Improvement of protein stability in protein microarrays

Protein stability in microarrays was improved using protein stabilizers. PEG 200 at 30% (w/v) was the most efficient stabilizer giving over 4-fold improvement in protein stability compared to without the stabilizer. PEG 200 above 10% (w/v) in the array solution prevented the evaporation of water in the sample and thereby improved protein stability in the microarray. When the streptavidin-biotin binding reaction was performed under optimized conditions, biotin-BSA-fluorescein isothiocyanate (FITC) was detected from 1 ng ml^–1 to 5 g ml^–1 by fluorescence analysis.     

Interplay between protein homeostasis networks in protein aggregation and proteotoxicity

The misfolding and aggregation of disease proteins is characteristic of numerous neurodegenerative diseases. Particular neuronal populations are more vulnerable to proteotoxicity while others are more apt to tolerate the misfolding and aggregation of disease proteins. Thus, the cellular environment must play a significant role in determining whether disease proteins are converted into toxic or benign forms. The endomembrane network of eukaryotes divides the cell into different subcellular compartments that possess distinct sets of molecular chaperones and protein interaction networks. Chaperones act as agonists and antagonists of disease protein aggregation to prevent the accumulation of toxic intermediates in the aggregation pathway. Interacting partners can also modulate the conformation and localization of disease proteins and thereby influence proteotoxicity. Thus, interplay between these protein homeostasis network components can modulate the self‐association of disease proteins and determine whether they elicit a toxic or benign outcome. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 229–236, 2010. 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     

High yield recombinant silk-like protein production in transgenic plants through protein targeting

DP1B is a synthetic analogue of spider dragline silk protein. It can be spun to form silk fiber. Previously, it had been expressed in transgenic plants, showing the general feasibility of the plant-based DP1B production. However, success of such a plant-based platform requires a great increase of DP1B productivity in plant cells to reduce production cost. This report describes a protein targeting approach to accumulate DP1B in apoplast, ER lumen, and vacuole in Arabidopsis cells, by utilizing appropriate combinations of sporamin-targeting determinant peptides and ER retention peptide. The approach has dramatically enhanced DP1B accumulation, resulting in high production yield. The accumulation can be as high as 8.5 and 6.7% total soluble protein in leaf tissue by targeting to apoplast and ER lumen, respectively, or as high as 18 and 8.2% total soluble protein in seeds by targeting to ER lumen and vacuole, respectively. However, the vacuole targeting in leaves and the apoplast targeting in seeds have failed to accumulate full length DP1B molecules or any DP1B at all, respectively, suggesting that they may not be suitable for applications in leaf tissues and seeds. Data in this study recommend a combination of seed-specific expression and ER-targeting as one of the best strategies for yield enhancement of plant-based DP1B production.     

ATP-dependent protein kinases in bacteria

Protein phosphorylation has been shown to occur in over fifty different bacterial species and, therefore, seems to be a universal device among prokaryotes. Most of the protein kinases responsible for this modification of proteins share the common property of using adenosine triphosphate as phosphoryl donnor. However, they differ from one another in a number of structural and functional aspects. Namely, they exhibit a varying acceptor amino acid specificity and can be classified, on this basis, in three main groups: protein-histidine kinases, protein-serine/threonine kinases and protein-tyrosine kinases. © 1993 Wiley-Liss, Inc.     

Structural motifs in protein cores and at protein–protein interfaces are different

Structures of proteins and protein–protein complexes are determined by the same physical principles and thus share a number of similarities. At the same time, there could be differences because in order to function, proteins interact with other molecules, undergo conformations changes, and so forth, which might impose different restraints on the tertiary versus quaternary structures. This study focuses on structural properties of protein–protein interfaces in comparison with the protein core, based on the wealth of currently available structural data and new structure‐based approaches. The results showed that physicochemical characteristics, such as amino acid composition, residue–residue contact preferences, and hydrophilicity/hydrophobicity distributions, are similar in protein core and protein–protein interfaces. On the other hand, characteristics that reflect the evolutionary pressure, such as structural composition and packing, are largely different. The results provide important insight into fundamental properties of protein structure and function. At the same time, the results contribute to better understanding of the ways to dock proteins. Recent progress in predicting structures of individual proteins follows the advancement of deep learning techniques and new approaches to residue coevolution data. Protein core could potentially provide large amounts of data for application of the deep learning to docking. However, our results showed that the core motifs are significantly different from those at protein–protein interfaces, and thus may not be directly useful for docking. At the same time, such difference may help to overcome a major obstacle in application of the coevolutionary data to docking—discrimination of the intramolecular information not directly relevant to docking.     

