Wheat seed proteins exhibit a complex mechanism of protein elasticity

Elastomeric proteins are found in a number of animal tissues (elastin, abductin and resilin), where they have evolved to fulfil a range of biological functions. All exhibit rubber-like elasticity, undergoing deformation without rupture, storing the energy involved in deformation, and then recovering to their initial state when the stress is removed. The second part of the process is passive, entropy decreasing when the proteins are deformed, with the higher entropy of the relaxed state providing the driving force for recoil. In plants there is only one well-documented elastomeric protein system, the alcohol-soluble seed storage proteins (gluten) of wheat. The elastic properties of these proteins have no known biological role, the proteins acting as a store for the germinating seed. Here we show that the modulus of elasticity of a group of wheat gluten subunits, when cross-linked by gamma-radiation, is similar to that of the cross-linked polypentapeptide of elastin. However, thermoelasticity studies indicate that the mechanism of elastic recoil is different from elastin and other characterized protein elastomers. Elastomeric force, f, has two components, an internal energy component, f(e), and an entropic component, f(s). The ratio f(e)/f can be determined experimentally; if this ratio is less than 0.5 the elastomeric force is predominantly entropic in origin. The ratio was determined as 5.6 for the cross-linked high M(r) subunits of wheat glutenin and near zero for the cross-linked polypentapeptide of elastin. Tensile stress must be entropic or energetic in origin, the results would suggest that elastic recoil in the wheat gluten subunits, in part, may be associated with extensive hydrogen bonding within and between subunits and that entropic and energetic mechanisms contribute to the observed elasticity.     

Structural insights into the elastin mimetic (LGGVG)6 using solid-state 13C NMR experiments and statistical analysis of the PDB

Elastin is a crosslinked hydrophobic protein found in abundance in vertebrate tissue and is the source of elasticity in connective tissues and blood vessels. The repeating polypeptide sequences found in the hydrophobic domains of elastin have been the focus of many studies that attempt to understand the function of the native protein on a molecular scale. In this study, the central residues of the (LGGVG)(6) elastin mimetic are targeted. Using a combination of a statistical analysis based on structures in the Brookhaven Protein Data Bank (PDB), 1D cross-polarization magic-angle-spinning (CPMAS) NMR spectroscopy, and 2D off-magic-angle-spinning (OMAS) spin-diffusion experiments, it is determined that none of the residues are found in a singular regular, highly ordered structure. Instead, like the poly(VPGVG) elastin mimetics, there are multiple conformations and significant disorder. Furthermore, the conformational ensembles are not reflective of proteins generally, as in the PDB, suggesting that the structure distributions in elastin mimetics are unique to these peptides and are a salient feature of the functional model of the native protein.     

Circular dichroism studies on repeating polypeptide sequences of abductin

The secondary structure of abductin was investigated by CD and NMR studies of several synthetic peptides. Results obtained with these peptides showed the dominant conformations to be the polyproline II (PPII) structure in aqueous solution and different types of beta-turns in the less polar solvent trifluoroethanol. Accordingly, a preliminary structure-elasticity relationship for abductin, not unlike that currently accepted for elastin, is proposed.     

Proline and glycine control protein self-organization into elastomeric or amyloid fibrils

Elastin provides extensible tissues, including arteries and skin, with the propensity for elastic recoil, whereas amyloid fibrils are associated with tissue-degenerative diseases, such as Alzheimer's. Although both elastin-like and amyloid-like materials result from the self-organization of proteins into fibrils, the molecular basis of their differing physical properties is poorly understood. Using molecular simulations of monomeric and aggregated states, we demonstrate that elastin-like and amyloid-like peptides are separable on the basis of backbone hydration and peptide-peptide hydrogen bonding. The analysis of diverse sequences, including those of elastin, amyloids, spider silks, wheat gluten, and insect resilin, reveals a threshold in proline and glycine composition above which amyloid formation is impeded and elastomeric properties become apparent. The predictive capacity of this threshold is confirmed by the self-assembly of recombinant peptides into either amyloid or elastin-like fibrils. Our findings support a unified model of protein aggregation in which hydration and conformational disorder are fundamental requirements for elastomeric function.     

