Determination of optimal non-denaturing elution conditions from affinity columns by a solid-phase screen

The purification of biological macromolecules by affinity chromatography is a widespread technique used to separate a protein from other biological components. However, this method may destroy the protein's physiological activity because elution conditions aimed to dissociate the protein of interest from the high-affinity matrix often irreversibly denature it. In the present work, we have developed a solid-phase assay to determine the optimal elution conditions for any buffer (in two steps) by determining (i) the lowest buffer concentration yielding maximum dissociation from the immobilized component and (ii) the highest buffer concentration that can be used without the loss of the protein's binding activity. Any buffer that can be reasonably used between these defined concentrations is suitable for elution within this interval. The screen is easily performed within a few hours and only requires nanograms to a few micrograms of protein. As an example, we demonstrate that more than 95% of the human transferrin receptor bound to a transferrin-sepharose ligand affinity column can be eluted with full binding activity at KSCN concentrations between 232 and 414 nM, whereas elution with urea is not suitable to purify fully functional protein.     

Functional modes and residue flexibility control the anisotropic response of guanylate kinase to mechanical stress

The coupling between the mechanical properties of enzymes and their biological activity is a well-established feature that has been the object of numerous experimental and theoretical works. In particular, recent experiments show that enzymatic function can be modulated anisotropically by mechanical stress. We study such phenomena using a method for investigating local flexibility on the residue scale that combines a reduced protein representation with Brownian dynamics simulations. We performed calculations on the enzyme guanylate kinase to study its mechanical response when submitted to anisotropic deformations. The resulting modifications of the protein's rigidity profile can be related to the changes in substrate binding affinity observed experimentally. Further analysis of the principal components of motion of the trajectories shows how the application of a mechanical constraint on the protein can disrupt its dynamics, thus leading to a decrease of the enzyme's catalytic rate. Eventually, a systematic probe of the protein surface led to the prediction of potential hotspots where the application of an external constraint would produce a large functional response both from the mechanical and dynamical points of view. Such enzyme-engineering approaches open the possibility to tune catalytic function by varying selected external forces.     

Neutral genetic drift can alter promiscuous protein functions, potentially aiding functional evolution

Background  

Many of the mutations accumulated by naturally evolving proteins are neutral in the sense that they do not significantly alter a protein's ability to perform its primary biological function. However, new protein functions evolve when selection begins to favor other, "promiscuous" functions that are incidental to a protein's original biological role. If mutations that are neutral with respect to a protein's primary biological function cause substantial changes in promiscuous functions, these mutations could enable future functional evolution.     

Automatic identification and representation of protein binding sites for molecular docking.

Molecular docking is a popular way to screen for novel drug compounds. The method involves aligning small molecules to a protein structure and estimating their binding affinity. To do this rapidly for tens of thousands of molecules requires an effective representation of the binding region of the target protein. This paper presents an algorithm for representing a protein's binding site in a way that is specifically suited to molecular docking applications. Initially the protein's surface is coated with a collection of molecular fragments that could potentially interact with the protein. Each fragment, or probe, serves as a potential alignment point for atoms in a ligand, and is scored to represent that probe's affinity for the protein. Probes are then clustered by accumulating their affinities, where high affinity clusters are identified as being the "stickiest" portions of the protein surface. The stickiest cluster is used as a computational binding "pocket" for docking. This method of site identification was tested on a number of ligand-protein complexes; in each case the pocket constructed by the algorithm coincided with the known ligand binding site. Successful docking experiments demonstrated the effectiveness of the probe representation.     

Novel role for pectin methylesterase in Arabidopsis: a new function showing ribosome-inactivating protein (RIP) activity

Ribosome-inactivating proteins (RIPs, EC 3.2.2.22) are plant enzymes that can inhibit the translation process by removing single adenine residues of the large rRNA. These enzymes are known to function in defense against pathogens, but their biological role is unknown, partly due to the absence of work on RIPs in a model plant. In this study, we purified a protein showing RIP activity from Arabidopsis thaliana by employing chromatography separations coupled with an enzymatic activity. Based on N-terminal and internal amino acid sequencing, the RIP purified was identified as a mature form of pectin methylesterase (PME, At1g11580). The purified native protein showed both PME and RIP activity. PME catalyzes pectin deesterification, releasing acid pectin and methanol, which cause cell wall changes. We expressed the full-length and mature form of cDNA clones into an expression vector and transformed it in Escherichia coli for protein expression. The recombinant PME proteins (full-length and mature) expressed in E. coli did not show either PME or RIP activity, suggesting that post-translational modifications are important for these enzymatic activities. This study demonstrates a new function for an old enzyme identified in a model plant and discusses the possible role of a protein's conformational changes corresponding to its dual enzymatic activity.     

