Structural changes accompanying the irreversible oxidation of the chromatophore membrane from Rhodospirillum rubrum G9

The structure of the chromatophore membrane of the carotenoid-free mutant Rhodospirillum rubrum G9 and the effect of irreversible photooxidation upon this structure have been investigated using several physical techniques. Native chromatophore membranes undergo endothermic transitions in two temperature regions; at temperatures of about 0°C a broad reversible transition and between approx. 50 and 90°C several endothermic transitions are observed which are irreversible. The first transition can be assigned to the gel-to-liquid crystal transition of the lipid bilayer present in chromatophores; the irreversible one is attributed to changes mainly in the quarternary and possibily tertiary structure of membrane proteins. CD measurements showed that heating of chromatophores up to 70°C has no effect upon the protein secondary structure. Photooxidation has little effect on the structure and dynamics of the lipid bilayer in the chromatophore membrane. The order (or average conformation) of both the lipid polar groups and the hydrocarbon chains is hardly changed. However, the lipid phase transition is dramatically broadened and the protein-associated endothermic transitions are greatly reduced. This indicates that the major effect of photooxidation is upon lipid-protein and protein-protein interactions. Electron microscopy studies support this interpretation. It can be shown that the dense and regular packing of protein particles observed in the chromatophore membrane is lost as an effect of photooxidation. Instead, randomly distributed particles of varying size and shape are seen. These results are interpreted to mean that pigment-protein interactions are responsible for maintaining the native long-range order in the chromatophore membrane of R. rubrum G9. Destruction of the pigments by photooxidation leads to irreversible protein dissociation which in turn is followed probably by random protein reaggregation.     

Adsorption and separation of proteins by a smectitic clay mineral

The adsorption of proteins by a smectitic clay mineral was investigated. The clay used in this study is a mixture of montmorillonite and amorphous SiO₂. Due to the high porosity the montmorillonite units are accessible for protein adsorption. The amorphous silica prevents the montmorillonite from swelling and allows column packing. Protein adsorption was performed at different pH under static conditions. Furthermore, static capacities were determined. The material reveals high adsorption capacities for proteins under static conditions (270–408 mg/g), whereby proteins are mainly adsorbed via electrostatic interactions. The Freundlich isotherm is suggested as an adsorption model. For desorption a pH shift was found to be most effective. Binding and elution of human serum albumin and ovalbumin were tested under dynamic conditions. Dynamic capacities of about 40 mg/g for ovalbumin at 764 cm/h were found. The clay mineral provides suitable properties for the application as cost-efficient, alternative separation material.     

A cytoplasmic protein, NfrC, is required for bacteriophage N4 adsorption.

At least four genes are required for irreversible adsorption of bacteriophage N4. nfrA and nfrB have been characterized previously and encode an outer membrane protein and inner membrane protein, respectively. The nfrC gene product is characterized in detail in this study. We have mapped the nfrD locus to min 52 on the Escherichia coli linkage map. Maxicell analysis of nfrC and a null allele (nfrC2) cloned into a high-copy-number plasmid shows its gene product to be 42 kDa in size. We determined the nfrC nucleotide sequence which predicts a gene product of 42 kDa. Western blots (immunoblots) of Escherichia coli proteins after cellular fractionation show NfrC to be a cytoplasmic protein which is required for irreversible bacteriophage N4 adsorption, an event occurring at the cell surface.     

Changes in crystallographic structure and thermostability of a Cu,Zn superoxide dismutase mutant resulting from the removal of a buried cysteine