Entropic elastic processes in protein mechanisms. II. Simple (passive) and coupled (active) development of elastic forces

The first part of this review on entropic elastic processes in protein mechanisms (Urry, 1988) demonstrated with the polypentapeptide of elastin (Val¹-Pro²-Gly³-Val⁴-Gly⁵)_n that elastic structure develops as the result of an inverse temperature transition and that entropic elasticity is due to internal chain dynamics in a regular nonrandom structure. This demonstration is contrary to the pervasive perspective of entropic protein elasticity of the past three decades wherein a network of random chains has been considered the necessary structural consequence of the occurrence of dominantly entropic elastomeric force. That this is not the case provides a new opportunity for understanding the occurrence and role of entropic elastic processes in protein mechanisms. Entropic elastic processes are considered in two classes: passive and active. The development of elastomeric force on deformation is class I (passive) and the development of elastomeric force as the result of a chemical process shifting the temperature of a transition is class II (active). Examples of class I are elastin, the elastic filament of muscle, elastic force changes in enzyme catalysis resulting from binding processes and resulting in the straining of a scissile bond, and in the turning on and off of channels due to changes in transmembrane potential. Demonstration of the consequences of elastomeric force developing as the result of an inverse temperature transition are seen in elastin, where elastic recoil is lost on oxidation, i.e., on decreasing the hydrophobicity of the chain and shifting the temperature for the development of elastomeric force to temperatures greater than physiological. This is relevant in general to loss of elasticity on aging and more specifically to the development of pulmonary emphysema. Since random chain networks are not the products of inverse temperature transitions and the temperature at which an inverse temperature transition occurs depends on the hydrophobicity of the polypeptide chain, it now becomes possible to consider chemical processes for turning elastomeric force on and off by reversibly changing the hydrophobicity of the polypeptide chain. This is herein called mechanochemical coupling of the first kind; this is the chemical modulation of the temperature for the transition from a less-ordered less elastic state to a more-ordered more elastic state. In the usual considerations to date, development of elastomeric force is the result of a standard transition from a more-ordered less elastic state to a less-ordered more elastic state. When this is chemically modulated, it is herein called mechanochemical coupling of the second kind. For elastin and the polypentapeptide of elastin, since entropic elastomeric force results on formation of a regular nonrandom structure and thermal randomization of chains results in loss of elastic modulus to levels of limited use in protein mechanisms, consideration of regular spiral-like structures rather than ramdom chain networks or random coils are proposed for mechanochemical coupling of the second kind. Chemical processes to effect mechanochemical coupling in biological systems are most obviously phosphorylation-dephosphorylation and changes in calcium ion activity but also changes in pH. These issues are considered in the events attending parturition in muscle contraction and in cell motility.     

Elastin: a representative ideal protein elastomer

During the last half century, identification of an ideal (predominantly entropic) protein elastomer was generally thought to require that the ideal protein elastomer be a random chain network. Here, we report two new sets of data and review previous data. The first set of new data utilizes atomic force microscopy to report single-chain force-extension curves for (GVGVP)(251) and (GVGIP)(260), and provides evidence for single-chain ideal elasticity. The second class of new data provides a direct contrast between low-frequency sound absorption (0.1-10 kHz) exhibited by random-chain network elastomers and by elastin protein-based polymers. Earlier composition, dielectric relaxation (1-1000 MHz), thermoelasticity, molecular mechanics and dynamics calculations and thermodynamic and statistical mechanical analyses are presented, that combine with the new data to contrast with random-chain network rubbers and to detail the presence of regular non-random structural elements of the elastin-based systems that lose entropic elastomeric force upon thermal denaturation. The data and analyses affirm an earlier contrary argument that components of elastin, the elastic protein of the mammalian elastic fibre, and purified elastin fibre itself contain dynamic, non-random, regularly repeating structures that exhibit dominantly entropic elasticity by means of a damping of internal chain dynamics on extension.     