Patterns of protein protein interactions in salt solutions and implications for protein crystallization

The second osmotic virial coefficients of seven proteins-ovalbumin, ribonuclease A, bovine serum albumin, alpha-lactalbumin, myoglobin, cytochrome c, and catalase-were measured in salt solutions. Comparison of the interaction trends in terms of the dimensionless second virial coefficient b(2) shows that, at low salt concentrations, protein-protein interactions can be either attractive or repulsive, possibly due to the anisotropy of the protein charge distribution. At high salt concentrations, the behavior depends on the salt: In sodium chloride, protein interactions generally show little salt dependence up to very high salt concentrations, whereas in ammonium sulfate, proteins show a sharp drop in b(2) with increasing salt concentration beyond a particular threshold. The experimental phase behavior of the proteins corroborates these observations in that precipitation always follows the drop in b(2). When the proteins crystallize, they do so at slightly lower salt concentrations than seen for precipitation. The b(2) measurements were extended to other salts for ovalbumin and catalase. The trends follow the Hofmeister series, and the effect of the salt can be interpreted as a water-mediated effect between the protein and salt molecules. The b(2) trends quantify protein-protein interactions and provide some understanding of the corresponding phase behavior. The results explain both why ammonium sulfate is among the best crystallization agents, as well as some of the difficulties that can be encountered in protein crystallization.     

Energetics of aliphatic deletions in protein cores

Although core residues can sometimes be replaced by shorter ones without introducing significant changes in protein structure, the energetic consequences are typically large and destabilizing. Many efforts have been devoted to understand and predict changes in stability from analysis of the environment of mutated residues, but the relationships proposed for individual proteins have often failed to describe additional data. We report here 17 apoflavodoxin large-to-small mutations that cause overall protein destabilizations of 0.6-3.9 kcal.mol(-1). By comparing two-state urea and three-state thermal unfolding data, the overall destabilizations observed are partitioned into effects on the N-to-I and on the I-to-U equilibria. In all cases, the equilibrium intermediate exerts a "buffering" effect that reduces the impact of the overall destabilization on the N-to-I equilibrium. The performance of several structure-energetics relationships, proposed to explain the energetics of hydrophobic shortening mutations, has been evaluated by using an apoflavodoxin data set consisting of 14 mutations involving branching-conservative aliphatic side-chain shortenings and a larger data set, including similar mutations implemented in seven model proteins. Our analysis shows that the stability changes observed for any of the different types of mutations (LA, IA, IV, and VA) in either data set are best explained by a combination of differential hydrophobicity and of the calculated volume of the modeled cavity (as previously observed for LA and IA mutations in lysozyme T4). In contrast, sequence conservation within the flavodoxin family, which is a good predictor for charge-reversal stabilizing mutations, does not perform so well for aliphatic shortening ones.     

Determining and visualizing flexibility in protein structures

How to compare the structures of an ensemble of protein conformations is a fundamental problem in structural biology. As has been previously observed, the widely used RMSD measure due to Kabsch, in which a rigid‐body superposition minimizing the least‐squares positional deviations is performed, has its drawbacks when comparing and visualizing a set of flexible protein structures. Here, we develop a method, fleximatch, of protein structure comparison that takes flexibility into account. Based on a distance matrix measure of flexibility, a weighted superposition of distance matrices rather than of atomic coordinates is performed. Subsequently, this allows a consistent determination of (a) a superposition of structures for visualization, (b) a partitioning of the protein structure into rigid molecular components (core atoms), and (c) an atomic mobility measure. The method is suitable for highlighting both particularly flexible and rigid parts of a protein from structures derived from NMR, X‐ray diffraction or molecular simulation. Proteins 2015; 83:820–826. © 2015 Wiley Periodicals, Inc.     