In principle, protein thermostability depends on efficient interior packing of apolar residues and on avoidance of irreversible denaturation in the unfolded state. To study these effects, the single free cysteine in the highly stable enzyme bovine Cu,Zn superoxide dismutase was mutated to alanine (Cys6----Ala), and the recombinant protein was expressed in yeast, purified, characterized for reversible and irreversible denaturation, crystallized isomorphously to the wild-type enzyme, and used to determine the atomic structure. Removal of the chemically reactive thiol significantly decreased the rate of irreversible denaturation (as monitored by thermal inactivation at 70 degrees C), but the observed energetic cost (delta delta G of 0.7-1.3 kcal/mol as determined by differential scanning calorimetry) was much less than predicted from either the change in hydrophobicity or packing due to removal of the interior sulfur atom. X-ray diffraction data were collected to 2.1-A resolution using an area detector, and the atomic model for the mutant enzyme was determined by fitting to electron density difference maps, followed by reciprocal space refinement both with stereochemical restraints using PROLSQ and with molecular dynamics using X-PLOR. The refined 2.1-A resolution crystallographic structure suggests that small concerted and compensating shifts (less than 0.5 A) of the surrounding side chains and of the adjacent N- and C-terminal beta-strands significantly reduced the energetic cost of the interior mutation by improving packing and stereochemistry in the mutant enzyme. Taken together, these results differentiate between the effects of reversible and irreversible processes as they impact the design of thermostable proteins and suggest that relatively subtle concerted shifts can significantly reduce the energetic cost of evolutionary variation in internal residues of proteins with Greek key beta-barrel folds.     

Effect of thermal stability on protein adsorption to silica using homologous aldo-keto reductases

Gaining more insight into the mechanisms governing the behavior of proteins at solid/liquid interfaces is particularly relevant in the interaction of high-value biologics with storage and delivery device surfaces, where adsorption-induced conformational changes may dramatically affect biocompatibility. The impact of structural stability on interfacial behavior has been previously investigated by engineering nonwild-type stability mutants. Potential shortcomings of such approaches include only modest changes in thermostability, and the introduction of changes in the topology of the proteins when disulfide bonds are incorporated. Here we employ two members of the aldo-keto reductase superfamily (alcohol dehydrogenase, AdhD and human aldose reductase, hAR) to gain a new perspective on the role of naturally occurring thermostability on adsorbed protein arrangement and its subsequent impact on desorption. Unexpectedly, we find that during initial adsorption events, both proteins have similar affinity to the substrate and undergo nearly identical levels of structural perturbation. Interesting differences between AdhD and hAR occur during desorption and both proteins exhibit some level of activity loss and irreversible conformational change upon desorption. Although such surface-induced denaturation is expected for the less stable hAR, it is remarkable that the extremely thermostable AdhD is similarly affected by adsorption-induced events. These results question the role of thermal stability as a predictor of protein adsorption/desorption behavior.     

Sodium dodecyl sulfate polyacrylamide gel electrophoresis for direct quantitation of protein adsorption

A simple method, sodium dodecyl sulfate polyacrylamide gel electrophoresis coupled with direct protein adsorption analysis (SDS–PAGE/DPA), is presented here for the quantitation of adsorption-caused protein loss. No complicated steps and expensive equipment are involved, and this method is capable of measuring proteins adsorbed on sample vials at extremely low concentrations (in pg/μl). We used this method to characterize the effects of concentration, time, and volume on adsorption. We also applied this method to discover differential sample loss in protein mixtures and its utility in developing preventive strategies of adsorption.     

Foreign protein degradation and instability in plants and plant tissue cultures

Low production cost is a key factor driving the development of plants and plant tissue cultures for the synthesis of therapeutic and other foreign proteins. Because product yield and concentration exert a major influence on process economics, improving foreign protein accumulation is crucial for enhancing the commercial success of plant-based production systems. Strategies aimed at increasing transgene expression have been effective; however, a critical but poorly understood factor contributing to low foreign protein yield is post-synthesis and/or post-secretion instability and degradation. Loss of foreign protein as result of biological and physical processes such as proteolytic destruction and irreversible surface adsorption can occur in plants and plant culture systems. This review highlights the need to consider such mechanisms and outlines a range of remedial strategies aimed at minimizing foreign protein degradation and loss.     