The structure and mechanical properties of the proteins of lamprey cartilage

The cyanogen bromide‐resistant proteins of lamprey cartilage are biochemically related to the mammalian elastic protein, elastin. This study investigates their mechanical properties and enquires whether, like elastin, long‐range elasticity arises in them from a combination of entropic and hydrophobic mechanisms. Branchial and pericardial proteins resembled elastin mechanically, with elastic moduli of 0.13–0.35 MPa, breaking strains of 50%, and low hysteresis. Annular and piston proteins had higher elastic moduli (0.27–0.75 MPa) and larger hysteresis. Exchanging solvent water for trifluoroethanol increased the elastic moduli, whereas increasing temperature lowered the elastic moduli. Raman microspectrometry showed small differences in side‐chain modes consistent with reported biochemical differences. Decomposition of the amide I band indicated that the secondary structures were like those of elastin, preponderantly unordered, which probably confer the conformational flexibility necessary for entropy elasticity. Piston and annular proteins showed the strongest interactions with water, suggesting, together with the mechanical testing data, a greater role of hydrophobic interactions in their mechanics. Two‐photon imaging of intrinsic fluorescence and dye injection experiments showed that annular and piston proteins formed closed‐cell honeycomb structures, whereas the branchial and pericardial proteins formed open‐cell structures, which may account for the differences in mechanical properties. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 187–202, 2015.     

Comparative genomics of elastin: Sequence analysis of a highly repetitive protein.

Due to the low complexity associated with their sequences, uncovering the evolutionary and functional relationships in highly repetitive proteins such as elastin, spider silks, resilin and abductin represents a significant challenge. Using the polymeric extracellular protein elastin as a model system, we present a novel computational approach to the study of sequence, function and evolutionary relationships in repetitive proteins. To address the absence of accurate sequence annotation for repetitive proteins such as elastin, we have constructed a new database repository, ElastoDB (http://theileria.ccb.sickkids.ca/elastin), dedicated to the storage and retrieval of elastin sequence- and meta-data. To analyse their sequence relationships we have devised an innovative new method, based on the identification of overrepresented 'fuzzy' motifs. Applying this method to elastin sequences derived from mammals, chicken, Xenopus and zebrafish resulted in the identification of both highly conserved, and taxon and species specific motifs that likely represent important functional and/or structural elements. The relative spacing and organization of these elements suggest that exon duplication events have played an important role in the evolution of elastin. Clustering of similarity profiles generated for sets of exons and introns, revealed a pattern of putative duplication events involving exons 15-30 in mammalian and chicken elastins, exons 20-31 in both zebrafish elastins, exons 15-20 in fugu elastin and exons 35-50 in Xenopus elastin 1. The success of this approach for elastin offers a promising route to the elucidation of sequence, structure, function and evolutionary relationships for many other proteins with sequences of low complexity.     

How sequence determines elasticity of disordered proteins

How nature tunes sequences of disordered protein to yield the desired coiling properties is not yet well understood. To shed light on the relationship between protein sequence and elasticity, we here investigate four different natural disordered proteins with elastomeric function, namely: FG repeats in the nucleoporins; resilin in the wing tendon of dragonfly; PPAK in the muscle protein titin; and spider silk. We obtain force-extension curves for these proteins from extensive explicit solvent molecular dynamics simulations, which we compare to purely entropic coiling by modeling the four proteins as entropic chains. Although proline and glycine content are in general indicators for the entropic elasticity as expected, divergence from simple additivity is observed. Namely, coiling propensities correlate with polyproline II content more strongly than with proline content, and given a preponderance of glycines for sufficient backbone flexibility, nonlocal interactions such as electrostatic forces can result in strongly enhanced coiling, which results for the case of resilin in a distinct hump in the force-extension curve. Our results, which are directly testable by force spectroscopy experiments, shed light on how evolution has designed unfolded elastomeric proteins for different functions.     

