Using protein templates to direct the formation of thin-film polymer surfaces

Protein imprinted electrodes formed by the cyclic voltammetric deposition of conductive polymers, on screen-printed platinum supports, in the presence of target proteins have been fabricated. An initial layer of polypyrrole was used as a supporting polymer layer, upon which were formed two layers of polyaminophenylboronic acid. The first of these layers was non-imprinted and formed a barrier between the polypyrrole and the outer layer, which was deposited in the presence of a protein template (lysozyme or cytochrome c). After protein extraction, re-binding of the template proteins to their respective imprinted electrodes showed a distinct two-phase binding profile; whereas, binding to control polymers, made in the same way but without the addition of protein templates, showed progressive binding typical of non-specific recognition. Reductions in the observed current transmission due to bonding to the polymer surface of non-conductive protein have been used as a measure of re-binding. It was found that when challenged with 1 part per million protein in solution, the current reductions for the lysozyme and cytochrome c imprinted electrodes were 30.3 and 66.2%, respectively, compared to 4.5 and 29.9% for their respective control electrodes. All measurements carried out at -0.1 V with Ag/AgCl reference.     

The selectivity of protein‐imprinted gels and its relation to protein properties: A computer simulation study

Polymer‐based protein recognition systems have enormous potential within clinical and diagnostic fields due to their reusability, biocompatibility, ease of manufacturing, and potential specificity. Imprinted polymer matrices have been extensively studied and applied as a simple technique for creating artificial polymer‐based recognition gels for a target molecule. Although this technique has been proven effective when targeting small molecules (such as drugs), imprinting of proteins have so far resulted in materials with limited selectivity due to the large molecular size of the protein and aqueous environment. Using coarse‐grained molecular simulation, we investigate the relation between protein makeup, polymer properties, and the selectivity of imprinted gels. Nonspecific binding that results in poor selectivity is shown to be strongly dependent on surface chemistry of the template and competitor proteins as well as on polymer chemistry. Residence time distributions of proteins diffusing within the gels provide a transparent picture of the relation between polymer constitution, protein properties, and the nonspecific interactions with the imprinted gel. The pronounced effect of protein surface chemistry on imprinted gel specificity is demonstrated.     

Kinetics, thermodynamics and evolution of non-native interactions in a protein folding nucleus

A lattice model with side chains was used to investigate protein folding with computer simulations. In this model, we rigorously demonstrate the existence of a specific folding nucleus. This nucleus contains specific interactions not present in the native state that, when weakened, slow folding but do not change protein stability. Such a decoupling of folding kinetics from thermodynamics has been observed experimentally for real proteins. From our results, we conclude that specific non-native interactions in the transition state would give rise to straight phi-values that are negative or larger than unity. Furthermore, we demonstrate that residue Ile 34 in src SH3, which has been shown to be kinetically, but not thermodynamically, important, is universally conserved in proteins with the SH3 fold. This is a clear example of evolution optimizing the folding rate of a protein independent of its stability and function.     

Stereoselective histidine sensor based on molecularly imprinted sol-gel films

A piezoelectric sensor coated with a thin molecularly imprinted sol-gel film has been developed for the determination of L-histidine in the liquid phase. Without preprotection, L-histidine was imprinted directly into silica sol-gel films that consisted of a hybrid mixture of functionalized organosilicon precursors (phenyltrimethoxysilane and methyltrimethoxysolane). The viscoelasticity of the film in the air and in buffer solution has been studied by the piezoelectric quartz crystal impedance technique. The binding of L-histidine to the imprinted film in the liquid phase was investigated by the piezoelectric microgravimetry and electrochemical impedance technique. Scatchard analysis showed that the maximum binding site (Qmax) of the L-histidine imprinted sol-gel film is about 23.7 micromol/g. A linear range from 5.0x10(-8) to 1.0x10(-4) M for a detection of L-histidine has been observed with a detection limit of 2.5x10(-8) M for S/N=3. The proposed imprinted sol-gel sensor exhibits good stability, high specificity, and excellent stereoselectivity.     

