structural basis for TPR domain self‐association and how such self‐association can be regulated in TPR domain‐containing proteins. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Morphology of self‐assembled structures formed by short peptides from the amyloidogenic protein tau depends on the solvent in which the peptides are dissolved

The aggregation behavior of peptides Ac‐VQIVYK‐amide (AcPHF6) and Ac‐QIVYK‐amide (AcPHF5) from the amyloidogenic protein tau was examined by atomic force microscopy (AFM) and fluorescence microscopy. Although AcPHF5 did not show enhancement of thioflavin T (ThT) fluorescence in aqueous buffer, distinct aggregates were discernible when peptide was dissolved in organic solvents such as methanol (MeOH), trifluoroethanol (TFE), and hexafluoroisopropanol (HFIP) dried on mica and examined by AFM. Self‐association was evident even though the peptide did not have the propensity to form secondary structures in the organic solvents. In dried films, the peptide adopts predominantly β‐conformation which results in the formation of distinct aggregates. ThT fluorescence spectra and fluorescence images indicate the formation of fibrils when AcPHF6 solutions in organic solvents were diluted into buffer. AcPHF6 had the ability to organize into fibrillar structures when AFM samples were prepared from peptide dissolved in MeOH, TFE, HFIP, and also when diluted into buffer. AcPHF6 showed propensity for β‐structure in aqueous buffer. In MeOH and TFE, AcPHF6 showed helical and β‐structure. Morphology of the fibrils was dependent on peptide conformation in the organic solvents. The structures observed for AcPHF6 are formed rapidly and long incubation periods in the solvents are not necessary. The structures with varying morphologies observed for AcPHF5 and AcPHF6 appear to be mediated by surfaces such as mica and the organic solvents used for dissolution of the peptides. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Coevolutionary constraints in the sequence‐space of macromolecular complexes reflect their self‐assembly pathways

Is the order in which biomolecular subunits self‐assemble into functional macromolecular complexes imprinted in their sequence‐space? Here, we demonstrate that the temporal order of macromolecular complex self‐assembly can be efficiently captured using the landscape of residue‐level coevolutionary constraints. This predictive power of coevolutionary constraints is irrespective of the structural, functional, and phylogenetic classification of the complex and of the stoichiometry and quaternary arrangement of the constituent monomers. Combining this result with a number of structural attributes estimated from the crystal structure data, we find indications that stronger coevolutionary constraints at interfaces formed early in the assembly hierarchy probably promotes coordinated fixation of mutations that leads to high‐affinity binding with higher surface area, increased surface complementarity and elevated number of molecular contacts, compared to those that form late in the assembly. Proteins 2017; 85:1183–1189. © 2017 Wiley Periodicals, Inc.     

Ion‐specific modulation of protein interactions: Anion‐induced,reversible oligomerization of a fusion protein

Ions can significantly modulate the solution interactions of proteins. We aim to demonstrate that the salt-dependent reversible heptamerization of a fusion protein called peptibody A or PbA is governed by anion-specific interactions with key arginyl and lysyl residues on its peptide arms. Peptibody A, an E. coli expressed, basic (pI = 8.8), homodimer (65.2 kDa), consisted of an IgG1-Fc with two, C-terminal peptide arms linked via penta-glycine linkers. Each peptide arm was composed of two, tandem, active sequences (SEYQGLPPQGWK) separated by a spacer (GSGSATGGSGGGASSGSGSATG). PbA was monomeric in 10 mM acetate, pH 5.0 but exhibited reversible self-association upon salt addition. The sedimentation coefficient (s_w) and hydrodynamic diameter (D_H) versus PbA concentration isotherms in the presence of 140 mM NaCl (A5N) displayed sharp increases in s_w and D_H, reaching plateau values of 9 s and 16 nm by 10 mg/mL PbA. The D_H and sedimentation equilibrium data in the plateau region (>12 mg/mL) indicated the oligomeric ensemble to be monodisperse (PdI = 0.05) with a z-average molecular weight (M_z) of 433 kDa (stoichiometry = 7). There was no evidence of reversible self-association for an IgG1-Fc molecule in A5N by itself or in a mixture containing fluorescently labeled IgG1-Fc and PbA, indicative of PbA self-assembly being mediated through its peptide arms. Self-association increased with pH, NaCl concentration, and anion size (I⁻ > Br⁻ > Cl⁻ > F⁻) but could be inhibited using soluble Trp-, Phe-, and Leu-amide salts (Trp > Phe > Leu). We propose that in the presence of salt (i) anion binding renders PbA self-association competent by neutralizing the peptidyl arginyl and lysyl amines, (ii) self-association occurs via aromatic and hydrophobic interactions between the..xx..xxx..xx.. motifs, and (iii) at >10 mg/mL, PbA predominantly exists as heptameric clusters.     