Elastomeric proteins: biological roles, structures and mechanisms

Elastomeric proteins are able to withstand significant deformations without rupture before returning to their original state when the stress is removed. Although elastomeric proteins differ considerably in their amino acid sequence, they all have a complex domain structure and share two common properties. Namely, they contain elastomeric domains, comprised of repeated sequences, and additional domains that form intermolecular crosslinks. Furthermore, several protein contain beta-turns as a structural motif within the elastomeric domains.     

A turning point in the knowledge of the structure-function-activity relations of elastin]

In this review are presented the last new results of our research group dealing with the molecular structures (atomic level) of tropoelastin, elastin and elastin derived peptides studied by using essentially methods of bioinformatics (theoretical predictions and molecular modelling) linked to experimental circular dichroism spectroscopic studies. We already had characterized both the local secondary structure and some parts of the tertiary structure of the tropoelastin and elastin molecules (human, bovine...), by using either theoretical predictions (local secondary structure, linear epitopes...) and/or experimental data (optical spectroscopic methods: Raman scattering, infrared absorption, circular dichroism). Except the cross-linking regions which are in helical conformations, the whole tropoelastin structure displays a lot of beta-reverse turns which usually belong to irregular structures in proteins. These turns play a key role in other regularly structures orientation (alpha-helix, beta-strand), thus they are very important in the native protein 3D architecture. It is particularly true for human tropoelastin, because its sequence is rich in glycines and prolines, and these residues are frequently met in beta-turns (a beta-turn is made of four consecutive residues which are stabilized by an hydrogen bond). Several types of beta-turns can be defined with the dihedral angles values phi and psi of the two central residues. Thus, by using a very recent updated set of propensities for the amino acid residues to belong to given types of reverse beta-turns (extracted from a reference set of known 3-D structures of globular proteins), we have determined, (by using our home made software COUDES), for all possible tetrapeptides of the human tropoelastin sequence, the distribution and the characterization of the possible type of turns. Thus, it is shown that the locations and/or the types of these reverse beta-turns reveal a regularity and are not all random. This confirms our hypothesis that intra-molecular elasticity of tropoelastin could be explained by the possibility of transitions between conformations involving short beta-strands and beta-turns. This result is of great interest in the construction (by using molecular biology) of elastic biomaterials derived from the elastin sequence (particularly, the elastin derived peptides corresponding to the sequence exon 21--(exon 24--exon 24...). Our study permit also to predict the conformations of specific elastin derived peptides which could have interesting biological activity. Peptides resulting from the degradation of elastin, the insoluble polymer of tropoelastin and responsible for the elasticity of vertebrate tissues, can induce biological effects and notably the regulation of matrix metalloproteinases (MMP-s) activity. Recently, it was proposed that some elastin derived hexapeptides resulting from circular permutations of VGVAPG (a three fold repetition sequence in exon 24 of human tropoelastin) possess MMP-1 production and activation regulation properties. This effect depends on the presence of the tropoelastin specific membraneous receptor 67 KDa EBP (Elastin Binding Protein). Our results obtained by using both circular dichroism spectroscopy and linear predictions confirmed the hypothesis of a structure dependent mechanism with a possibly occurring type VIII beta-turn on the first four residues of the GXXPG sequence consensus which is only present among all active peptides. Thus, we have performed extensive molecular dynamics studies, in both implicit and explicit solvent, on these active and inactive elastin derived hexapeptides. Using our own analysis method of pattern recognition of the types of the beta-reverse-turns followed during the molecular dynamics trajectory, we found that active and inactive peptides effectively form two well distinct conformational groups in which active peptides preferentially adopt conformation close to type VIII GXXP (beta-reverse-turn. The structural role of the C terminal G residue could also be explained. Additional molecular simulations on (VGVAPG)2 and (VGVAPG)3 show the formation of two or three GXXP tetrapeptides adopting a structure close to type VIII beta-reverse-turn, suggesting a local conformational preference for this motif. This observation of a specific structural single and/or repeated motif is in agreement with the circular dichroism spectra of the involved (VGVAPG)1, (VGVAPG)2 and (VGVAPG)3 peptides and then it can be proposed that their biological activities have to be linear. The final aim of this type of work is to understand more about the sequence/structure/function/activity relationships of those structured peptides in order to propose specific sequences (corresponding to specific structures) for best biological activity results.     

