Protein Chemistry

In this study, the effect of increasing mat open assembly time on the strength properties of oriented strandboards bonded with polymeric methylene diphenol di-isocyanate (pMDI) resin was examined. Isocyanates are more sensitive to open assembly times than other resin ...

Denaturation of human seminal transferrin (HSmT) compared with human serum transferrin (HSrT) was followed to check structural differences between these two proteins. Second derivative UV spectroscopy indicated that treatment with 6 M guanidine hydrochloride (Gnd.HCI) induced greater structural changes in HSrT than in HSmT and, in particular; (i) the exposure value of tyrosinyl residues was almost 2.5-fold higher in native HSmT than in native HSrT; and (ii) a much more pronounced movement of tryptophanyl residues toward a higher polar environment could be noticed in HSrT after incubation with denaturating agent. Fluorescence measurements showed that: (i) a shift of the maximum emission wavelength of HSmT occurred (maximum emission was centered at 333 nm instead of 323 nm as for HSrT; excitation= 280 nm) ; (ii) the intrinsic tryptophan fluorescence intensity of HSmT increased after 36 hr in the range of 1.5-4.0 M of denaturant, whereas an opposite behavior was found for HSrT in the range 0.0 2.0 M; and (iii) the wavelength maximum of fluorescence emission changed in a biphasic manner for HSrT and, conversely, under the same experimental conditions, HSmT gave a linear and parallel increase of fluorescence emission after 1 and 36 hr. We carl conclude that this different behavior of HSmT with respect to HSrT might be due mainly to the fact that both the number and the exposure of tyrosinyl and tryptophanyl residues are different. Lately, these effects are discussed in relationship with the fact that HSmT contains less than half disulphide bridges than HSrT.

beta H-crystallin was exposed to radiolytically generated hydroxyl radicals at defined radical concentrations, and its capacity to act as an amine-acceptor substrate and as an amine-donor substrate for transglutaminase were investigated. [14C]Methylamine was used as a probe for labelling amine-acceptor sites; a novel biotinylated hexapeptide was used to label amine-donor sites. The results demonstrate that both primary amine incorporation and hexapeptide incorporation by transglutaminase are considerably increased after oxidative attack on the crystallin. The identity of the labelled subunits was established, and it is shown that, in both cases, this increased incorporation is not due to the production of new substrates, but that the existing incorporation sites become more susceptible. Moreover, using the newly developed probe, we could identify, for the first time, the major crystallin subunits active as amine-donor substrates (both before and after treatment) to be beta B1-, beta...

In recent years, dihydrodipicolinate synthase (DHDPS; EC 4.2.1.52) has received considerable attention from both mechanistic and structural viewpoints. DHDPS is part of the diaminopimelate pathway leading to lysine, coupling (S)-aspartate--semialdehyde with pyruvate via a Schiff base to a conserved active-site lysine. In this paper, the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DHDPS from methicillin-resistant Staphylococcus aureus, an important bacterial pathogen, are reported. The enzyme was crystallized in a number of forms, predominantly from PEG precipitants, with the best crystal diffracting to beyond 1.45 A resolution. The space group was P1 and the unit-cell parameters were a = 65.4, b = 67.6, c = 78.0 A ˚, = 90.1, = 68.9, = 72.3 . The crystal volume per protein weight (V M ) was 2.34 A ˚3 Da À1 , with an estimated solvent content of 47% for four monomers per asymmetric unit. The structure of the enzyme will help to guide the design of novel therapeutics against the methicillin-resistant S. aureus pathogen.

Rice is the staple food of half of the world. Rice is easily digestible, rich source of starch followed by proteins and lipids as major components. Rice proteins are having great nutritional value and show many health benefits. Rice has significant amount of protein which is available in mainly four fractions such as Globulins, Albumins, Glutelins and Prolamins. Rice protein is high in Sulphur containing aminoacids. Rice protein is the major factor for determining texture, pasting capacity and sensory characteristics there by determines the cooking and eating qualities of rice. The yield of utilizable protein is higher in rice. The proportion of protein classes varies among genotypes and even among brown rice, milled rice and rice bran. The present study can be used to know the total protein content and amount of protein classes among Samba Mahsuri and 97 mutants due to its novelty to screen quality and its wider applications in food industry.

