Trypsinogen and chymotrypsinogen: the mysterious hyper-reactivity of selected cysteines is still present after their divergent evolution.

An enigmatic and never described hyper-reactivity of most of the cysteines resident in the reduced, molten globule-like intermediate of a few proteins has been recently discovered. In particular, all ten cysteines of chymotrypsinogen showed hundred times increased reactivity against hydrophobic reagents. A single cysteine (Cys1) was also found thousand times more reactive toward GSSG, making speculate that a single glutathionylation could represent the primordial event of its oxidative folding. In the present study, we compare these kinetic properties with those present in trypsinogen taken in its reduced, molten globule-like intermediate and identify the origin of these unusual properties. Despite the divergent evolution of these two proteins, the different amount of disulfides and the very different 3D localization of three disulfides, their hyper-reactivity toward hydrophobic thiol reagents and disulfides is very similar. Mass spectrometry identifies two cysteines in trypsinogen, Cys148 and Cys197, 800 times more reactive toward GSSG than an unperturbed protein cysteine. These results point toward a stringent and accurate preservation of these peculiar kinetic properties during a divergent evolution suggesting some important role, which at the present can only be hypothesized. Similar extraordinary hyper-reactivity has been found also in albumin, ribonuclease, and lysozyme confirming that it cannot be considered a kinetic singularity of a single protein. Interestingly, the very flexible and fluctuating structures like those typical of the molten globule status prove capable of enabling sophisticated actions typical of enzymes such as binding to GSSG with relevant specificity and high affinity (KD  = 0.4 mm) and accelerating the reaction of its cysteines by thousands of times.

The digestive system of the stony coral Stylophora pistillata.

Because hermatypic species use symbiotic algal photosynthesis, most of the literature in this field focuses on this autotrophic mode and very little research has studied the morphology of the coral's digestive system or the digestion process of particulate food. Using histology and histochemestry, our research reveals that Stylophora pistillata's digestive system is concentrated at the corals' peristome, actinopharynx and mesenterial filaments (MF). We used in-situ hybridization (ISH) of the RNA transcript of the gene that codes for the S. pistillata digestive enzyme, chymotrypsinogen, to shed light on the functionality of the digestive system. Both the histochemistry and the ISH pointed to the MF being specialized digestive organs, equipped with large numbers of acidophilic and basophilic granular gland cells, as well as acidophilic non-granular gland cells, some of which produce chymotrypsinogen. We identified two types of MF: short, trilobed MF and unilobed, long and convoluted MF. Each S. pistillata polyp harbors two long convoluted MF and 10 short MF. While the short MF have neither secreting nor stinging cells, each of the convoluted MF display gradual cytological changes along their longitudinal axis, alternating between stinging and secreting cells and three distinctive types of secretory cells. These observations indicate the important digestive role of the long convoluted MF. They also indicate the existence of novel feeding compartments in the gastric cavity of the polyp, primarily in the nutritionally active peristome, in the actinopharynx and in three regions of the MF that differ from each other in their cellular components, general morphology and chymotrypsinogen excretion.

Interactions of urea with native and unfolded proteins: a volumetric study.

We describe a statistical thermodynamic approach to analyzing urea-dependent volumetric properties of proteins. We use this approach to analyze our urea-dependent data on the partial molar volume and adiabatic compressibility of lysozyme, apocytochrome c, ribonuclease A, and α-chymotrypsinogen A. The analysis produces the thermodynamic properties of elementary urea-protein association reactions while also yielding estimates of the effective solvent-accessible surface areas of the native and unfolded protein states. Lysozyme and apocytochrome c do not undergo urea-induced transitions. The former remains folded, while the latter is unfolded between 0 and 8 M urea. In contrast, ribonuclease A and α-chymotrypsinogen A exhibit urea-induced unfolding transitions. Thus, our data permit us to characterize urea-protein interactions in both the native and unfolded states. We interpreted the urea-dependent volumetric properties of the proteins in terms of the equilibrium constant, k, and changes in volume, ΔV0, and compressibility, ΔKT0, for a reaction in which urea binds to a protein with a concomitant release of two waters of hydration to the bulk. Comparison of the values of k, ΔV0, and ΔKT0 with the similar data obtained on small molecules mimicking protein groups reveals lack of cooperative effects involved in urea-protein interactions. In general, the volumetric approach, while providing a unique characterization of cosolvent-protein interactions, offers a practical way for evaluating the effective solvent accessible surface area of biologically significant fully or partially unfolded polypeptides.