Thermochemical study of amino acid imprinted polymer films

Molecularly imprinted polymers provide an alternative to traditional methods of amino acid analysis. The imprinted polymers are more robust and significantly less expensive than, for example, ELISA analysis. Amino acid imprinted nylon‐6 thin films were studied by differential scanning calorimetry and scanning electron microscopy. Endothermic peaks were observed for imprinted films at temperatures higher than that for pure nylon, indicating the formation of a more‐ordered, hydrogen bonded polymer. Removal of the amino acid from the imprinted film resulted in reversion to the peak observed for pure nylon‐6. Additives, β‐cyclodextrin and multiwalled carbon nanotubes, were added to the imprinted polymer solutions as a means to increase the porosity of the films. These studies resulted in alternative morphologies and calorimetric results that provide additional functionalities and applications for imprinted polymers. Copyright © 2015 John Wiley & Sons, Ltd.     

Protein interactions with urea and guanidinium chloride. A calorimetric study.

The interaction of urea and guanidinium chloride with proteins has been studied calorimetrically by titrating protein solutions with denaturants at various fixed temperatures, and by scanning them with temperature at various fixed concentrations of denaturants. It has been shown that the observed heat effects can be described in terms of a simple binding model with independent and similar binding sites. Using the calorimetric data, the number of apparent binding sites for urea and guanidinium chloride have been estimated for three proteins in their unfolded and native states (ribonuclease A, hen egg white lysozyme and cytochrome c). The intrinsic and total thermodynamic characteristics of their binding (the binding constant, the Gibbs energy, enthalpy, entropy and heat capacity effect of binding) have also been determined. It is found that the binding of urea and guanidinium chloride by protein is accompanied by a significant decrease of enthalpy and entropy. At all concentrations of denaturants the enthalpy term slightly dominates the entropy term in the Gibbs energy function. Correlation analysis of the number of binding sites and structural characteristics of these proteins suggests that the binding sites for urea and guanidinium chloride are likely to be formed by several hydrogen bonding groups. This type of binding of the denaturant molecules should lead to a significant restriction of conformational freedom within the polypeptide chain. This raises a doubt as to whether a polypeptide chain in concentrated solutions of denaturants can be considered as a standard of a random coil conformation.     

Improved Gō-like models demonstrate the robustness of protein folding mechanisms towards non-native interactions

The use of simple theoretical models has provided a considerable contribution to our present understanding of the means by which proteins adopt their native fold from the plethora of available unfolded states. A common assumption in building computationally tractable models has been the neglect of stabilizing non-native interactions in the class of models described as "Gō-like." The focus of this study is the characterization of the folding of a number of proteins via a Gō-like model, which aims to map a maximal amount of information reflecting the protein sequence onto a "minimalist" skeleton. This model is shown to contain sufficient information to reproduce the folding transition states of a number of proteins, including topologically analogous proteins that fold via different transition states. Remarkably, these models also demonstrate consistency with the general features of folding transition states thought to be stabilized by non-native interactions. This suggests that native interactions are the primary determinant of most protein folding transition states, and that non-native interactions lead only to local structural perturbations. A prediction is also included for an asymmetrical folding transition state of bacteriophage lambda protein W, which has yet to be subjected to experimental characterization.     

Thermodynamics of ubiquitin unfolding

The energetics of ubiquitin unfolding have been studied using differential scanning microcalorimetry. For the first time it has been shown directly that the enthalpy of protein unfolding is a nonlinear function of temperature. Thermodynamic parameters of ubiquitin unfolding were correlated with the structure of the protein. The enthalpy of hydrogen bonding in ubiquitin was calculated and compared to that obtained for other proteins. It appears that the energy of hydrogen bonding correlates with the average length of the hydrogen bond in a given protein structure. © 1994 John Wiley & Sons, Inc.     

Hsp26: a temperature-regulated chaperone

Small heat shock proteins (sHsps) are a conserved protein family, with members found in all organisms analysed so far. Several sHsps have been shown to exhibit chaperone activity and protect proteins from irreversible aggregation in vitro. Here we show that Hsp26, an sHsp from Saccharomyces cerevisiae, is a temperature-regulated molecular chaperone. Like other sHsps, Hsp26 forms large oligomeric complexes. At heat shock temperatures, however, the 24mer chaperone complex dissociates. Interestingly, chaperone assays performed at different temperatures show that the dissociation of the Hsp26 complex at heat shock temperatures is a prerequisite for efficient chaperone activity. Binding of non-native proteins to dissociated Hsp26 produces large globular assemblies with a structure that appears to be completely reorganized relative to the original Hsp26 oligomers. In this complex one monomer of substrate is bound per Hsp26 dimer. The temperature-dependent dissociation of the large storage form of Hsp26 into a smaller, active species and the subsequent re-association to a defined large chaperone-substrate complex represents a novel mechanism for the functional activation of a molecular chaperone.     