Measuring “Unmeasurable” Folding Kinetics of Proteins by Single-Molecule Force Spectroscopy

Folding and unfolding are fundamental biological processes in cell and are important for the biological functions of proteins. Characterizing the folding and unfolding kinetics of proteins is important for understanding the energetic landscape leading to the active native conformations of these molecules. However, the thermal or chemical-induced unfolding of many proteins is irreversible in vitro, precluding characterization of the folding kinetics of such proteins, just as it is impossible to “un-boil” an egg. Irreversible unfolding often manifests as irreversible aggregation of unfolded polypeptide chains, which typically occurs between denatured protein molecules in response to the exposure of hydrophobic residues to solvent. An example of such a protein where thermal denaturation results in irreversible aggregation is the β-1,4 endoxylanase from Bacillus circulans (BCX). Here, we report the use of single-molecule atomic force microscopy to directly measure the folding kinetics of BCX in vitro. By mechanically unfolding BCX, we essentially allowed only one unfolded molecule to exist in solution at a given time, effectively eliminating the possibility for aggregation. We found that BCX can readily refold back to the native state, allowing us to measure its folding kinetics for the first time. Our results demonstrate that single-molecule force-spectroscopy-based methods can adequately tackle the challenge of “un-boiling eggs”, providing a general methodology to characterize the folding kinetics of many proteins that suffer from irreversible denaturation and thus cannot be characterized using traditional equilibrium methodologies.     

Effect of pH on rate of interfacial inactivation of serine proteases in aqueous-organic systems

We studied the inactivation of trypsin and alpha- and beta-chymotrypsin by passage of droplets of tridecane though their aqueous solutions. The mechanism involves contact with the interface, because the loss of activity is proportional to the total area exposed. The rates of inactivation vary up to fivefold over the pH range 3 to 10. However, there is no clear maximum at the isoelectric point (pI) of each enzyme, where the amount of protein adsorbed is usually found to be highest. This is probably because, at the pI, there is also a minimum in structural alteration on adsorption. There may be a weak correlation with pH effects on foamability of the enzyme solutions, a parameter reported to reflect the "hardness" of different proteins, which controls their interfacial unfolding. The pH dependence of both inactivation and hardness cautions against attempts to correlate inactivation of different enzymes with a single value of a parameter such as adiabatic compressibility. There is no correlation between the effects of pH on interfacial inactivation and those reported in the literature on irreversible inactivation in concentrated urea or at high temperature.     

Irreversible Denaturation of Maltodextrin Glucosidase Studied by Differential Scanning Calorimetry,Circular Dichroism,and Turbidity Measurements

Thermal denaturation of Escherichia coli maltodextrin glucosidase was studied by differential scanning calorimetry, circular dichroism (230 nm), and UV-absorption measurements (340 nm), which were respectively used to monitor heat absorption, conformational unfolding, and the production of solution turbidity. The denaturation was irreversible, and the thermal transition recorded at scan rates of 0.5–1.5 K/min was significantly scan-rate dependent, indicating that the thermal denaturation was kinetically controlled. The absence of a protein-concentration effect on the thermal transition indicated that the denaturation was rate-limited by a mono-molecular process. From the analysis of the calorimetric thermograms, a one-step irreversible model well represented the thermal denaturation of the protein. The calorimetrically observed thermal transitions showed excellent coincidence with the turbidity transitions monitored by UV-absorption as well as with the unfolding transitions monitored by circular dichroism. The thermal denaturation of the protein was thus rate-limited by conformational unfolding, which was followed by a rapid irreversible formation of aggregates that produced the solution turbidity. It is thus important to note that the absence of the protein-concentration effect on the irreversible thermal denaturation does not necessarily means the absence of protein aggregation itself. The turbidity measurements together with differential scanning calorimetry in the irreversible thermal denaturation of the protein provided a very effective approach for understanding the mechanisms of the irreversible denaturation. The Arrhenius-equation parameters obtained from analysis of the thermal denaturation were compared with those of other proteins that have been reported to show the one-step irreversible thermal denaturation. Maltodextrin glucosidase had sufficiently high kinetic stability with a half-life of 68 days at a physiological temperature (37°C).     