Protein-containing PEGylated cubosomic particles: freeze-fracture electron microscopy and synchrotron radiation circular dichroism study.

The purpose of this work is to investigate the entrapment of protein molecules in cubosomic nanocarriers that are sterically stabilized by an amphiphilic poly(ethylene glycol) (PEG) derivative. Toward that aim, the mechanism of fragmentation of a self-assembled, PEGylated cubic lipid phase into nanoparticles (NPs) is investigated in excess aqueous medium. The molar ratio between the cubic-phase-forming lipid monoolein (MO) and its PEGylated derivative (MO-PEG(2000)) is selected as to favor the formation of inverted-type liquid-crystalline (LC) structures (permitting one to reveal the stages of the fragmentation and bicontinuous membrane NP assembly process) rather than a phase transformation to lamellar or micellar phases. The PEGylated amphiphile considerably affects the interfacial curvature of the cubic lipid membrane and, under agitation, contributes to the fragmentation of the bicontinuous cubic lattice into NPs. Freeze-fracture electron microscopy (FF-EM), quasi-elastic light scattering (QELS), and confocal laser scanning fluorescence microscopy (CLSFM) are applied for determination of the NPs' sizes, inner organization, and stability with regard to a thermal stimulus. Entrapped protein molecules can essentially stabilize the cubosomic particles (proteocubosomes), which display well-defined inner organization of nanochannels in their freeze-fracture planes. The protein α-chymotrypsinogen A is studied in proteocubosome dispersions by means of far-UV synchrotron radiation circular dichroism (SRCD) spectroscopy. It is suggested that the protein molecules are entrapped in the interior of the PEGylated cubosomes via a "nanopockets" mechanism. The LC PEGylated proteocubosomes offer new possibilities for investigation of protein loading in sterically stabilized ("Stealth") nanostructured lipid carriers, which differ from Poloxamer-stabilized isasomes.

Positive cooperative mechanistic binding of proteins at low concentrations: a comparison of poly (sodium N-undecanoyl sulfate) and sodium dodecyl sulfate.

The interactions of the negatively charged achiral molecular micelle, poly (sodium N-undecanoyl sulfate) (poly-SUS), with four different proteins using intrinsic and extrinsic fluorescence spectroscopic probes, are studied. A comparison of poly-SUS with the conventional surfactant, sodium dodecyl sulfate (SDS), and the monomeric species, SUS, is also reported. In this work, we observed that poly-SUS preferentially binds to acidic proteins, exhibiting positive cooperativity at concentrations less than 1 mM for all proteins studied. Moreover, it appears that the hydrophobic microdomain formed through polymerization of the terminal vinyl group of the monomer, SUS, is largely responsible for the superior binding capacity of poly-SUS. From these results, we conclude that the interactions of poly-SUS with the acidic proteins are predominantly hydrophobic and postulate that poly-SUS would produce superior interactions relative to SDS at low concentrations in polyacrylamide gel electrophoresis (PAGE). As predicted, use of poly-SUS allowed separation of the His-tagged tumor suppressor protein, p53, at sample buffer concentrations as low as 0.08% w/v (2.9 mM), which is 24 times lower than required for SDS in the standard reducing PAGE protocol. This work highlights the use of poly-SUS as an effective surfactant in 1D biochemical analysis.

Molecular level insights into thermally induced α-chymotrypsinogen A amyloid aggregation mechanism and semiflexible protofibril morphology.

Understanding nonnative protein aggregation is critical not only to a number of amyloidosis disorders but also for the development of effective and safe biopharmaceuticals. In a series of previous studies [Weiss et al. (2007) Biophys. J. 93, 4392-4403; Andrews et al. (2007) Biochemistry 46, 7558-7571; Andrews et al. (2008) Biochemistry 47, 2397-2403], α-chymotrypsinogen A (aCgn) and bovine granulocyte colony stimulating factor (bG-CSF) have been shown to exhibit the kinetic and morphological features of other nonnative aggregating proteins at low pH and ionic strength. In this study, we investigated the structural mechanism of aCgn aggregation. The resultant aCgn aggregates were found to be soluble and exhibited semiflexible filamentous aggregate morphology under transmission electron microscopy. In addition, the filamentous aggregates were demonstrated to possess amyloid characteristics by both Congo red binding and X-ray diffraction. Peptide level hydrogen exchange (HX) analysis suggested that a buried native ß-sheet comprised of three peptide segments (39-46, 51-64, and 106-114) reorganizes into the cross-ß amyloid core of aCgn aggregates and that at least ∼50% of the sequence adopts a disordered structure in the aggregates. Furthermore, the equimolar, bimodal HX labeling distribution observed for three reported peptides (65-102, 160-180, and 229-245) suggested a heterogeneous assembly of two molecular conformations in aCgn aggregates. This demonstrates that extended ß-sheet interactions typical of the amyloid are sufficiently strong that a relatively small fraction of polypeptide sequence can drive formation of filamentous aggregates even under conditions favoring colloidal stability.