Loss of intramolecular electrostatic interactions and limited conformational ensemble may promote self‐association of cis–tau peptide

Self‐association of proteins can be triggered by a change in the distribution of the conformational ensemble. Posttranslational modification, such as phosphorylation, can induce a shift in the ensemble of conformations. In the brain of Alzheimer's disease patients, the formation of intra‐cellular neurofibrillary tangles deposition is a result of self‐aggregation of hyper‐phosphorylated tau protein. Biochemical and NMR studies suggest that the cis peptidyl prolyl conformation of a phosphorylated threonine‐proline motif in the tau protein renders tau more prone to aggregation than the trans isomer. However, little is known about the role of peptidyl prolyl cis/trans isomerization in tau aggregation. Here, we show that intra‐molecular electrostatic interactions are better formed in the trans isomer. We explore the conformational landscape of the tau segment containing the phosphorylated‐Thr²³¹‐Pro²³² motif using accelerated molecular dynamics and show that intra‐molecular electrostatic interactions are coupled to the isomeric state of the peptidyl prolyl bond. Our results suggest that the loss of intra‐molecular interactions and the more restricted conformational ensemble of the cis isomer could favor self‐aggregation. The results are consistent with experiments, providing valuable complementary atomistic insights and a hypothetical model for isomer specific aggregation of the tau protein. Proteins 2015; 83:436–444. © 2014 Wiley Periodicals, Inc.     

A tale of two tails: The importance of unstructured termini in the aggregation pathway of β2‐microglobulin

The identification of intermediate states for folding and aggregation is important from a fundamental standpoint and for the design of novel therapeutic strategies targeted at conformational disorders. Protein human β2‐microglobulin (HB2m) is classically associated with dialysis‐related amyloidosis, but the single point mutant D76N was recently identified as the causative agent of a hereditary systemic amyloidosis affecting visceral organs. Here, we use D76N as a model system to explore the early stage of the aggregation mechanism of HB2m by means of an integrative approach framed on molecular simulations. Discrete molecular dynamics simulations of a structured‐based model predict the existence of two intermediate states populating the folding landscape. The intermediate I₁ features an unstructured C‐terminus, while I₂, which is exclusively populated by the mutant, exhibits two unstructured termini. Docking simulations indicate that I₂ is the key species for aggregation at acidic and physiological pH contributing to rationalize the higher amyloidogenic potential of D76N relative to the wild‐type protein and the ΔN6 variant. The analysis carried out here recapitulates the importance of the DE‐loop in HB2m self‐association at a neutral pH and predicts a leading role of the C‐terminus and the adjacent G‐strand in the dimerization process under acidic conditions. The identification of aggregation hot‐spots is in line with experimental results that support the importance of Phe56, Asp59, Trp60, Phe62, Tyr63, and Tyr66 in HB2m amyloidogenesis. We further predict the involvement of new residues such as Lys94 and Trp95 in the aggregation process.     

Nanoscale elongating control of the self‐assembled protein filament with the cysteine‐introduced building blocks

Self‐assembly of artificially designed proteins is extremely desirable for nanomaterials. Here we show a novel strategy for the creation of self‐assembling proteins, named “Nanolego.” Nanolego consists of “structural elements” of a structurally stable symmetrical homo‐oligomeric protein and “binding elements,” which are multiple heterointeraction proteins with relatively weak affinity. We have established two key technologies for Nanolego, a stabilization method and a method for terminating the self‐assembly process. The stabilization method is mediated by disulfide bonds between Cysteine‐residues incorporated into the binding elements, and the termination method uses “capping Nanolegos,” in which some of the binding elements in the Nanolego are absent for the self‐assembled ends. With these technologies, we successfully constructed timing‐controlled and size‐regulated filament‐shape complexes via Nanolego self‐assembly. The Nanolego concept and these technologies should pave the way for regulated nanoarchitecture using designed proteins.     