Analyte separation by OMNiMIPs imprinted with multiple templates

Changes detected in the imprinting effect by OMNiMIPs imprinted with multiple templates appear to be a function of the maximum template loading. Below the maximum template loading, the polymers imprinted with multiple compounds provide molecular recognition close to the polymers imprinted with single compounds, for each template compound tested. However, template loading past this point can result in significant lowering of the imprinting effect. For example, 1,1′-bi-2-naphthol enantiomers showed nearly a 60% loss in enantioselectivity on OMNiMIP 8 (imprinted with four templates); yet still maintained a separation factor of 3.7 allowing baseline separation of enantiomers. Similar behavior was seen for the other three template molecules, although losses in enantioselectivity were less severe. The multi-analyte imprinted OMNiMIP 8 was shown to be capable of separating a template molecule individually from a mixture, including enantiomers, but not all four concurrently. With respect to physical properties of the different OMNiMIPs, gradual trends in porosity and surface area correlated to the concentration of the templates, independent of their molecular structure.     

Structural basis for the molecular memory of imprinted proteins in anhydrous media

Fourier-transform infrared (FTIR) spectroscopy has been used to quantitatively examine the secondary structure of imprinted (i.e., lyophilized in the presence of multifunctional ligands followed by removal of the latter) proteins in anhydrous media. Lysozyme, chymotrypsinogen, and bovine serum albumin, imprinted with L-malic acid, all exhibited significant differences in the secondary structure compared to that of their nonimprinted counterparts. A rise in the beta-sheet content, which invariably occurs upon lyophilization, is substantially lower for imprinted proteins. Alterations in the alpha-helix contents of these proteins have also been observed upon imprinting, although these changes are specific to the protein. A structural explanation has been obtained herein for other previously observed aspects of the protein imprinting phenomenon, including the effects of the ligand and the solvent and the lack of enantioselectivity. Exposure to aqueous solution, but not to anhydrous solvents, results in the disappearance of imprinting-induced changes in the secondary structure of proteins. (c) 1996 John Wiley & Sons, Inc.     

Energy barriers, cooperativity, and hidden intermediates in the folding of small proteins

Current theoretical views of the folding process of small proteins (< approximately 100 amino acids) postulate that the landscape of potential mean force (PMF) for the formation of the native state has a funnel shape and that the free energy barrier to folding arises from the chain configurational entropy only. However, recent theoretical studies on the formation of hydrophobic clusters with explicit water suggest that a barrier should exist on the PMF of folding, consistent with the fact that protein folding generally involves a large positive activation enthalpy at room temperature. In addition, high-resolution structural studies of the hidden partially unfolded intermediates have revealed the existence of non-native interactions, suggesting that the correction of the non-native interactions during folding should also lead to barriers on PMF. To explore the effect of a PMF barrier on the folding behavior of proteins, we modified Zwanzig's model for protein folding with an uphill landscape of PMF for the formation of transition states. We found that the modified model for short peptide segments can satisfy the thermodynamic and kinetic criteria for an apparently two-state folding. Since the Levinthal paradox can be solved by a stepwise folding of short peptide segments, a landscape of PMF with a locally uphill search for the transition state and cooperative stabilization of folding intermediates/native state is able to explain the available experimental results for small proteins. We speculate that the existence of cooperative hidden folding intermediates in small proteins could be the consequence of the highly specific structures of the native state, which are selected by evolution to perform specific functions and fold in a biologically meaningful time scale.     

Recognition of conformational changes in beta-lactoglobulin by molecularly imprinted thin films

Pathogenesis in protein conformational diseases is initiated by changes in protein secondary structure. This molecular restructuring presents an opportunity for novel shape-based detection approaches, as protein molecular weight and chemistry are otherwise unaltered. Here we apply molecular imprinting to discriminate between distinct conformations of the model protein beta-lactoglobulin (BLG). Thermal- and fluoro-alcohol-induced BLG isoforms were imprinted in thin films of 3-aminophenylboronic acid on quartz crystal microbalance chips. Enhanced rebinding of the template isoform was observed in all cases when compared to the binding of nontemplate isoforms over the concentration range of 1-100 microg mL(-1). Furthermore, it was observed that the greater the changes in the secondary structure of the template protein the lower the binding of native BLG challenges to the imprint, suggesting a strong steric influence in the recognition system. This feasibility study is a first demonstration of molecular imprints for recognition of distinct conformations of the same protein.     

