Hydrolysis of chimeric proteins by enteropeptidase at the specific linker (Asp)4Lys depending on refolding conditions

Refolding from inclusion bodies of chimeric proteins containing the enteropeptidase-specific linker (Asp)4Lys was carried out. It was shown that, depending on the refolding conditions, chimeric proteins function as substrates or inhibitors of the enteropeptidase. The efficiency of the enteropeptidase hydrolysis of chimeric proteins containing the (Asp)4Lys linker may depend not only on the amino acid sequence of the protein binding site for the enzyme but also on the site conformation.     

Identification of a novel set of scaffolding residues that are instrumental for the inhibitory property of Kunitz (STI) inhibitors

For canonical serine protease inhibitors (SPIs), scaffolding spacer residue Asn or Arg religates cleaved scissile peptide bond to offer efficient inhibition. However, several designed “mini‐proteins,” containing the inhibitory loop and the spacer(s) with trimmed scaffold behave like substrates, indicating that scaffolding region beyond the spacer is also important in the inhibitory process. To understand the loop‐scaffold compatibility, we prepared three chimeric proteins ECI^L‐WCI^S, ETI^L‐WCI^S, and STI^L‐WCI^S, where the inhibitory loop of ECI, ETI, and STI is placed on the scaffold of their homolog WCI. Results show that although ECI^L‐WCI^S and STI^L‐WCI^S behave like good inhibitors, ETI^L‐WCI^S behaves like a substrate. That means a set of loop residues (SRLRSAFI), offering strong trypsin inhibition in ETI, act as a substrate when they seat on the scaffold of WCI. Crystal structure of ETI^L‐WCI^S shows that the inhibitory loop is of noncanonical conformation. We identified three novel scaffolding residues Trp88, Arg74, and Tyr113 in ETI that act as barrier to confine the inhibitory loop to canonical conformation. Absence of this barrier in the scaffold of WCI makes the inhibitory loop flexible in ETI^L‐WCI^S leading to a loss of canonical conformation, explaining its substrate‐like behavior. Incorporation of this barrier back in ETI^L‐WCI^S through mutations increases its inhibitory power, supporting our proposition. Our study provides structural evidence for the contribution of remote scaffolding residues in the inhibitory process of canonical SPIs. Additionally, we rationalize why the loop‐scaffold swapping is not permitted even among the members of highly homologous inhibitors, which might be important in the light of inhibitor design.     

Limited proteolysis of natively unfolded protein 4E‐BP1 in the presence of trifluoroethanol

Natively unfolded (intrinsically disordered (ID) proteins) have been attracting an increasing attention due to their involvement in many regulatory processes. Natively unfolded proteins can fold upon binding to their metabolic partners. Coupled folding and binding events usually involve only relatively short motifs (binding motifs). These binding motifs which are able to fold should have an increased propensity to form a secondary structure. The aim of the present work was to probe the conformation of the intrinsically disordered protein 4E‐BP1 in the native and partly folded states by limited proteolysis and to reveal regions with a high propensity to form an ordered structure. Trifuoroethanol (TFE) in low concentrations (up to 15 vol%) was applied to increase the helical population of protein regions with a high intrinsic propensity to fold. When forming helical structures, these regions lose mobility and become more protected from proteases than random/unfolded protein regions. Limited proteolysis followed by mass spectrometry analysis allows identification of the regions with decreased mobility in TFE solutions. Trypsin and V8 proteases were used to perform limited proteolysis of the 4E‐BP1 protein in buffer and in solutions with low TFE concentrations at 37°C and at elevated temperatures (42 and 50°C). Comparison of the results obtained with the previously established 4E‐BP1 structure and the binding motif illustrates the ability of limited proteolysis in the presence of a folding assistant (TFE) to map the regions with high and low propensities to form a secondary structure revealing potential binding motifs inside the intrinsically disordered protein. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 591–602, 2014.     