Carbonylated proteins accumulated as vitality decreases during long-term storage of beech (Fagus sylvatica L.) seeds

Key message

Carbonylation of proteins associated with a stress response may contribute to the lowered viability of naturally aged beech seeds, especially the desiccation tolerance-associated proteins and USP-like protein.

Abstract

Proteins are modified by a large number of reactions that involve reactive oxygen species-mediated oxidation. The direct oxidation of amino acids produces 2,4-dinitrophenylhydrazine-detectable protein products. Carbonylation is irreversible, and carbonylated proteins are marked for proteolysis or can escape degradation and form high molecular weight aggregates, which accumulate with age. Beech (Fagus sylvatica L.) seeds stored under optimal conditions for different periods of time, ranging from 2 to 13 years, were analyzed. Protein carbonylation was examined as a potential cause for the loss of viability of beech seeds, and the characteristic spots of protein carbonyls were identified. Here, we present and discuss the role of carbonylation in the proteome of beech seeds that contribute to the loss of seed viability during natural aging. The long-term storage of beech seeds is intricate because their germination capacity decreases with age and is negatively correlated with the level of protein carbonyls that accumulate in the seeds. We establish that protein synthesis, folding and degradation are the most affected biochemical traits in long-term stored beech seeds. In addition, we suggest that proteins associated with the stress response may have contributed to the lowered viability of beech seeds, especially the desiccation tolerance-associated proteins that include T-complex protein 1 and the universal stress protein (USP)-like protein, which is identified as carbonylated for first time here.     

Affine magnetic sorbents supported on coal ash microspheres for recombinant protein isolation

The results of the development and utilization of an affine magnetic sorbent with Ni²⁺ ions immobilized on coal ash microspheres are reported. The applicability of the material in the isolation of Histag proteins is demonstrated by examples of the recombinant green fluorescent protein from Clytia gregaria and the Ca²⁺ regulated photoprotein obelin from Obelia longissima. The specific sorption capacity of the sorbent was 2–7 mg/cm³ for medium-size proteins (20–30 kDa). The particles are suitable for chromatography with the presence of chaotropic agents and EDTA. They are easy to manipulate as isolation of a target protein takes 30–35 min. On the one hand, the elevated affinity of the sorbent to proteins rich in native histidines may result in a high degree of irreversible sorption; on the other hand, it allows isolation of such proteins without the introduction of artificial polyhistidine fragments.     

Breakdown of Ribulose Bisphosphate Carboxylase and Change in Proteolytic Activity during Dark-induced Senescence of Wheat Seedlings

When 8-day-old wheat seedlings (Triticum aestivum L. var. Chris) are placed in the dark the fully expanded primary leaves undergo the normal changes associated with senescence, for example, loss of chlorophyll, soluble protein, and photosynthetic capacity (Wittenbach 1977 Plant Physiol. 59: 1039-1042). Senescence in this leaf is completely reversible when plants are transferred to the light during the first 2 days, but thereafter it becomes an irreversible process. During the reversible stage of senescence the loss of ribulose bisphosphate carboxylase (RuBPCase) quantitated immunochemically, accounted for 80% of the total loss of soluble protein. There was no significant change in RuBPCase activity per milligram of antibody-recognized carboxylase during this stage despite an apparent decline in specific activity on a milligram of soluble protein basis. With the onset of the irreversible stage of senescence there was a rapid decline in activity per milligram of carboxylase, suggesting a loss of active sites. There was no increase in total proteolytic activity during the reversible stage of senescence despite the loss of carboxylase, indicating that this initial loss was not due to an increase in total activity. An 80% increase in proteolytic activity was correlated with the onset of the irreversible stage and the rapid decline in RuBPCase activity per milligram of carboxylase. Delaying senescence with zeatin reduced the rate of loss of carboxylase and delayed both the onset of the irreversible stage and the increase in proteolytic activity to the same degree, suggesting that these events are closely related. The main proteinases present in wheat and responsible for the increase in activity are the thiol proteinases. These proteinases have a high affinity for RuBPCase, exhibiting an apparent K_m at 38 C of 1.8 × 10⁻⁷ m. The K_m for casein was 1.1 × 10⁻⁶ m. If casein is representative of noncarboxylase protein, then the higher affinity for carboxylase may provide an explanation for its apparent preferential loss during the reversible stage of senescence.     