A striated muscle fiber consists of thousands of myofibrils with crystalline hexagonal myofilament lattices. Because the lattices are randomly oriented, the fiber gives rise to an equatorial x-ray diffraction pattern, which is essentially a rotary-averaged "powder diffraction," carrying only information about the distance between the lattice planes. We were able to record an x-ray diffraction pattern from a single myofilament lattice, very likely originating from a single myofibril from the flight muscle of a bumblebee, by orienting the incident x-ray microbeam along the myofibrillar axis (end-on diffraction). The pattern consisted of a number of hexagonally symmetrical diffraction spots whose originating lattice planes were readily identified. This also held true for some of the weak higher order reflections. The spot-like appearance of reflections implies that the lattice order is extremely well maintained for a distance of millimeters, covering up to a thousand of ϳ2.5-m-long sarcomeres connected in series. The results open the possibility of applying the x-ray microdiffraction technique to study many other micrometer-sized assemblies of functional biomolecules in the cell.

Here we report the identification of protein targets of chemopreventive phenethyl isothiocyanate (PEITC) via "click" chemistry in the A549 human lung cancer cell line, using a novel alkynetagged PEITC which was also found to show potent in vitro anticancer activity. Isothiocyanates (ITCs) derived from cruciferous vegetables 1 are shown to inhibit carcinogenesis in animal models. Inverse associations between cruciferous vegetable/ ITCs intake and cancers have been demonstrated in epidemiological studies. Inducing apoptosis 7-9 is considered as an important mechanism for their cancer preventive effects because it may eliminate the initiated cells. 10 However, the molecular target(s) for inducing apoptosis by ITCs is still under investigation. It is known that ITCs can conjugate with glutathione (GSH) and induce cellular oxidative stress; ITCs can also bind to intracellular proteins (e.g. tubulin) which can induce apoptosis. We reported previously that phenethyl isothiocyanate (PEITC), an ITC rich in watercress, induced apoptosis underlies a mechanism of inhibiting tumorigenesis, and direct covalent binding to cellular proteins which may constitute an important early event in the induction of apoptosis by PEITC in A549 human lung cancer cells. As electrophiles, ITCs can form covalent bond with the thiol groups in proteins. To fully understand the cellular responses to PETIC, it is crucial to develop an unbiased method that can rapidly identify their protein targets. Compared with the twodimensional gel electrophoresis method using radiolabeled PEITC reported previously, 12 the major advantage offered by the click chemistry method are: (1) convenient and simple to perform; (2) easy to enrich targeted protein(s); (3) more cost-effective; (4) higher specificity; and (5) safer for the researchers. "Click" chemistry 15 was introduced in 2001 by K.B. Sharpless and associates. It has emerged as a powerful tool in drug discovery, chemical biology 7 and proteomic

The objective of this study was to evaluate the effect of addition of CO(2) to raw milk on UHT milk quality during storage. Control milk (without CO(2) addition) and treated milk (with CO(2) addition up to pH 6.2) were stored in bulk tanks at 4°C for 6d. After storage, both samples were UHT processed using indirect heating (140°C for 5s). Samples were aseptically packed in low-density polyethylene pouches and stored in the dark at room temperature. Raw milk was evaluated upon receipt for physicochemical composition, proteolysis, lipolysis, standard plate count, psychrotrophic bacteria, and Pseudomonas spp. counts, and after 6d of storage for proteolysis, lipolysis, and microbial counts. After processing, UHT milk samples were evaluated for physicochemical composition, proteolysis, and lipolysis. Samples were evaluated for proteolysis and lipolysis twice a month until 120d. Peptides from pH 4.6-soluble N filtrates were performed by reversed-phase HPLC after 1 and 120d of storage. A s...