Influence of the amino acid residue downstream of (Asp)4Lys on enterokinase cleavage of a fusion protein

We have studied the cleavage efficiency of the protease enterokinase (EK) using the novel vector pESP4. pESP4 is a yeast expression vector equipped with ligation-independent cloning sites, a GST purification tag, and a FLAG epitope tag. EK is used to cleave the FLAG and GST tags leaving the protein of interest without any extraneously added amino acids. We have found that EK is relatively permissive of the amino acid residue downstream of the recognition sequence (the P'1 position). This makes EK an ideal choice to use as a protease to cleave any protein of interest cloned within the pESP4 yeast expression vector.     

Purified chaperonin 60 (groEL) interacts with the nonnative states of a multitude of Escherichia coli proteins.

In vitro experiments employing the soluble proteins from Escherichia coli reveal that about half of them, in their unfolded or partially folded states, but not in their native states, can form stable binary complexes with chaperonin 60 (groEL). These complexes can be isolated by gel filtration chromatography and are efficiently discharged upon the addition of Mg.ATP. Binary complex formation is substantially reduced if chaperonin 60 is presaturated with Rubisco-I, the folding intermediate of Rubisco, but not with native Rubisco. Binary complex formation is also reduced if the transient species that interact with chaperonin 60 are permitted to progress to more stable states. This implies that the structural elements or motifs that are recognized by chaperonin 60 and that are responsible for binary complex formation are only present or accessible in the unfolded states of proteins or in certain intermediates along their respective folding pathways. Given the high-affinity binding that we have observed in the present study and the normal cellular abundance of chaperonin 60, we suspect that the folding of most proteins in E. coli does not occur in free solution spontaneously, but instead takes place while they are associated with molecular chaperones.     

Defect in modification at the anticodon wobble nucleotide of mitochondrial tRNA(Lys) with the MERRF encephalomyopathy pathogenic mutation

Hen lysozyme single-disulfide variants were constructed to characterize the structures associated with the formation of individual native disulfide bonds. Circular dichroism spectra and the effective concentration of protein thiol groups showed that the propensity for structure formation was relatively high for Cys-6–Cys-127 and Cys-30–Cys-115 disulfides. The urea concentration dependence of individual effective concentrations showed that the apparent sizes of the structures were 14–50% of the whole molecule. The intrinsic stability of each submolecular structure in a reduced form of protein, obtained by subtracting the entropic contribution of cross-linking, was highest for Cys-64–Cys-80 and lowest for Cys-76–Cys-94 disulfide bonds.     

Multidomain Structure of the Rapana thomasiana (Gastropod) Hemocyanin Structural Subunit RHSS1

The Rapana thomasiana hemocyanin structural subunit RHSS1 is composed of eight functional dioxygen-binding domains. To determine the multidomain structure, the polypeptide chain of RHSS1 was subjected to limited proteolysis with TPCK-trypsin, elastase and other proteinases. Individual functional units and fragments, containing two or three domains, were isolated and characterized. All domains and fragments were N-terminally sequenced and the order of the dioxygen-binding units in the polypeptide chain of RHSS1 was established.     

Modeling of the structural features of integral-membrane proteins reverse-environment prediction of integral membrane protein structure (REPIMPS)

The Profiles-3D application, an inverse-folding methodology appropriate for water-soluble proteins, has been modified to allow the determination of structural properties of integral-membrane proteins (IMPs) and for testing the validity of solved and model structures of IMPs. The modification, known as reverse-environment prediction of integral membrane protein structure (REPIMPS), takes into account the fact that exposed areas of side chains for many residues in IMPs are in contact with lipid and not the aqueous phase. This (1) allows lipid-exposed residues to be classified into the correct physicochemical environment class, (2) significantly improves compatibility scores for IMPs whose structures have been solved, and (3) reduces the possibility of rejecting a three-dimensional structure for an IMP because the presence of lipid was not included. Validation tests of REPIMPS showed that it (1) can locate the transmembrane domain of IMPs with single transmembrane helices more frequently than a range of other methodologies, (2) can rotationally orient transmembrane helices with respect to the lipid environment and surrounding helices in IMPs with multiple transmembrane helices, and (3) has the potential to accurately locate transmembrane domains in IMPs with multiple transmembrane helices. We conclude that correcting for the presence of the lipid environment surrounding the transmembrane segments of IMPs is an essential step for reasonable modeling and verification of the three-dimensional structures of these proteins.     