The use of vascular endothelial growth factor functionalized agarose to guide pluripotent stem cell aggregates toward blood progenitor cells.

The developmental potential of pluripotent stem cells is influenced by their local cellular microenvironment. To better understand the role of vascular endothelial growth factor (VEGFA) in the embryonic cellular microenvironment, we synthesized an artificial stem cell niche wherein VEGFA was immobilized in an agarose hydrogel. Agarose was first modified with coumarin-protected thiols. Upon exposure to ultra-violet excitation, the coumarin groups were cleaved leaving reactive thiols to couple with maleimide-activated VEGFA. Mouse embryonic stem cells (ESC) aggregates were encapsulated in VEGFA immobilized agarose and cultured for 7 days as free-floating aggregates under serum-free conditions. Encapsulated aggregates were assessed for their capacity to give rise to blood progenitor cells. In the presence of bone morphogenetic protein-4 (BMP-4), cells exposed to immobilized VEGFA upregulated mesodermal markers, brachyury and VEGF receptor 2 (T+VEGFR2+) by day 4, and expressed CD34 and CD41 (CD34+CD41+) on day 7. It was found that immobilized VEGFA treatment was more efficient at inducing blood progenitors (including colony forming cells) on a per molecule basis than soluble VEGFA. This work demonstrates the use of functionalized hydrogels to guide encapsulated ESCs toward blood progenitor cells and introduces a tool capable of recapitulating aspects of the embryonic microenvironment.

Effects of immobilization onto aluminum hydroxide particles on the thermally induced conformational behavior of three model proteins.

Although the thermal unfolding/aggregation behavior of proteins in solution has been extensively studied, little is known about proteins immobilized on the surface of nanoparticles and other solid-phase materials. In this study we carefully monitor and analyze the thermal denaturation process of three model proteins adsorbed onto aluminum hydroxide as a function of temperature by FT-IR spectroscopy. The results reveal that the proteins immobilized onto aluminum hydroxide retain their native conformation at lower temperatures (<45 degrees C). Upon thermal denaturation, the structural transition between the native and denatured states is very similar, in terms of disappearance of the major native secondary structural elements, between the proteins adsorbed onto aluminum hydroxide adjuvant and in solution. This result suggests that the thermal stability of proteins is not significantly affected, or marginally affected at most, by the adsorption onto aluminum hydroxide adjuvant, considering a 5 degrees C temperature interval used for data collection. However, the adsorption rate and crowding of proteins on aluminum hydroxide particles have a profound effect on the aggregation behavior of the proteins, hydrogen bonding strength of intermolecular beta-sheet aggregates and conformation of intermediate states.

Characterization of a three-component coupling reaction on proteins by isotopic labeling and nuclear magnetic resonance spectroscopy.

A three-component Mannich-type electrophilic aromatic substitution reaction was previously developed to target the phenolic side chain of tyrosine residues on proteins. This reaction proceeds under mild conditions and provides a convenient alternative to lysine-targeting strategies. However, the use of reactive aldehydes, such as formaldehyde, warrants careful inspection of the reaction products to ensure that other modifications have not occurred. Through the use of isotopically enriched reagents, nuclear magnetic resonance (NMR)-based studies were used to obtain structural confirmation of the tyrosine-modification products. These experiments also revealed the formation of a reaction byproduct arising from the indole ring of tryptophan residues. Cysteine residues were shown to not participate in the reaction, except in the case of a reduced disulfide, which formed a dithioacetal. We anticipate that this analysis method will prove useful for the detailed study of a number of bioconjugation reactions.

Attachment of peptide building blocks to proteins through tyrosine bioconjugation.