An innovative approach to molecularly imprinted capillaries for polar templates by grafting polymerization

Molecularly imprinted polymers have been successfully used as selective stationary phases in capillary electrophoresis. Notwithstanding, this technique suffers from several drawbacks as the loss of molecular recognition properties in aqueous media and the lack of feasibility for imprinted systems directed towards highly polar templates soluble in aqueous environments only. Thus, the preparation of imprinted polymers for highly polar, water-soluble analytes, represents a challenge. In this work, we present an innovative approach to overcome these drawbacks. It is based on a surface molecular imprinting technique that uses preformed macromonomers as both functional recognition elements and cross-linking agents. A poly-2-hydroxyethyl-co-methacrylic acid linear polymer was grafted from the surface of silica capillaries. The grafted polymer was exhaustively esterified with methacrylic anhydride to obtain polyethylendimethacrylate-co-methacrylic acid linear chains. Then, as a proof of concept, an adequate amount of a very polar template like penicillin V was added in a hydro-organic mixture, and a thin layer of imprinted polymer was obtained by cross-linking the polymer linear chains. The binding behaviour of the imprinted and non-imprinted capillaries was evaluated in different separation conditions in order to assess the presence of template selectivity and molecular recognition effects. The experimental results clearly show that this innovative kind of imprinted material can be easily obtained in very polar polymerization environments and that it is characterized by enhanced molecular recognition properties in aqueous buffers and good selectivity towards the template and strictly related molecules.     

Atomic-level structure characterization of an ultrafast folding mini-protein denatured state

Atomic-level analyses of non-native protein ensembles constitute an important aspect of protein folding studies to reach a more complete understanding of how proteins attain their native form exhibiting biological activity. Previously, formation of hydrophobic clusters in the 6 M urea-denatured state of an ultrafast folding mini-protein known as TC5b from both photo-CIDNP NOE transfer studies and FCS measurements was observed. Here, we elucidate the structural properties of this mini-protein denatured in 6 M urea performing (15)N NMR relaxation studies together with a thorough NOE analysis. Even though our results demonstrate that no elements of secondary structure persist in the denatured state, the heterogeneous distribution of R(2) rate constants together with observing pronounced heteronuclear NOEs along the peptide backbone reveals specific regions of urea-denatured TC5b exhibiting a high degree of structural rigidity more frequently observed for native proteins. The data are complemented with studies on two TC5b point mutants to verify the importance of hydrophobic interactions for fast folding. Our results corroborate earlier findings of a hydrophobic cluster present in urea-denatured TC5b comprising both native and non-native contacts underscoring their importance for ultra rapid folding. The data assist in finding ways of interpreting the effects of pre-existing native and/or non-native interactions on the ultrafast folding of proteins; a fact, which might have to be considered when defining the starting conditions for molecular dynamics simulation studies of protein folding.     

Proteins containing non-native disulfide bonds generated by oxidative stress can act as signals for the induction of the heat shock response

While oxidative stress can induce a heat shock response, the primary signals that initiate activation have not been identified. To identify such signals, HepG2 and V 79 cells were exposed to menadione, a compound that redox-cycles to generate superoxide. The oxidative stress generated by menadione resulted in oxidation of protein thiols in a dose-dependent manner. This was followed by protein destabilization and denaturation, as determined by differential scanning calorimetry of whole cells. To directly evaluate the effect of non-native disulfides on protein conformation, Ca²⁺-ATPase, isolated from rabbit sarcoplasmic reticulum, was chemically modified to contain non-native intermolecular or glutathione (GHS)-mixed disulfides. Differential scanning calorimetry profiles and 1-anilinonaphthalene-8-sulfonic acid fluorescence indicated that formation of non-native disulfides produced protein destabilization, denaturation, and exposure of hydrophobic domains. Cellular proteins shown to contain oxidized thiols formed detergent-insoluble aggregates. Cells treated with menadione exhibited activation of HSF-1, accumulated Hsp 70 mRNA, and increased synthesis of Hsp 70. This work demonstrates that formation of physiologically relevant, non-native intermolecular and GSH-mixed disulfides causes proteins to destabilize, unfold such that hydrophobic domains are exposed, and initiate a signal for induction of the heat shock response. J. Cell. Physiol. 171:143–151, 1997. © 1997 Wiley-Liss, Inc.     