Functional display of proteins, mutant proteins, fragments of proteins and peptides on the surface of filamentous (bacterio) phages: A review

Cytoplasmic expression of complex eukaryotic proteins inEscherichia coli usually yields inactive protein preparations. In some cases, (part) of the biological activity can be recovered by rather inefficient denaturation-renaturation procedures. Recently, novel concepts have been developed for the expression of fully functional eukaryotic proteins inE. coli. Essential to the success of these procedures is the transport of such proteins across the inner membrane to the periplasmic space, allowing proper folding and the establishment of disulfide bonding. Subsequently, fully functional proteins can be exposed on the surface of filamentous (bacterio)phages, provided a system is employed that consists of a cloning vector (e.g. the phagemid pComb3, Barbas et al., 1991) that generates phage particles in the presence of a helper phage. The main advantage of surface display of recombinant proteins is to facilitate the screening of very large numbers of different molecules by simple selection methods (panning). In addition, periplasmic expression yields relatively large quantities (e.g. 1 mg l^–1 of culture) soluble protein. In this review, the principle aspects of this novel expression system based on the phagemid pComb3 will be discussed. Two examples for functional periplasmic expression of human proteins inE. coli will be presented, namely i) the antigen-binding moiety (Fab fragment) of human immunoglobulins (IgGs) and ii) the human plasminogen activator inhibitor 1, an essential regulator of the plasminogen activation system. Finally, perspectives for the application of this system to express mutant proteins, fragments of proteins and peptides are indicated.Abbreviations Ap^R ampicillin resistance - cfu colony forming unit(s) - cpIII gene III-encoded coat protein of M13 - cpVIII gene VIII-encoded coat protein of M13 - ER endoplasmic reticulum - Fab fragment of Ig containing light chain, variable region and first constant region of heavy chain - Fd variable region and first constant region of the heavy chain - Fv fragment containing variable regions of heavy and light chain - Ig immunoglobulin - Km^R kanamycin resistance - kb kilobase or 1000 basepairs - PAI-1 plasminogen activator inhibitor 1 - t-PA tissue-type plasminogen activator - u-PA urokinase-type plasminogen activator     

Effect of ammonia-stress on the total autolytic levels of proteins in tissues of an air-breathing fish,Channa punctatus (Bloch)

G season-specific effect of a sub-lethal ambient ammonium chloride concentration on the total autolysis of protein in different tissues of the Indian air-breathing murrel,Chauna punctatos (Bloch), has been demonstrated. While its effect on the autotytie levels of protein of different tissues (except white muscle) was marginal in the winter-adapted fish, the same in the summer-adapted fish was more pronounced. In general a reduction in the autolysic levels of the tissue protein of the ammonium chloride-stressed fish was observed. In certain tissues like white muscle of an ammonium chloride-stressed fish and gill of the ammonium hydroxide-stressed fish, augmented levels of autolysis were noticed. The present findings suggest that the accumulation of amino acids in different tissues of the ammonia-stressed fish, as observed earlier, can be explained best by mechanisms other than ammonia induced increased breakdown of proteins.     