Recent efforts have yielded a number of short peptide sequences with useful binding, sensing, and cellular uptake properties. In order to attach these sequences to tyrosine residues on intact proteins, a three-component Mannich-type strategy is reported. Two solid phase synthetic routes were developed to access peptides up to 20 residues in length with anilines at either the N- or C-termini. In the presence of 20 mM formaldehyde, these functional groups were coupled to tyrosine residues on proteins under mild reaction conditions. The identities of the resulting bioconjugates were confirmed using mass spectrometry and immunoblot analysis. Screening experiments have demonstrated that the method is compatible with substrates containing all of the amino acids, including lysine and cysteine residues. Importantly, tyrosine residues on proteins exhibit much faster reaction rates, allowing short peptides containing this residue to be coupled without cross reactions.

Compatible solutes as protectants for zymogens against proteolysis.

Compatible solutes are small organic osmoprotectants that have the capability to stabilize proteins. In coupled assays, the effect of the solutes ectoine, hydroxyectoine and betaine on the activation of the zymogens trypsinogen and chymotrypsinogen, catalyzed by enteropeptidase and trypsin, respectively, was studied. To different extents, all solutes protected the zymogens against activation. Ectoine (800 mM) was the most potent solute in reducing the formation of trypsin to 4% of the control value and of chymotrypsin to 23%. In separate experiments, the ability of the solutes to preserve proteolytic activity during incubation was investigated. After 4 h, trypsin and chymotrypsin completely lost their activity, but in the presence of ectoine, approximately 50% residual activity was maintained. It is proposed that a conformational shift of the protein towards folded, native-like states induced by preferential exclusion of the solute is responsible for the stabilizing and chaperone-like effects.

Improving disulfide connectivity prediction with sequential distance between oxidized cysteines.

SUMMARY: Predicting disulfide connectivity precisely helps towards the solution of protein structure prediction. In this study, a descriptor derived from the sequential distance between oxidized cysteines (denoted as DOC) is proposed. An approach using support vector machine (SVM) method based on weighted graph matching was further developed to predict the disulfide connectivity pattern in proteins. When DOC was applied, prediction accuracy of 63% for our SVM models could be achieved, which is significantly higher than those obtained from previous approaches. The results show that using the non-local descriptor DOC coupled with local sequence profiles significantly improves the prediction accuracy. These improvements demonstrate that DOC, with a proper scaling scheme, is an effective feature for the prediction of disulfide connectivity. The method developed in this work is available at the web server PreCys (prediction of cys-cys linkages of proteins).

A three-component Mannich-type reaction for selective tyrosine bioconjugation.

A new selective bioconjugation reaction is described for the modification of tyrosine residues on protein substrates. The reaction uses imines formed in situ from aldehydes and electron-rich anilines to modify phenolic side chains through a Mannich-type electrophilic aromatic substitution pathway. The reaction takes place under mild pH and temperature conditions and can modify protein substrates at concentrations as low as 20 muM. Using an efficient fluorescence-based assay, we demonstrated the reaction using a number of aldehydes and protein targets. Importantly, proteins lacking surface-accessible tyrosines remained unmodified. It was also demonstrated that enzymatic activity is preserved under the mild reaction conditions. This strategy represents one of the first carbon-carbon bond-forming reactions for protein modification and provides an important complement to more commonly used lysine- and cysteine-based methods.

Why is trehalose an exceptional protein stabilizer? An analysis of the thermal stability of proteins in the presence of the compatible osmolyte trehalose.

Trehalose, a naturally occurring osmolyte, is known to be an exceptional stabilizer of proteins and helps retain the activity of enzymes in solution as well as in the freeze-dried state. To understand the mechanism of action of trehalose in detail, we have conducted a thorough investigation of its effect on the thermal stability in aqueous solutions of five well characterized proteins differing in their various physico-chemical properties. Among them, RNase A has been used as a model enzyme to investigate the effect of trehalose on the retention of enzymatic activity upon incubation at high temperatures. 2 m trehalose was observed to raise the transition temperature, Tm of RNase A by as much as 18 degrees C and Gibbs free energy by 4.8 kcal mol-1 at pH 2.5. There is a decrease in the heat capacity of protein denaturation (DeltaCp) in trehalose solutions for all the studied proteins. An increase in the DeltaG and a decrease in the DeltaCp values for all the proteins points toward a general mechanism of stabilization due to the elevation and broadening of the stability curve (DeltaG versus T). A direct correlation of the surface tension of trehalose solutions and the thermal stability of various proteins has been observed. Wyman linkage analysis indicates that at 1.5 m concentration 4-7 molecules of trehalose are excluded from the vicinity of protein molecules upon denaturation. We further show that an increase in the stability of proteins in the presence of trehalose depends upon the length of the polypeptide chain. The pH dependence data suggest that even though the charge status of a protein contributes significantly, trehalose can be expected to work as a universal stabilizer of protein conformation due to its exceptional effect on the structure and properties of solvent water compared with other sugars and polyols.