A comparative study has been performed on five native laccases purified from the three basidiomycete fungi Pleurotus ostreatus, Rigidoporus lignosus, and Trametes trogii to relate their different catalytic capacities to their structural properties. Spectroscopic absorption features and EPR spectra at various pH values of the five enzymes are very similar and typical of the blue oxidases. The analysis of the dependence of kinetic parameters on pH suggested that a histidine residue is involved in the binding of nonphenolic substrates, whereas both a histidine and an acidic residue may be involved in the binding of phenolic compounds. His and an Asp residue are indeed found at the bottom of a cavity which may be regarded as a suitable substrate channel for approaching to type 1 copper in the 3D homology models of the two laccases from Pleuorotus ostreatus (POXC and POXA1b) whose sequences are known.

A new protease (araujiain h I) was purified to mass spectroscopy homogeneity from the latex of Araujia hortorum Fourn. (Asclepiadaceae) fruits by ultracentrifugation and ion exchange chromatography. The enzyme has a molecular mass of 24,031 (mass spectrometry) and an isoelectric point higher than 9.3. The optimum pH range for casein hydrolysis was 8.0 -9.5. The enzyme showed remarkable caseinolytic activity at high temperatures, although its thermal stability decayed rapidly. The proteinase was activated by thiol compounds and inhibited by common thiol-blocking reagents, particularly E-64 and HgCl 2 , suggesting the enzyme belongs to the cysteine protease family. The concentration of active sites as determined by titration with E-64 was 3.3 µM. When assayed on N-α-CBZ-amino acid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative, followed by those of alanine, asparagine, glycine, and leucine, in decreasing order. Partial homology (36-48%) with other plant cysteine proteinases was observed in an internal fragment obtained by Protease V8 treatment.

Asclepias fruticosa L. is a small shrub containing latex with proteolytic activity. The crude extract (latex diluted 1:250 and ultracentrifuged) contained 276 mg of protein/mL and the proteolytic activity reached 1.2 caseinolytic U/mL. This enzyme preparation was very stable even after 2 hours at 45°C, but was quickly inactivated after 5 minutes at 80°C. Chromatographic purification was achieved by FPLC using a cation exchanger (SP-Sepharose FF). Thus, a unique proteolitically active fraction could be isolated, being homogeneous by bidimensional electrophoresis and mass spectrometry (M r 5 23,652). The optimum pH range was achieved at 8.5-10.5. The enzyme activity was completely inhibited by specific cysteine peptidases inhibitors. Isoelectric focusing followed by zymogram showed the enzyme had a pI greater than 9.3. The N-terminus sequence (LPDSVD-WREKGVVFPIRNQGK) shows a great deal of similarity to those of the other cysteine endopeptidases isolated from latices of Asclepiadaceae even when a high degree of homology could be observed with other plant cysteine endopeptidases.

A new cysteine endopeptidase (morrenain b I) has been purified and characterized from the latex of stems and petiols of Morrenia brachystephana Griseb. (Asclepiadaceae). Morrenain b I was the minor proteolytic component in the latex but showed higher specific activity than morrenain b II, which was the main active fraction. Both enzymes showed similar pH profiles and molecular masses, but kinetic parameters and N-terminal sequences were quite distinct, demonstrating that they are different enzymes instead of different forms of the same enzyme.