Arginine Inhibits Adsorption of Proteins on Polystyrene Surface

Nonspecific adsorption of protein on solid surfaces causes a reduction of concentration as well as enzyme inactivation during purification and storage. However, there are no versatile inhibitors of the adsorption between proteins and solid surfaces at low concentrations. Therefore, we examined additives for the prevention of protein adsorption on polystyrene particles (PS particles) as a commonly-used material for vessels such as disposable test tubes and microtubes. A protein solution was mixed with PS particles, and then adsorption of protein was monitored by the concentration and activity of protein in the supernatant after centrifugation. Five different proteins bound to PS particles through electrostatic, hydrophobic, and aromatic interactions, causing a decrease in protein concentration and loss of enzyme activity in the supernatant. Among the additives, including arginine hydrochloride (Arg), lysine hydrochloride, guanidine hydrochloride, NaCl, glycine, and glucose, Arg was most effective in preventing the binding of proteins to PS particles as well as activity loss. Moreover, even after the mixing of protein and PS particles, the addition of Arg caused desorption of the bound protein from PS particles. This study demonstrated a new function of Arg, which expands the potential for application of Arg to proteins.     

A radiochemical study of irreversible protein loss on high-performance liquid chromatography column frits

Much success has been achieved in the separation and purification of a wide range of proteins using various high-pressure liquid chromatography techniques. Quantitative analyses of proteins which require 100% mass recovery of the protein are still beset with problems, especially when the total injected amount of protein decreases to below 10 micrograms. Stainless-steel frits have been cited for their deleterious effects on chromatography in general. In addition, the frits have specifically been found to be a significant contributor to irreversible protein loss--particularly when protein sample sizes are on the order of 1 microgram or less. The findings presented below should therefore be of concern to those using HPLC for protein work.     

Quantitative fluorescence correlation spectroscopy reveals a 1000-fold increase in lifetime of protein functionality

We have investigated dilute protein solutions with fluorescence correlation spectroscopy (FCS) and have observed that a rapid loss of proteins occurs from solution. It is commonly assumed that such a loss is the result of protein adsorption to interfaces. A protocol was developed in which this mode of protein loss can be prevented. However, FCS on fluorescent protein (enhanced green fluorescent protein, mCherry, and mStrawberry) solutions enclosed by adsorption-protected interfaces still reveals a decrease of the fluorescent protein concentration, while the diffusion time is stable over long periods of time. We interpret this decay as a loss of protein functionality, probably caused by denaturation of the fluorescent proteins. We show that the typical lifetime of protein functionality in highly dilute, approximately single molecule per femtoliter solutions can be extended more than 1000-fold (typically from a few hours to >40 days) by adding compounds with surfactant behavior. No direct interactions between the surfactant and the fluorescent proteins were observed from the diffusion time measured by FCS. A critical surfactant concentration of more than 23 μM was required to achieve the desired protein stabilization for Triton X-100. The surfactant does not interfere with DNA-protein binding, because similar observations were made using DNA-cutting restriction enzymes. We associate the occurrence of denaturation of proteins with the activity of water at the water-protein interface, which was recently proposed in terms of the “water attack model”. Our observations suggest that soluble biomolecules can extend an influence over much larger distances than suggested by their actual volume.     