Gene controlled by promoter--P_(TH4) depending on whiG of Streptomyces coelicolor

The downstream gene controlled by promoter--PTH4 which is related to Streptomycesdifferentiation was cloned, and its sequence was determined by the dideoxy chain termination method. The results indicated that the 1597 bp of DNA fragment conferred a complete open reading frame (ORF). In searches of databases, the deduced product of the ORF was not homologous with any known proteins; it may be a new protein. The function of the gene was studied using the strategy of gene disruption; the actinorhodin could not be produced when this gene was disrupted. Therefore, this gene may be related to actinorhodin biosynthesis in Streptomyces coelicolor, and the result also shows that this gene may play a role in multiple level regulation of differentiation genes in Streptomyces.     

Thermodynamic, enzymatic and structural effects of removing a salt bridge at the base of loop 4 in (pro)caspase-3

Interactions between loops 2, 2′ and 4, known as the loop bundle, stabilize the active site of caspase-3. Loop 4 (L4) is of particular interest due to its location between the active site and the dimer interface. We have disrupted a salt bridge between K242 and E246 at the base of L4 to determine its role in overall conformational stability and in maintaining the active site environment. Stability measurements show that only the K242A single mutant decreases stability of the dimer, whereas both single mutants and the double mutant demonstrate much lower activity compared to wild-type caspase-3. Structural studies of the caspase-3 variants show the involvement of K242 in hydrophobic interactions that stabilize helix 5, near the dimer interface, and the role of E246 appears to be to neutralize the positive charge of K242 within the hydrophobic cluster. Overall, the results suggest E246 and K242 are important in procaspase-3 for their interaction with neighboring residues, not with one another. Conversely, formation of the K242–E246 salt bridge in caspase-3 is needed for an accurate, stable conformation of loop L4 and proper active site formation in the mature enzyme.     

Influence of a sugar moiety (rhamnosylglucoside) at 3-O position on the reactivity of quercetin with whey proteins

The reaction of whey proteins (WP) with quercetin and rutin (quercetin-3-O-rhamnosylglucoside) is influenced by the glycosidic bound sugar moiety. The protein derivatives formed showed a blocking of tryptophan (max. 49%), free amino (max. 32%) and thiol groups (max. 24%). The amount of quercetin and rutin bound covalently (up to 94 and 31 nmol mg⁻¹, respectively) was estimated by their characteristic absorbance between 300 and 340 nm. At least one molecule of the phenolic reactant was covalently bound to a β-lactoglobulin molecule (β-Lg). High molecular protein fractions were detected by SDS-PAGE (cross-linking with quercetin). All results confirm that quercetin is more reactive than rutin. The pH-dependent solubility of the derivatives decreased, although their hydrophilic character increased. The structural changes (circular dichroism (CD)) showed that especially rutin causes perturbation of the secondary (decrease of -helix elements accompanied by an increase in random coil) and tertiary structure. The in vitro proteolytic digestibility, especially of the rutin derivatives was elevated, due to an increase in denaturation of the derivatives.     

Domain organization of XAF1 and the identification and characterization of XIAP(RING) -binding domain of XAF1

X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) has been implicated as a novel tumor suppressor, which was proposed to exert pro-apoptotic effect by antagonizing the anticaspase activity of XIAP. Here, we delineated the domain architecture of XAF1 by applying limited proteolysis and peptide mass fingerprinting analysis. Our results indicated that XAF1 has a distinct domain organization, with a highly compact N-terminal domain (XAF1(NTD) ) followed by a middle domain (XAF1(MD) ), a 42-residue unstructured linker and a C-terminal domain (XAF1(CTD) ). The search of XIAP binding region within XAF1 revealed that a modest affinity XIAP(RING) binding site (dissociation constant, K(d) , ～18 μM) is located at the C-terminal portion of XAF1. This C-terminal region, embracing XAF1(CTD) and a flexible tail at C-terminus (residue Thr251-Ser301), is functionally identified as XIAP(RING) -binding domain of XAF1 (XAF1(RBD) ) in the present study. We have also mapped the interaction sites for XAF1(RBD) on XIAP(RING) by using NMR spectroscopy. By applying in vitro ubiquitination assay, we observed that XAF1(RBD) /XIAP interaction is essential for the ubiquitination of GST-XAF1(RBD) fusion protein. In addition, the C-terminal XAF1 fragment harboring XAF1(RBD) was found to be substantially ubiquitinated by XIAP(RING) . Base on these observations, we speculate a possible role of XAF1(RBD) in targeting XAF1 for XIAP-mediated ubiquitination.     