Spectroscopic evidence revealing polyproline II structure in hydrophobic, putatively elastomeric sequences encoded by specific exons of human tropoelastin

Elastin, the protein responsible for tissue elasticity, is contained in arterial walls, lungs, and skin. Given the cassette like organization of the human tropoelastin gene, giving rise to alternating exons encoding for crosslink domains and elastomeric domains, it is tempting to suggest that polypeptides encoded by different exons could adopt structures independent of the other exons. The results obtained with the polypeptide sequences encoded by exons 3, 7, and 30 of human tropoelastin are described. It is shown that these hydrophobic exons may partly assume the polyproline II (PPII) structure, as found by circular dichroism studies in aqueous solution. Classical Raman spectroscopy evidences a specific sharp band at 1314 cm(-1), which is assigned to the PPII structure adopted by these exons in the solid state. As these sequences are among those putatively responsible for elastomeric properties, these findings are of particular interest in relation to the current models of the elasticity of elastin.     

Expression of a new chimeric protein with a highly repeated sequence in tobacco cells

In wheat, the high-molecular weight (HMW) glutenin subunits are known to contribute to gluten viscoelasticity, and show some similarities to elastomeric animal proteins as elastin. When combining the sequence of a glutenin with that of elastin is a way to create new chimeric functional proteins, which could be expressed in plants. The sequence of a glutenin subunit was modified by the insertion of several hydrophobic and elastic motifs derived from elastin (elastin-like peptide, ELP) into the hydrophilic repetitive domain of the glutenin subunit to create a triblock protein, the objective being to improve the mechanical (elastomeric) properties of this wheat storage protein. In this study, we investigated an expression model system to analyze the expression and trafficking of the wild-type HMW glutenin subunit (GS_W) and an HMW glutenin subunit mutated by the insertion of elastin motifs (GS_M-ELP). For this purpose, a series of constructs was made to express wild-type subunits and subunits mutated by insertion of elastin motifs in fusion with green fluorescent protein (GFP) in tobacco BY-2 cells. Our results showed for the first time the expression of HMW glutenin fused with GFP in tobacco protoplasts. We also expressed and localized the chimeric protein composed of plant glutenin and animal elastin-like peptides (ELP) in BY-2 protoplasts, and demonstrated its presence in protein body-like structures in the endoplasmic reticulum. This work, therefore, provides a basis for heterologous production of the glutenin-ELP triblock protein to characterize its mechanical properties.     

Sequences and domain structures of mammalian, avian, amphibian and teleost tropoelastins: Clues to the evolutionary history of elastins

Tropoelastin is the monomeric form of elastin, a polymeric extracellular matrix protein responsible for properties of extensibility and elastic recoil in connective tissues of most vertebrates. As an approach to investigate how sequence and structural characteristics of tropoelastin assist in polymeric assembly and account for the elastomeric properties of this polymer, and to better understand the evolutionary history of elastin, we have identified and characterized tropoelastins from frog (Xenopus tropicalis) and zebrafish (Danio rerio), comparing these to their mammalian and avian counterparts. Unlike other species, two tropoelastin genes were expressed in zebrafish. All tropoelastins shared a predominant and characteristic alternating domain arrangement, as well as the fundamental crosslinking sequence motifs. However, zebrafish and frog tropoelastins had several unusual characteristics, including increased exon numbers and protein molecular weights, and decreased hydropathies. For all tropoelastins there was evidence of evolutionary expansion of the proteins by extensive replication of a hydrophobic-crosslinking exon pair. This was particularly apparent for zebrafish and frog tropoelastin genes, where remnants of sequence similarity were also seen in introns flanking the replicated exon pair. While overall alignment of mammalian, avian, frog and zebrafish tropoelastin sequences was not possible because of sequence variability, the C-terminal exon was well-conserved in all species. In addition, good sequence alignment was possible for several exons just upstream of the putative region of replication, suggesting that these conserved domains may represent 'primordial' core sequences present in the ancestral sequence common to all tropoelastins and in some way essential to the structure/function of elastin.     