A circumventing role for the non-native intermediate in the folding of β-lactoglobulin

Folding experiments have suggested that some proteins have kinetic intermediates with a non-native structure. A simple G ?o model does not explain such non-native intermediates. Therefore, the folding energy landscape of proteins with non-native intermediates should have characteristic properties. To identify such properties, we investigated the folding of bovine β-lactoglobulin (βLG). This protein has an intermediate with a non-native α-helical structure, although its native form is predominantly composed of β-structure. In this study, we prepared mutants whose α-helical and β-sheet propensities are modified and observed their folding using a stopped-flow circular dichroism apparatus. One interesting finding was that E44L, whose β-sheet propensity was increased, showed a folding intermediate with an amount of β-structure similar to that of the wild type, though its folding took longer. Thus, the intermediate seems to be a trapped intermediate. The high α-helical propensity of the wild-type sequence likely causes the folding pathway to circumvent such time-consuming intermediates. We propose that the role of the non-native intermediate is to control the pathway at the beginning of the folding reaction.     

Conformational fluctuations and protein reactivity. Determination of the rate-constant spectrum and consequences in elementary biochemical processes

When a protein's active site happens to be strongly coupled with the protein structure, the rate constant of the reaction may eventually be modulated by the conformational fluctuations. Evidence for this effect has long been provided by extensive flash photolysis investigations of liganded hemoproteins and more recently of the non-heme respiratory protein hemerythrin in hydro-organic solvents. Within a given protein conformational substate, an elementary reaction step is characterized by one single free energy barrier and by a first-order rate constant, k, which changes with temperature according to an Arrhenius law. At physiological temperature and low viscosity, ultrafast conformational relaxation causes efficient averaging of the reaction rates and the protein displays exponential kinetics with an average rate constant (k). Under sufficiently general conditions, it can be shown that (k) also follows a simple Arrhenius law with 'effective' values of the pre-exponential factor Aeff and activation enthalpy Heff. It is found that Aeff strongly depends on the overall shape of the rate constant distribution and that Heff actually corresponds to the lower limit of the enthalpy of activation, i.e. the value associated with the highest possible reaction rate. The underlying distribution of rate constants can be reconstructed from a set of experiments in which the kinetics depart from an exponential, i.e. at low temperature and high viscosity. The most probable distribution of exponentials consistent with the observed kinetics of the geminate recombinations of oxygen with photodissociated hemerythrin has been determined by using a new approach, known as the maximum entropy method. The results are consistent with a single pre-exponential value and a distributed enthalpy spectrum. As expected, Heff does not coincide either with the most probable nor with the average value of the enthalpy. The most salient findings are that the probability for any protein molecule to have an enthalpy of activation equal to the effective value Heff vanishes and that Aeff differs by nearly three orders of magnitude from the true value A0. Biochemical reaction rates are actually average values, since protein reactions are measured under physiological conditions, where conformational relaxation is always fast. Our understanding of the significance of Aeff and Heff is therefore entirely dependent on the knowledge of the distribution function of the rate constants. In particular, enthalpy and entropy terms of similar reactions performed by different proteins cannot be compared as long as the distribution of the rate constants remains unknown.     

Why water-soluble, compact, globular proteins have similar specific enthalpies of unfolding at 110 degrees C.

The changes in free energy, enthalpy, and entropy of unfolding have been measured for many water-soluble, compact, globular proteins by a number of workers. In principle, a wide range in stability could be achieved by proteins, as measured by the free energy of unfolding; in practice, evolution only allows a narrow range in this quantity. Proteins are only marginally stable at room temperature for many possible reasons, including ensuring that folding is reversible and polypeptide chains are not trapped in incorrectly folded structures. Many of these proteins have approximately the same values of enthalpy of unfolding around 110 degrees C. We show here that this arises because the change in entropy of unfolding at room temperature and the change in heat capacity on unfolding, which governs the temperature variation of the enthalpy and entropy, both vary with the magnitude of the hydrophobic effect in the protein. As all these proteins have evolved to achieve similar stabilities at room temperature, the enthalpy of unfolding will also vary with the size of the hydrophobic effect in the protein. A consequence of this is that curves of the specific unfolding enthalpy against temperature for different proteins intersect around 110 degrees C. A similar conclusion, on the basis of similar melting points rather than similar free energies of unfolding, has been reached independently by Baldwin and Muller (R. L. Baldwin, personal communication).