蛋白质的棕榈酰化修饰

棕榈酰化修饰是蛋白质翻译后脂质修饰的重要形式,是调控蛋白质的转运、稳定、定位和功能的重要机制,同时,棕榈酰化修饰还参与多种细胞生物学进程,与许多疾病的发生发展密切相关。本文主要就蛋白质棕榈酰化及其修饰酶与蛋白质功能、相关疾病的关系做一综述。     

古菌蛋白激酶的研究进展

磷酸化是蛋白质翻译后修饰(post-translational modification)的主要方式,可由蛋白激酶、磷酸转移酶、磷酸化酶等多种方式催化进行。其中,由蛋白激酶(protein kinases)/磷酸酶(protein phosphatases)介导的可逆的蛋白磷酸化是细胞中信号转导的重要机制,在DNA复制、转录、蛋白质翻译、DNA损伤修复等生命过程中起广泛的调节作用。目前,古菌中蛋白激酶的研究尚属于初期阶段。虽然磷酸化蛋白质组学研究表明,古菌中存在大量的磷酸化蛋白质,但是我们对其具体催化作用的酶及调控机制尚不清楚。本文总结了古菌中已报道的蛋白激酶所参与的生命过程,包括古菌的DNA代谢、细胞代谢、细胞周期和运动机制等四个方面,并对今后的研究提出展望。     

Design and structure of two new protein cages illustrate successes and ongoing challenges in protein engineering

In recent years, new protein engineering methods have produced more than a dozen symmetric, self‐assembling protein cages whose structures have been validated to match their design models with near‐atomic accuracy. However, many protein cage designs that are tested in the lab do not form the desired assembly, and improving the success rate of design has been a point of recent emphasis. Here we present two protein structures solved by X‐ray crystallography of designed protein oligomers that form two‐component cages with tetrahedral symmetry. To improve on the past tendency toward poorly soluble protein, we used a computational protocol that favors the formation of hydrogen‐bonding networks over exclusively hydrophobic interactions to stabilize the designed protein–protein interfaces. Preliminary characterization showed highly soluble expression, and solution studies indicated successful cage formation by both designed proteins. For one of the designs, a crystal structure confirmed at high resolution that the intended tetrahedral cage was formed, though several flipped amino acid side chain rotamers resulted in an interface that deviates from the precise hydrogen‐bonding pattern that was intended. A structure of the other designed cage showed that, under the conditions where crystals were obtained, a noncage structure was formed wherein a porous 3D protein network in space group I2₁3 is generated by an off‐target twofold homomeric interface. These results illustrate some of the ongoing challenges of developing computational methods for polar interface design, and add two potentially valuable new entries to the growing list of engineered protein materials for downstream applications.     

Structure-function relationships in a bacterial DING protein

A recombinant DING protein from Pseudomonas fluorescens has been previously shown to have a phosphate-binding site, and to be mitogenic for human cells. Here we report the three-dimensional structure of the protein, confirming a close similarity to the "Venus flytrap" structure seen in other human and bacterial phosphate-binding proteins. Site-directed mutagenesis confirms the role of a key residue involved in phosphate binding, and that the mitogenic activity is not dependent on this property. Deletion of one of the two hinged domains that constitute the Venus flytrap also eliminates phosphate binding whilst enhancing mitogenic activity.     

In-cell protein NMR and protein leakage

In-cell nuclear magnetic resonance spectroscopy is a tool for studying proteins under physiologically relevant conditions. In some instances, however, protein signals from leaked protein are observed in the liquid surrounding the cells. Here, we examine the expression of four proteins in Escherichia coli. We describe the controls that should be used for in-cell NMR experiments and show that leakage is likely when the protein being studied exceeds ～20% of the total cellular protein.     

Mechanisms of myelin basic protein and proteolipid protein targeting in oligodendrocytes (Review)

Summary

The segregation of proteins to specific cellular membranes is recognized as a common phenomenon. In oligodendrocytes of the central nervous system, localization of certain proteins to select regions of the plasma membrane gives rise to the myelin membrane. Whilst the fundamental structure and composition of myelin is well understood, less is known of the mechanisms by which the constituent proteins are specifically recruited to those regions of plasma membrane that are forming myelin. The two principal proteins of myelin, the myelin basic protein and proteolipid protein, differ greatly in character and sites of synthesis. The message for myelin basic protein is selectively translocated to the ends of the cell processes, where it is translated on free ribosomes and is incorporated directly into the membrane. Proteolipid protein synthesized at the rough endoplasmic reticulum, processed through the Golgi apparatus, and presumably transported via vesicles to the myelin membrane. This review examines the mechanisms by which these two proteins are targeted to the myelin membrane.     