Relationship between protein structure and methionine oxidation in recombinant human alpha 1-antitrypsin

alpha 1-Antitrypsin is a metastable and conformationally flexible protein that belongs to the serpin family of protease inhibitors. Although it is known that methionine oxidation in the protein's active site results in a loss of biological activity, there is little specific knowledge regarding the reactivity of each of the protein's methionine residues. In this study, we have used peptide mapping to study the oxidation kinetics of each of alpha 1-antitrypsin's methionines in alpha 1-AT((C232S)) as well as M351L and M358V mutants. These kinetic studies establish that Met1, Met226, Met242, Met351, and Met358 are reactive with hydrogen peroxide at neutral pH and that each reactive methionine is oxidized in a bimolecular, rather than coupled, mechanism. Analysis of Met226, Met351, and Met358 oxidation provides insights regarding the structure of alpha 1-antitrypsin's active site that allow us to relate conformation to experimentally observed reactivity. The relationship between solution pH and methionine oxidation was also examined to evaluate methionine reactivity under conditions that perturb the native structure. Methionine oxidation data show that at pH 5, global conformational changes occur that alter the oxidation susceptibility of each of alpha 1-antitrypsin's 10 methionine residues. Between pH 6 and 9, however, more localized conformational changes occur that affect primarily the reactivity of Met242. In sum, this work provides a detailed analysis of methionine oxidation in alpha 1-antitrypsin and offers new insights into the protein's solution structure.     

Applications of analytical ultracentrifugation to protein size-and-shape distribution and structure-and-function analyses

The rebirth of modern analytical ultracentrifugation (AUC) began in 1990s. Since then many advanced AUC detectors have been developed that provide a vast range of versatile choices when characterizing the physical and chemical features of macromolecules. In addition, there have been remarkable advances in software that allow the analysis of AUC data using more sophisticated models, including quaternary structures, conformational changes, and biomolecular interactions. Here we report the application of AUC to protein size-and-shape distribution analysis and structure-and-function analysis in the presence of ligands or lipids. Using band-sedimentation velocity, quaternary structural changes and an enzyme's catalytic activity can be observed simultaneously. This provides direct insights into the correlation between quaternary structure and catalytic activity of the enzyme. On the other hand, also in this study, we have applied size-and-shape distribution analysis to a lipid-binding protein in either an aqueous or lipid environment. The sedimentation velocity data for the protein with or without lipid were evaluated using the c(s,f(r)) two-dimensional distribution model, which provides a precise and quantitative means of analyzing the protein's conformational changes.     

Quantifying the kinetic stability of hyperstable proteins via time-dependent SDS trapping

Globular proteins are usually in equilibrium with unfolded conformations, whereas kinetically stable proteins (KSPs) are conformationally trapped by their high unfolding transition state energy. Kinetic stability (KS) could allow proteins to maintain their activity under harsh conditions, increase a protein's half-life, or protect against misfolding-aggregation. Here we show the development of a simple method for quantifying a protein's KS that involves incubating a protein in SDS at high temperature as a function of time, running the unheated samples on SDS-PAGE, and quantifying the bands to determine the time-dependent loss of a protein's SDS resistance. Six diverse proteins, including two monomer, two dimers, and two tetramers, were studied by this method, and the kinetics of the loss of SDS resistance correlated linearly with their unfolding rate determined by circular dichroism. These results imply that the mechanism by which SDS denatures proteins involves conformational trapping, with a trapping rate that is determined and limited by the rate of protein unfolding. We applied the SDS trapping of proteins (S-TraP) method to superoxide dismutase (SOD) and transthyretin (TTR), which are highly KSPs with native unfolding rates that are difficult to measure by conventional spectroscopic methods. A combination of S-TraP experiments between 75 and 90 °C combined with Eyring plot analysis yielded an unfolding half-life of 70 ± 37 and 18 ± 6 days at 37 °C for SOD and TTR, respectively. The S-TraP method shown here is extremely accessible, sample-efficient, cost-effective, compatible with impure or complex samples, and will be useful for exploring the biological and pathological roles of kinetic stability.     

High affinity ligand binding is not essential for granulocyte-macrophage colony-stimulating factor receptor activation.