Proline‐poor hydrophobic domains modulate the assembly and material properties of polymeric elastin

Elastin is a self‐assembling extracellular matrix protein that provides elasticity to tissues. For entropic elastomers such as elastin, conformational disorder of the monomer building block, even in the polymeric form, is essential for elastomeric recoil. The highly hydrophobic monomer employs a range of strategies for maintaining disorder and flexibility within hydrophobic domains, particularly involving a minimum compositional threshold of proline and glycine residues. However, the native sequence of hydrophobic elastin domain 30 is uncharacteristically proline‐poor and, as an isolated polypeptide, is susceptible to formation of amyloid‐like structures comprised of stacked β‐sheet. Here we investigated the biophysical and mechanical properties of multiple sets of elastin‐like polypeptides designed with different numbers of proline‐poor domain 30 from human or rat tropoelastins. We compared the contributions of these proline‐poor hydrophobic sequences to self‐assembly through characterization of phase separation, and to the tensile properties of cross‐linked, polymeric materials. We demonstrate that length of hydrophobic domains and propensity to form β‐structure, both affecting polypeptide chain flexibility and cross‐link density, play key roles in modulating elastin mechanical properties. This study advances the understanding of elastin sequence‐structure‐function relationships, and provides new insights that will directly support rational approaches to the design of biomaterials with defined suites of mechanical properties. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 563–573, 2015.     

Polytetrapeptide of elastin. Temperature-correlated elastomeric force and structure development

High molecular weight polytetrapeptide of elastin, (L.Val1-L.Pro2-Gly3-Gly4)n, was synthesized using activation of the (GGVP) permutation for polymerization. The temperature-dependence of aggregation was characterized as a function of concentration and the circular dichroism spectra were obtained in the 20 degrees to 70 degrees C temperature range. The latter showed an inverse temperature transition centered near 50 degrees C in which polypeptide order increased on raising the temperature. A concentration of 0.6 g of polytetrapeptide in 1 g of water was gamma irradiation cross-linked (20 Mrad) to form an elastomeric matrix. A study of the temperature-dependence of elastomeric force demonstrated a transition toward increased force on raising the temperature with a midpoint of the transition near 50 degrees C. Thus, there is a correlation between increase in intramolecular order and elastomeric force development. These results are compared to previous results on the polypentapeptide of elastin, (VPGVG)n and on an analog, (IPGVG)n, to demonstrate that the temperature of the transition is proportional to the hydrophobicity of the repeating unit. The point is noted that the elastomeric force development correlates better with intramolecular ordering than with intermolecular processes.     

Structure of the induced antibacterial protein from tasar silkworm, Antheraea mylitta. Implications to molecular evolution

The crystal structure of an antibacterial protein of immune origin (TSWAB), purified from tasar silkworm (Antheraea mylitta) larvae after induction by Escherichia coli infection, has been determined. This is the first insect lysozyme structure and represents induced lysozymes of innate immunity. The core structure of TSWAB is similar to c-type lysozymes and alpha-lactalbumins. However, TSWAB shows significant differences with respect to the other two proteins in the exposed loop regions. The catalytic residues in TSWAB are conserved with respect to the chicken lysozyme, indicating a common mechanism of action. However, differences in the noncatalytic residues in the substrate binding groove imply subtle differences in the specificity and the level of activity. Thus, conformational differences between TSWAB and chicken lysozyme exist, whereas functional mechanisms appear to be similar. On the other hand, alpha-lactalbumins and c-type lysozymes exhibit drastically different functions with conserved molecular conformation. It is evident that a common molecular scaffold is exploited in the three enzymes for apparently different physiological roles. It can be inferred on the basis of the structure-function comparison of these three proteins having common phylogenetic origin that the conformational changes in a protein are minimal during rapid evolution as compared with those in the normal course of evolution.     