MIAX: a new paradigm for modeling biomacromolecular interactions and complex formation in condensed phases.

A new paradigm is proposed for modeling biomacromolecular interactions and complex formation in solution (protein-protein interactions so far in this report) that constitutes the scaffold of the automatic system MIAX (acronym for Macromolecular Interaction Assessment X). It combines in a rational way a series of computational methodologies, the goal being the prediction of the most native-like protein complex that may be formed when two isolated (unbound) protein monomers interact in a liquid environment. The overall strategy consists of first inferring putative precomplex structures by identification of binding sites or epitopes on the proteins surfaces and a simultaneous rigid-body docking process using geometric instances alone. Precomplex configurations are defined here as all those decoys the interfaces of which comply substantially with the inferred binding sites and whose free energy values are lower. Retaining all those precomplex configurations with low energies leads to a reasonable number of decoys for which a flexible treatment is amenable. A novel algorithm is introduced here for automatically inferring binding sites in proteins given their 3-D structure. The procedure combines an unsupervised learning algorithm based on the self-organizing map or Kohonen network with a 2-D Fourier spectral analysis. To model interaction, the potential function proposed here plays a central role in the system and is constituted by empirical terms expressing well-characterized factors influencing biomacromolecular interaction processes, essentially electrostatic, van der Waals, and hydrophobic. Each of these procedures is validated by comparing results with observed instances. Finally, the more demanding process of flexible docking is performed in MIAX embedding the potential function in a simulated annealing optimization procedure. Whereas search of the entire configuration hyperspace is a major factor precluding hitherto systems from efficiently modeling macromolecular interaction modes and complex structures, the paradigm presented here may constitute a step forward in the field because it is shown that a rational treatment of the information available from the 3-D structure of the interacting monomers combined with conveniently selected computational techniques can assist to elude search of regions of low probability in configuration space and indeed lead to a highly efficient system oriented to solve this intriguing and fundamental biologic problem.

Catalysis of reduction of disulfide by selenol.

The reduction of a disulfide to thiols, using sodium borohydride or dithiothreitol as reductant, is catalyzed by selenol. The rates of reduction of oxidized glutathione and bis(2-hydroxyethyl) disulfide by sodium borohydride are accelerated using a catalytic amount of selenol (8 mol% of the disulfide), by factors of 10 and 12, respectively. A rapid and convenient assay for disulfide has been developed, which involves its reduction with sodium borohydride in the presence of a catalytic amount of selenol, followed by acid quench and measurement of the resulting thiol by Ellman's assay. The disulfide bonds in proteins are reduced rapidly using excess dithiothreitol (5 mM) and selenol (0.8 mM), which can be separated conveniently from the reduced protein by gel filtration. The rates of reduction of disulfide bonds in immunoglobulin and alpha-chymotrypsinogen A by dithiothreitol are enhanced by a factor of 90 in the presence of selenol. The rate-determining step is the reaction of selenolate anion with disulfide. Selenocystamine, a commercially available diselende, is used as the precursor of the catalyst; it is reduced to its selenol in situ.

Three-dimensional structure of the complexes between bovine chymotrypsinogen A and two recombinant variants of human pancreatic secretory trypsin inhibitor (Kazal-type).

Variants of the human pancreatic secretory trypsin inhibitor (PSTI) have been created during a protein design project to generate a high-affinity inhibitor with respect to some serine proteases other than trypsin. Two modified versions of human PSTI with high affinity for chymotrypsin were crystallized as a complex with chymotrypsinogen. Both crystallize isomorphously in space group P4(1)2(1)2 with lattice constants a = 84.4 A, c = 86.7 A and diffract to 2.3 A resolution. The structure was solved by molecular replacement. The final R-value after refinement with 8.0 to 2.3 A resolution data was 19.5% for both complexes after inclusion of about 50 bound water molecules. The overall three-dimensional structure of PSTI is similar to the structure of porcine PSTI in the trypsinogen complex (1TGS). Small differences in the relative orientation of the binding loop and the core of the inhibitors indicate flexible adaptation to the proteases. The chymotrypsinogen part of the complex is similar to chymotrypsin. After refolding induced by binding of the inhibitor the root-mean-square difference of the active site residues A186 to A195 and A217 to A222 compared to chymotrypsin was 0.26 A.