The high affinity receptor of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is a heterodimer composed of two members of the cytokine receptor superfamily. GM-CSF binds to the alpha-subunit (GM-R alpha) with low affinity and to the receptor alpha beta complex (GM-R alpha beta) with high affinity. The GM-CSF.GM-R alpha beta complex is responsible for biological activity. Interactions of the N-terminal helix of mouse GM-CSF with mGM-R alpha beta were examined by introducing single alanine substitutions of hydrophilic residues in this region of mGM-CSF. The consequences of these substitutions were evaluated by receptor binding and biological assays. Although all mutant proteins exhibited near wild-type biological activity, most were defective in high affinity receptor binding. In particular, substitution of Glu-21 with alanine abrogated high affinity binding leaving low affinity binding unaffected. Despite near wild-type biological activity, no detectable binding interaction of this mutant with mGM-R beta in the context of mGM-R alpha beta was observed. Cross-linking studies showed an apparent interaction of this mutant protein with mGM-R alpha beta. The deficient receptor binding characteristics and near wild-type biological activity of this mutant protein demonstrate that mGM-CSF receptor activation can occur independently of high affinity binding, suggesting that conformational changes in the receptor induced by mGM-CSF binding generate an active ligand-receptor complex.     

A 63-kDa protein with androgen-binding activity is not from the androgen receptor.

We have found a new protein in the heart of rat and mice that can be selectively and covalently labelled with the synthetic androgen analog mibolerone. Binding is specific as it can be displaced by excess radioinert ligand. The protein is prominently expressed in liver, kidney, and heart, but not in skeletal muscle. It is water soluble and found in the cytosol. Under denaturing conditions it has a molecular weight of 63,000 and appears on two-dimensional gels with an isoelectric point of 6.3. The protein's affinity for androgen is lower than that of the androgen receptor and it is about 100-fold more abundant than the receptor in the heart. Expression of the protein is not induced by androgen. The presence of this protein in testicular feminization (tfm) mice with a genetical defect of the androgen receptor rules out that it is the androgen receptor or a portion thereof. The biological role of this protein is not yet known.     

Effects of D-myo-inositol 1-phosphate on the transfer function of phosphatidylinositol transfer protein alpha

The lipid metabolite D-myo-inositol-1-phosphate is shown to increase the phospholipid transfer activity of phosphatidylinositol transfer protein alpha from liposomal and liver microsomal membranes. Dose-response curves indicated substantial enhancements of transfer in the low mM range that upon normalization were independent of membrane composition or the identity of the transferred phospholipid. The unnormalized effect is potentiated by anionic membrane surface charge and substantial membrane phosphatidylethanolamine content consistent with alterations of the protein's membrane binding affinity and alterations of surface electrostatic interactions as contributing factors.     

Bioactive peptide design using the Resonant Recognition Model

With a large number of DNA and protein sequences already known, the crucial question is to find out how the biological function of these macromolecules is "written" in the sequence of nucleotides or amino acids. Biological processes in any living organism are based on selective interactions between particular bio-molecules, mostly proteins. The rules governing the coding of a protein's biological function, i.e. its ability to selectively interact with other molecules, are still not elucidated. In addition, with the rapid accumulation of databases of protein primary structures, there is an urgent need for theoretical approaches that are capable of analysing protein structure-function relationships. The Resonant Recognition Model (RRM) [1, 2] is one attempt to identify the selectivity of protein interactions within the amino acid sequence. The RRM [1, 2] is a physico-mathematical approach that interprets protein sequence linear information using digital signal processing methods. In the RRM the protein primary structure is represented as a numerical series by assigning to each amino acid in the sequence a physical parameter value relevant to the protein's biological activity. The RRM concept is based on the finding that there is a significant correlation between spectra of the numerical presentation of amino acids and their biological activity. Once the characteristic frequency for a particular protein function/interaction is identified, it is possible then to utilize the RRM approach to predict the amino acids in the protein sequence, which predominantly contribute to this frequency and thus, to the observed function, as well as to design de novo peptides having the desired periodicities. As was shown in our previous studies of fibroblast growth factor (FGF) peptidic antagonists [2, 3] and human immunodeficiency virus (HIV) envelope agonists [2, 4], such de novo designed peptides express desired biological function. This study utilises the RRM computational approach to the analysis of oncogene and proto-oncogene proteins. The results obtained have shown that the RRM is capable of identifying the differences between the oncogenic and proto-oncogenic proteins with the possibility of identifying the "cancer-causing" features within their protein primary structure. In addition, the rational design of bioactive peptide analogues displaying oncogenic or proto-oncogenic-like activity is presented here.     