Novel phosphorylation-dependent regulation in an unstructured protein

This work explores how phosphorylation of an unstructured protein region in inhibitor-2 (I2) regulates protein phosphatase-1 (PP1) enzyme activity using molecular dynamics (MD). Free I2 is largely unstructured; however, when bound to PP1, three segments adopt a stable structure. In particular, an I2 helix (i-helix) blocks the PP1 active site and inhibits phosphatase activity. I2 phosphorylation in the PP1-I2 complex activates phosphatase activity without I2 dissociation. The I2 Thr74 regulatory phosphorylation site is in an unstructured domain in PP1-I2. PP1-I2 MD demonstrated that I2 phosphorylation promotes early steps of PP1-I2 activation in explicit solvent models. Moreover, phosphorylation-dependent activation occurred in PP1-I2 complexes derived from I2 orthologs with diverse sequences from human, yeast, worm, and protozoa. This system allowed exploration of features of the 73-residue unstructured human I2 domain critical for phosphorylation-dependent activation. These studies revealed that components of I2 unstructured domain are strategically positioned for phosphorylation responsiveness including a transient α-helix. There was no evidence that electrostatic interactions of I2 phosphothreonine74 influenced PP1-I2 activation. Instead, phosphorylation altered the conformation of residues around Thr74. Phosphorylation uncurled the distance between I2 residues Glu71 to Tyr76 to promote PP1-I2 activation, whereas reduced distances reduced activation. This I2 residue Glu71 to Tyr76 distance distribution, independently from Thr74 phosphorylation, controls I2 i-helix displacement from the PP1 active site leading to PP1-I2 activation.     

Sub-microsecond protein folding

We have investigated the structure, equilibria, and folding kinetics of an engineered 35-residue subdomain of the chicken villin headpiece, an ultrafast-folding protein. Substitution of two buried lysine residues by norleucine residues stabilizes the protein by 1 kcal/mol and increases the folding rate sixfold, as measured by nanosecond laser T-jump. The folding rate at 300 K is (0.7 micros)(-1) with little or no temperature dependence, making this protein the first sub-microsecond folder, with a rate only twofold slower than the theoretically predicted speed limit. Using the 70 ns process to obtain the effective diffusion coefficient, the free energy barrier height is estimated from Kramers theory to be less than approximately 1 kcal/mol. X-ray crystallographic determination at 1A resolution shows no significant change in structure compared to the single-norleucine-substituted molecule and suggests that the increased stability is electrostatic in origin. The ultrafast folding rate, very accurate X-ray structure, and small size make this engineered villin subdomain an ideal system for simulation by atomistic molecular dynamics with explicit solvent.     

Computational methods in protein structure prediction

This review presents the advances in protein structure prediction from the computational methods perspective. The approaches are classified into four major categories: comparative modeling, fold recognition, first principles methods that employ database information, and first principles methods without database information. Important advances along with current limitations and challenges are presented.     

Monitoring of conformational change in maltose binding protein using split green fluorescent protein

In this study, we describe a novel method for the detection of conformational changes in proteins, which is predicated on the reconstitution of split green fluorescent protein (GFP). We employed fluorescence complementation assays for the monitoring of the conformationally altered proteins. In particular, we used maltose binding protein (MBP) as a model protein, as MBP undergoes a characteristic hinge-twist movement upon substrate binding. The common feature of this approach is that GFP, as a reporter protein, splits into two non-fluorescent fragments, which are genetically fused to the N- and C-termini of MBP. Upon binding to maltose, the chromophores move closer together, resulting in the generation of fluorescence. This split GFP method also involves the reconstitution of GFP, which is determined via observations of the degree to which fluorescence intensity is restored. As a result, reconstituted GFP has been observed to generate fluorescence upon maltose binding in vitro, thereby allowing for the direct detection of changes in fluorescence intensity in response to maltose, in a concentration- and time-dependent fashion. Our findings showed that the fluorescence complementation assay can be used to monitor the conformational alterations of a target protein, and this ability may prove useful in a number of scientific and medical applications.