Fmoc‐based peptide thioester synthesis with self‐purifying effect: heading to native chemical ligation in parallel formats

The chemical synthesis of proteins has facilitated functional studies of proteins due to the site‐specific incorporation of post‐translational modifications, labels, and non‐proteinogenic amino acids. Moreover, native chemical ligation provides facile access to proteins by chemical means. However, the application of the native chemical ligation reaction in the synthesis of parallel formats such as protein arrays has been complicated because of the often cumbersome and time‐consuming synthesis of the required peptide thioesters. An Fmoc‐based peptide thioester synthesis with self‐purification on the sulfonamide ‘safety‐catch’ linker widens this bottleneck because HPLC purification can be avoided. The method is based on an on‐resin cyclization–thiolysis reaction sequence. A macrocyclization via the N‐terminus of the full‐length peptide followed by a thiolytic C‐terminal ring opening allows selective detachment of the truncation products and the full‐length peptide. A brief overview of the chemical aspects of this method is provided including the optimization steps and the automation process. Furthermore, the application of the cyclization–thiolysis approach combined with the native chemical ligation reaction in the parallel synthesis of a library of 16 SH3‐domain variants of SHO1 in yeast is described, demonstrating the value of this new technique for the chemical synthesis of protein arrays. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Increasing protein stability by improving beta‐turns

Our goal was to gain a better understanding of how protein stability can be increased by improving β‐turns. We studied 22 β‐turns in nine proteins with 66–370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some β‐turn positions. We studied: Cold shock protein B (CspB), Histidine‐containing phosphocarrier protein, Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase α‐subunit, and Maltose binding protein. Of the 15 single proline mutations, 11 increased stability (Average = 0.8 ± 0.3; Range = 0.3–1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. On the basis of this and our previous work, we conclude that proteins can generally be stabilized by replacing nonproline residues with proline residues at the i + 1 position of Type I and II β‐turns and at the i position in Type II β‐turns. Other turn positions can sometimes be used if the φ angle is near ?60° for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in β‐turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in β‐turns that could be replaced by Gly to increase protein stability. Improving β‐turns by substituting Pro residues is a generally useful way of increasing protein stability. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Structural requirements essential for elastin coacervation: favorable spatial arrangements of valine ridges on the three‐dimensional structure of elastin‐derived polypeptide (VPGVG)n

The elastin precursor tropoelastin possesses a number of polymeric peptides with repeating 3–9 mer sequences. One of these is the pentapeptide Val‐Pro‐Gly‐Val‐Gly (VPGVG) present in almost all animal species, and its polymer (VPGVG)n coacervates just as does tropoelastin. In the present study, in order to explore the structural requirements essential for coacervation, (VPGVG)n and its shortened repeat analogs (VPGV)n, (VPG)n, and (PGVG)n were synthesized and their structural properties were investigated. In our turbidity measurements, (VPGVG)n demonstrated complete reversible coacervation in agreement with previous findings. The Gly⁵‐deleted polymer (VPGV)n also achieved self‐association, though the onset of self‐association occurred at a lower temperature. However, the dissociation of (VPGV)n upon temperature lowering was found to occur in a three‐step process; the Val_i⁴‐Val_i+1¹ structure arising in the VPGV polypeptide appeared to perturb the dissociation. No self‐association was observed for (VPG)n or (PGVG)n repeats. Spectroscopic measurements by CD, FT‐IR, and ¹H‐NMR showed that the (VPGV)n and (VPG)n both assumed ordered structures similar to that of (VPGVG)n. These results demonstrated that VPGVG is a structural element essential to achieving the β‐spiral structure required for self‐association followed by coacervation, probably due to the ideal spatial arrangement of the hydrophobic Val residues. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.