Protein elasticity based on conformations of sequential polypeptides: The biological elastic fiber

Fibrous elastin of the biological elastic fiber is a cross-linked condensed state in which there is roughly one-half polypeptide and one-half water. The precursor protein tropoelastin, a chemical fragmentation product -elastin, and a sequential polypeptide (l·Val¹-l·Pro²-Gly³-l·Val⁴-Gly⁵) _n , which is a prominent primary structural feature of tropoelastin, are each soluble in all proportions in water at 20°C. On heating to physiological temperatures, each undergoes aggregation and forms a dense viscoelastic phase, which as the fiber itself, is about 60% water. This reversible heat-elicited condensed phase is called the coacervate. Circular dichroism studies show coacervation to be a process of increasing intramolecular order. Electron microscopy (light, scanning, and transmission) shows coacervation to be a process of increasing order intermolecularly. Thus a rise in temperature between 20 and 40°C results in an increase in order of the polypeptide. Coacervation is an inverse temperature transition, and the condensed state is anisotropic at the molecular level. Thermoelasticity studies in water on bovine ligamentum nuchae fibrous elastin and on -irradiation cross-linked polypentapeptide coacervates show increases in elastomeric force,f, over the same 20–40°C temperature range in which the inverse temperature transition gives rise to the coacervate, and the constancy off/T with temperature, once the transition is effectively completed, suggests a high-entropy component to the elastomeric force. Thus the data argue for an anisotropic-entropic elastomer.Detailed conformational studies on the polypentapeptide result in the development of a -spiral conformation in which there are regularly recurring -turns in loose helical array (a structure that forms on raising the temperature) and in which there are recurring dynamic suspended segments that are the focal point of large, low-energy oscillatory motions called librations. The structure gives rise to a librational entropy mechanism of elasticity wherein the amplitudes of the rocking motions become damped on stretching. This perspective is substantiated by dielectric relaxation studies on the coacervate state and by characterization of synthetic analogs of the polypentapeptide. Dielectric relaxation studies on a concentrated state of about 60% water show the development of a regular structure over the same temperature range as for the development of the coacervate state, and the development of the regular structure with increasing temperature is seen to parallel the development of elastomeric force with increasing temperature. Increasing elastomeric force coincides with increasing regularity of structure! Synthetic analogs of the polypentapeptide, designed to interfere with the librational processes of the suspended segment, impair elastic function, and an analog that makes the -turn more rigid results in increased elastic modulus. This development of a librational entropy mechanism for protein elasticity is a departure from the kinetic theory of rubber elasticity, the random network perspective that has dominated the traditional view of biological elasticity for the past several decades. The new perspective opens the way to insightful consideration of new elastomeric biomaterials with numerous biomedical applications.     

Towards the molecular mechanism of Na(+)/solute symport in prokaryotes

The Na(+)/solute symporter family (SSF, TC No. 2.A.21) contains more than 40 members of pro- and eukaryotic origin. Besides their sequence similarity, the transporters share the capability to utilize the free energy stored in electrochemical Na(+) gradients for the accumulation of solutes. As part of catabolic pathways most of the transporters are most probably involved in the acquisition of nutrients. Some transporters play a role in osmoadaptation. With a high resolution structure still missing, a combination of genetic, protein chemical and spectroscopic methods has been used to gain new insights into the structure and molecular mechanism of action of the transport proteins. The studies suggest a common 13-helix motif for all members of the SSF according to which the N-terminus is located in the periplasm and the C-terminus is directed into the cytoplasm (except for proteins containing a N- or C-terminal extension). Furthermore, an amino acid substitution analysis of the Na(+)/proline transporter (PutP) of Escherichia coli, a member of the SSF, has identified regions of particular functional importance. For example, amino acids of TM II of PutP proved to be critical for high affinity binding of Na(+) and proline. In addition, it was shown that ligand binding induces widespread conformational alterations in the transport protein. Taken together, the studies substantiate the common idea that Na(+)/solute symport is the result of a series of ligand-induced structural changes.