Endo-b-1,4-mannanase from Thermotoga petrophila (TpMan) is a hyperthermostable enzyme that catalyzes the hydrolysis of b-1,4-mannoside linkages in various mannan-containing polysaccharides. A recent study reported that TpMan is composed of a GH5 catalytic domain joined by a linker to a carbohydrate-binding domain. However, at this moment, there is no threedimensional structure determined for TpMan. Little is known about the conformation of the TpMan as well as the role of the length and flexibility of the linker on the spatial arrangement of the constitutive domains. In this study, we report the first structural characterization of the entire TpMan by small-angle X-ray scattering combined with the three-dimensional structures of the individual domains in order to shed light on the low-resolution model, overall dimensions, and flexibility of this modular enzyme at different temperatures. The results are consistent with a linker with a compact structure and that occupies a small volume with respect to its large number of amino acids. Furthermore, at 20uC the results are consistent with a model where TpMan is a molecule composed of three distinct domains and that presents some level of molecular flexibility in solution. Even though the full enzyme has some degree of molecular flexibility, there might be a preferable conformation, which could be described by the rigid-body modeling procedure. Finally, the results indicate that TpMan undergoes a temperature-driven transition between conformational states without a significant disruption of its secondary structure. Our results suggest that the linker can optimize the geometry between the other two domains with respect to the substrate at high temperatures. These studies should provide a useful basis for future biophysical studies of entire TpMan.

Amaranth storage proteins begin to be hydrolyzed immediately following the completion of germination. Albumins and globulins (7S globulin, 11S-globulin and globulin-p) were formerly modified, and glutelins, the most aggregated fraction, later. Globulins mobilization starts with the proteolysis of the 7S like-globulin polypeptides and the propolypeptide and acid (A) polypeptides of 11S-globulin and globulin-p. This pattern of 11S-globulin mobilization is accounted by the structural model with propolypeptide and A polypeptides exposed to the outside. Amaranth globulin molecules showed minor changes in their sizes in spite of having some of their polypeptides cleaved. Although globulin-p is more aggregated than 11S-globulin, it showed greater conformational changes. Considering the high susceptibility of the propolypeptide to enzymatic hydrolysis, the higher content of this polypeptide in globulin-p molecules might explain their higher structural changes. According to the results, the order of mobilization of storage proteins depends on the combination of two structural characteristics, the state of aggregation and the presence on the surface of polypeptides susceptible to cleavage.

Protein denaturation is a fundamental phenomenon in biochemistry, wherein proteins lose their native conformational structure under stress conditions, thereby impairing their biological functions. This study investigates the denaturation behavior of egg albumin, primarily composed of ovalbumin, in response to elevated temperature and alterations in pH. Protein samples were subjected to controlled heat treatment and exposed to acidic and alkaline environments to assess conformational changes. The Biuret assay, a qualitative method to detect peptide bonds, was employed to gauge the accessibility of protein functional groups post-treatment. Observable changes in turbidity, coagulation, and colorimetric response were documented and analyzed. Results indicated significant structural alterations, especially under heat stress, suggesting that thermal denaturation exerts more severe effects compared to pH-induced denaturation. The findings underline the sensitivity of protein tertiary and quaternary structures to environmental perturbations and reinforce core principles of protein chemistry relevant to enzymology, food science, and molecular biology.

Differential chemical modification of E. coil chaperonin 60 (cpn60) was achieved by using one of several sulfhydryl-directed reagents. For native cpn60, the three cysteines were accessible for reaction with N-ethylmaleimide (NEM), while only two of them are accessible to the larger reagent 4,4'-dipyridyl disulfide (4-PDS). However, no sulfhydryl groups were modified when the even larger reagents 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) or 2-(4'-(iodoacetamido)anilino) naphthalene-6-sulfonic acid (IAANS), were employed, unless the chaperonin was unfolded. The cpn60 that had been covalently modified with NEM or IAAN$, was not able to support the chaperonin-assisted refolding of the mitochondrial enzyme rhodanese, which also requires cpnl0 and ATP hydrolysis. However, both modified forms of cpn60 were able to form binary complexes with rhodanese, as demonstrated by their ability to arrest the spontaneous refolding of the enzyme. That is, chemical modification with these sulfhydryldirected reagents produced a species that was not prevented from interaction with partially folded rhodanese, but that was prevented from supporting a subsequent step(s) during the chaperonin-assisted refolding process.