Outer Membrane Proteins Ail and OmpF of Yersinia pestis Are Involved in the Adsorption of T7-Related Bacteriophage Yep-phi

Yep-phi is a T7-related bacteriophage specific to Yersinia pestis, and it is routinely used in the identification of Y. pestis in China. Yep-phi infects Y. pestis grown at both 20°C and 37°C. It is inactive in other Yersinia species irrespective of the growth temperature. Based on phage adsorption, phage plaque formation, affinity chromatography, and Western blot assays, the outer membrane proteins of Y. pestis Ail and OmpF were identified to be involved, in addition to the rough lipopolysaccharide, in the adsorption of Yep-phi. The phage tail fiber protein specifically interacts with Ail and OmpF proteins, and residues 518N, 519N, and 523S of the phage tail fiber protein are essential for the interaction with OmpF, whereas residues 518N, 519N, 522C, and 523S are essential for the interaction with Ail. This is the first report to demonstrate that membrane-bound proteins are involved in the adsorption of a T7-related bacteriophage. The observations highlight the importance of the tail fiber protein in the evolution and function of various complex phage systems and provide insights into phage-bacterium interactions.     

Analysis and prediction of inter-strand packing distances between beta-sheets of globular proteins

Any two beta-strands belonging to two different beta-sheets in a protein structure are considered to pack interactively if each beta-strand has at least one residue that undergoes a loss of one tenth or more of its solvent contact surface area upon packing. A data set of protein 3-D structures (determined at 2.5 A resolution or better), corresponding to 428 protein chains, contains 1986 non-identical pairs of beta-strands involved in interactive packing. The inter-axial distance between these is significantly correlated to the weighted sum of the volumes of the interacting residues at the packing interface. This correlation can be used to predict the changes in the inter-sheet distances in equivalent beta-sheets in homologous proteins and, therefore, is of value in comparative modelling of proteins.     

De novo design of the hydrophobic core of ubiquitin.

We have previously reported the development and evaluation of a computational program to assist in the design of hydrophobic cores of proteins. In an effort to investigate the role of core packing in protein structure, we have used this program, referred to as Repacking of Cores (ROC), to design several variants of the protein ubiquitin. Nine ubiquitin variants containing from three to eight hydrophobic core mutations were constructed, purified, and characterized in terms of their stability and their ability to adopt a uniquely folded native-like conformation. In general, designed ubiquitin variants are more stable than control variants in which the hydrophobic core was chosen randomly. However, in contrast to previous results with 434 cro, all designs are destabilized relative to the wild-type (WT) protein. This raises the possibility that beta-sheet structures have more stringent packing requirements than alpha-helical proteins. A more striking observation is that all variants, including random controls, adopt fairly well-defined conformations, regardless of their stability. This result supports conclusions from the cro studies that non-core residues contribute significantly to the conformational uniqueness of these proteins while core packing largely affects protein stability and has less impact on the nature or uniqueness of the fold. Concurrent with the above work, we used stability data on the nine ubiquitin variants to evaluate and improve the predictive ability of our core packing algorithm. Additional versions of the program were generated that differ in potential function parameters and sampling of side chain conformers. Reasonable correlations between experimental and predicted stabilities suggest the program will be useful in future studies to design variants with stabilities closer to that of the native protein. Taken together, the present study provides further clarification of the role of specific packing interactions in protein structure and stability, and demonstrates the benefit of using systematic computational methods to predict core packing arrangements for the design of proteins.     

The Adsorption of Bacteriophage ϕX174 to its Host

The adsorption of purified ϕX174 to E. coli C and to E. coli C cell walls was investigated. Adsorption was measured by assaying for unadsorbed plaque formers. The amount of irreversible and reversible adsorption depends upon pH and divalent ion concentration. Maximum irreversible adsorption occurs in 0.1 M CaCl₂ at 36°C. There is no detectable reversible adsorption at conditions of pH and CaCl₂ concentration optimum for irreversible adsorption. Under these optimum conditions, diffusion is not the rate-limiting factor, and the encounter efficiency appears to be low. The rate constant is 1.0 × 10^-10 ml/sec. Phages adsorbed irreversibly to live cells cause infection and to the isolated cell walls apparently cause release of DNA. There is a specific ϕX174 receptor site on the mucocomplex portion of the cell wall.