Tyrosine kinase catalyzed phosphorylation and inactivation of the inhibitor protein of the cAMP-dependent protein kinase

The inhibitor protein of the cAMP-dependent protein kinase is a potential high affinity regulator of cAMP function. We now show that it is phosphorylated in Tyr7 by the intrinsic tyrosine kinase activity of epidermal growth factor receptor. The phosphorylated form can be readily separated from the unphosphorylated protein by high pressure liquid chromatography which has permitted the isolation of stoichiometrically phosphorylated protein. Using this method, it has been demonstrated that this phosphorylation, which occurs within the inhibitor protein's active domain, results in a 6 to 9-fold decrease in inhibitory potency. Possibly, a component of growth control could be the coupling of tyrosine kinase activity to cAMP-mediated cellular proliferation via the regulation of the efficacy of the inhibitor protein.     

Mutagenesis of bacterial elongation factor Tu at lysine 136. A conserved amino acid in GTP regulatory proteins

We have studied the effects of specific amino acid replacements in EF-Tu upon the protein's interactions with guanine nucleotides and elongation factor Ts (EFTs). We found that alterations at the lysine residue of the Asn-Lys-Cys-Asp sequence, the guanine ring-binding sequence, differentially affect the protein's ability to bind guanine nucleotides. Wild type EF-Tu (Lys-136) binds GDP and GTP much more tightly than do many of the altered proteins. Replacing lysine by arginine lowers the protein's affinity for GDP by about 20-fold relative to the change in its affinity for EF-Ts. Substitutions at residue 136 by glutamine (K136Q) and glutamic acid (K136E) further lower the protein relative affinity for GDP by factors of about 4 and 10, respectively. In contrast, replacement of the residue by isoleucine (K136I) eliminates guanine nucleotide binding as well as EF-Ts binding. Apparently, the distortion of this loop by substitution at residue 136 of a bulky hydrophobic residue can hamper the binding for both substrates or disrupt the folding of the protein. All altered proteins except EF-Tu(K136I) are able to bind tRNA(Phe); however, they require much higher concentrations of GTP than wild type EF-Tu. In minimal media, Escherichia coli cells harboring plasmids encoding EF-Tu(K136E) or EF-Tu(K136Q) suffer growth retardation relative to cells bearing the same plasmid encoding wild type EF-Tu. Co-transformation of these cells with a compatible plasmid bearing the EF-Ts gene reverses this growth problem. The growth retardation effect of some of the altered proteins can be explained by their sequestering EF-Ts. These results indicate that EF-Ts is essential to the growth of E. coli and suggest a technique for studying EF-Ts mutants as well as for identifying other guanine nucleotide exchange enzymes.     

Enzymatic mechanism controls redox-mediated protein-DNA interactions at the replication origin of kinetoplast DNA minicircles

Kinetoplast DNA (kDNA) is the mitochondrial DNA of trypanosomatids. Its major components are several thousand topologically interlocked DNA minicircles. Their replication origins are recognized by universal minicircle sequence-binding protein (UMSBP), a CCHC-type zinc finger protein, which has been implicated with minicircle replication initiation and kDNA segregation. Interactions of UMSBP with origin sequences in vitro have been found to be affected by the protein's redox state. Reduction of UMSBP activates its binding to the origin, whereas UMSBP oxidation impairs this activity. The role of redox in the regulation of UMSBP in vivo was studied here in synchronized cell cultures, monitoring both UMSBP origin binding activity and its redox state, throughout the trypanosomatid cell cycle. These studies indicated that UMSBP activity is regulated in vivo through the cell cycle dependent control of the protein's redox state. The hypothesis that UMSBP's redox state is controlled by an enzymatic mechanism, which mediates its direct reduction and oxidation, was challenged in a multienzyme reaction, reconstituted with pure enzymes of the trypanosomal major redox-regulating pathway. Coupling in vitro of this reaction with a UMSBP origin-binding reaction revealed the regulation of UMSBP activity through the opposing effects of tryparedoxin and tryparedoxin peroxidase. In the course of this reaction, tryparedoxin peroxidase directly oxidizes UMSBP, revealing a novel regulatory mechanism for the activation of an origin-binding protein, based on enzyme-mediated reversible modulation of the protein's redox state. This mode of regulation may represent a regulatory mechanism, functioning as an enzyme-mediated, redox-based biological switch.     