Effect of the manganese complex on the binding of the extrinsic proteins (17, 23 and 33 kDa) of Photosystem II

Selective extraction-reconstitution experiments with the extrinsic Photosystem II polypeptides (33 kDa, 23 kDa and 17 kDa) have demonstrated that the manganese complex and the 33 kDa polypeptide are both necessary structural elements for the tight binding of the water soluble 17 and 23 kDa species. When the manganese complex is intact the 33 kDa protein interacts strongly with the rest of the photosynthetic complex. Destruction of the Mn-complex has two dramatic effects: i) The binding of the 33 kDa polypeptide is weaker, since it can be removed by exposure of the PS II system to 2 M NaCl, and ii) the 17 and 23 kDa species do not rebind to Mn-depleted Photosystem II membranes that retain the 33 kDa protein.Abbreviations Chl chlorophyll - HQ hydroquinone - MES 2(N-morpholino)ethanesulfonic acid - PS II Photosystem II - Tris 2-amino-2-hydroxymethylpropane-1,3-diol     

A defective seed coat pattern (Net) is correlated with the post-transcriptional abundance of soluble proline-rich cell wall proteins

The pigmented seed coats of several soybean (Glycine max (L.) Merr.) plant introductions and isolines have unusual defects that result in cracking of the mature seed coat exposing the endosperm and cotyledons. It has previously been shown that the T (tawny) locus that controls the color of trichomes on stems and leaves also has an effect on both the structure and pigmentation of the seed coat. Distribution of pigmentation on the seed coat is controlled by alleles of the I (inhibitor) locus. It was also found that total seed coat proteins were difficult to extract from pigmented seed coats with i T genotypes because they have procyanidins that exhibit tannin properties. We report that the inclusion of poly-L-proline in the extraction buffer out-competes proteins for binding to procyanidins. Once this problem was solved, we examined expression of the proline-rich cell wall proteins PRP1 and PRP2 in pigmented genotypes with the dominant T allele. We found that both homozygous i T and i t genotypes have reduced soluble PRP1 levels. The epistatic interaction of the double recessive genotype at both loci is necessary to produce the pigmented, defective seed coat phenotype characteristic of seed coats with the double recessive i and t alleles. This implies a novel effect of an enzyme in the flavonoid pathway on seed coat structure in addition to its effect on flavonoids, anthocyanidins, and proanthocyanidins. No soluble PRP1 polypeptides were detectable in pigmented seed coats (i T genotypes) of isolines that also display a net-like pattern of seed coat cracking, known as the Net defect. PRP2 was also absent in one of the these lines. However, both PRP1 and PRP2 cytoplasmic mRNAs were found in the Net-defective seed coats. Together with in vitro translation studies, these results suggest that the absence of soluble PRP polypeptides in the defective Net lines is post-translational and could be due to a more rapid or premature insolubilization of PRP polypeptides within the cell wall matrix.     

Engineering a CD4 mimetic inhibiting the binding of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein gp120 to human lymphocyte CD4 by the transfer of a CD4 functional site to a small natural scaffold

Summary The solvent-exposed CDR2-like region of human CD4 was transferred to the structural scaffold of scorpion charybdotoxin, as a means to reproduce that site in a native-like conformation. The chimeric mini-protein (33 amino acids long), obtained by solid-phase synthesis, is able to specifically prevent the interaction of HIV-1 gp120 with CD4. This CD4 mimetic may represent a valuable tool in the study of the HIV-cell interactions and as a lead in the development of antiviral drugs.