Regulatory role for a conserved motif adjacent to the homeodomain of Hox10 proteins

Development of the vertebrate axial skeleton requires the concerted activity of several Hox genes. Among them, Hox genes belonging to the paralog group 10 are essential for the formation of the lumbar region of the vertebral column, owing to their capacity to block rib formation. In this work, we explored the basis for the rib-repressing activity of Hox10 proteins. Because genetic experiments in mice demonstrated that Hox10 proteins are strongly redundant in this function, we first searched for common motifs among the group members. We identified the presence of two small sequences flanking the homeodomain that are phylogenetically conserved among Hox10 proteins and that seem to be specific for this group. We show here that one of these motifs is required but not sufficient for the rib-repressing activity of Hox10 proteins. This motif includes two potential phosphorylation sites, which are essential for protein activity as their mutation to alanines resulted in a total loss of rib-repressing properties. Our data indicates that this motif has a significant regulatory function, modulating interactions with more N-terminal parts of the Hox protein, eventually triggering the rib-repressing program. In addition, this motif might also regulate protein activity by alteration of the protein's DNA-binding affinity through changes in the phosphorylation state of two conserved tyrosine residues within the homeodomain.     

Comparing the effect on protein stability of methionine oxidation versus mutagenesis: steps toward engineering oxidative resistance in proteins.

The biological activity of some proteins is known to be sensitive to oxidative damage caused by a variety of oxidants. The model protein staphylococcal nuclease was used to explore the effect on protein structural stability of oxidizing methionine to the sulfoxide form. These effects were compared with the effects of substituting methionines with isoleucine and leucine, a potential strategy for stabilizing proteins against oxidative damage. Wild-type nuclease and various mutants were oxidized with hydrogen peroxide. Stabilities of both oxidized and unoxidized proteins were determined by guanidine hydrochloride denaturation. Oxidation destabilized the wild-type protein by over 4 kcal/mol. This large loss of stability supports the idea that in some cases loss of biological activity is linked to disruption of the protein native state. Comparison of mutant protein's stability losses upon oxidation showed that methionines 65 and 98 had a much greater destabilizing effect when oxidized than methionines 26 or 32. While substitution of methionine 98 carried as great an energetic penalty as oxidation, substitution at position 65 was less disruptive than oxidation. Thus a simple substitution mutagenesis strategy to protect a protein against oxidative destabilization is practical for some methionine residues.     

Fine-structure map of the human ribosomal protein gene RPS14.

We have used polymerase chain reaction-mediated chemical mutagenesis (J.-J. Diaz, D. D. Rhoads, and D. J. Roufa, BioTechniques 11:204-211, 1991) to analyze the genetic fine structure of a human ribosomal protein gene, RPS14. Eighty-three DNA clones containing 158 random single-base substitution mutations were isolated. Mutant RPS14 alleles were tested for biological activity by transfection into cultured Chinese hamster cells. The resulting data permitted us to construct a map of the S14-coding sequence that is comparable to available fine-structure genetic maps of many prokaryotic and lower eukaryotic gene loci. As predicted from the multiplicity of protein-protein and protein-RNA interactions required for ribosomal protein transport and assembly into functional ribosomal subunits, the distribution of null mutations indicated that S14 is composed of multiple, functionally distinct polypeptide domains. Two of the protein's internal domains, designated domains B and D, were essential for S14 biological activity. In contrast, mutations which altered or deleted S14's amino-terminal 20 amino acid residues (domain A) had no observable effect on the protein's assembly and function in mammalian ribosomes. Interestingly, S14 structural domains deduced by in vitro mutagenesis correlate well with the RPS14 gene's exon boundaries.     

Monte Carlo sampling of near-native structures of proteins with applications

Since a protein's dynamic fluctuation inside cells affects the protein's biological properties, we present a novel method to study the ensemble of near-native structures (NNS) of proteins, namely, the conformations that are very similar to the experimentally determined native structure. We show that this method enables us to (i) quantify the difficulty of predicting a protein's structure, (ii) choose appropriate simplified representations of protein structures, and (iii) assess the effectiveness of knowledge-based potential functions. We found that well-designed simple representations of protein structures are likely as accurate as those more complex ones for certain potential functions. We also found that the widely used contact potential functions stabilize NNS poorly, whereas potential functions incorporating local structure information significantly increase the stability of NNS.