Rhodanese (thiosulfate cyanide sulfurtransferase; E.C. 2.8.1.1) is a mitochondrial enzyme that is unprocessed after import. We describe in vitro experiments showing that partially folded rhodanese can interact with lipid bilayers. The interaction was monitored by measuring the ability of rhodanese to disrupt small unilamellar vesicles composed of phosphatidylserine and to release 6-carboxyfluorescein that was trapped in the liposomes. Partially folded rhodanese, derived by dilution of urea-unfolded enzyme, efficiently induced liposome leakage. Native rhodanese had no effect on liposome integrity. Liposome disruption progressively decreased as rhodanese was given the opportunity to refold or aggregate before introduction of the liposomes. A synthetic 23 amino acid peptide representing the N-terminal sequence of rhodanese was very efficient at disrupting the liposomes. Shorter peptides chosen from within this sequence (residues 11-23 or residues 1-17) had no effect on liposome disruption. A peptide representing the tether region that connects the domains of the enzyme was also without effect. These results are consistent with the hypothesis that the N-terminal sequence of rhodanese is an uncleaved leader sequence, and can interact with membrane components that are involved in the mitochondrial uptake of this protein.

Molecular dynamics calculations have been performed to determine the average structures of ras-gene-encoded p21 proteins bound to GTP, i.e., the normal (wild-type) protein and two oncogenic forms of this protein, the Val 12-and Leu 61-p21 proteins. We find that the average structures for all of these proteins exhibit low coordinate fluctuations (which are highest for the normal protein), indicating convergence to specific structures. From previous dynamics calculations of the average structures of these proteins bound to GDP, major regional differences were found among these proteins , J. Protein Chem., in press]. We now find that the average structures of the oncogenic proteins are more similar to one another when the proteins are bound to GTP than when they are bound to GDP [Monaco et al. (1995), J. Protein Chem., in press]. However, they still differ in structure at specific amino acid residues rather than in whole regions, in contradistinction to the results found for the p21-GDP complexes. Two exceptions are the regions 25-32, in an a-helical region, and 97-110. The two oncogenic (Val 12-and Leu 61-) proteins have similar structures which differ significantly in the region of residues 97-110. This region has recently been identified as being critical in the interaction of p21 with kinase target proteins. The differences in structure between the oncogenic proteins suggest the existence of more than one oncogenic form of the p21 protein that can activate different signaling pathways.

Bovine @-lactoglobulin (BLG) was solubilized in solvent systems of varying polarity and hydrophobicity. An amount of 2 mg/mL initially added could be solubilized up to 70% (1.4 mg/mL) in CHC13/CH30H (7/3, v/v) acidified with HC1 and up to 40% (0.8 mg/mL) in CHC13/CH30H (3/7, v/v) in the presence of triethylamine. After solubilization in organic solvents, the yield of reductive alkylation is much higher (98%) than that in aqueous conditions (75%). Acylation of BLG with stearic acid anhydride was also performed in binary mixtures of methanol and chloroform. The efficiency of substitutions of c-amino groups in studied organic systems amounted to 80% compared to none in water. Besides, the emulsifying activity of 40% stearylated BLG was 3-6-fold superior (depending on the pH) than that of unmodified BLG, even after a 30-min heating at 80 "C.

The kinetics of the partial digestion of bovine a-lactalbumin (a-LA) by trypsin, a-chymotrypsin, and pepsin was monitored by lactose synthase activity, HPLC, and difference spectrophotometry. The relative stabilities of the various metal-bound states of a-LA to trypsin and chymotrypsin at 37 and 5°C decrease in the following order: Ca(II)-a-LA > Zn(II), Ca(II)a-LA> apo-a-LA. The HPLC digestion patterns of Ca(II)-a-LA and Zn(II), Ca(II)-a-LA at 5 and 37°C were similar, while the corresponding digestion patterns for apo-a-LA were quite different, reflecting the existence of the thermally induced denaturation states of apo-a-LA within this temperature region. Occupation of the first Zn(Ii)-binding site in Ca(II)-loaded a-LA slightly alters the HPLC digestion patterns at both temperatures and accelerates the digestion at 37°C due to Zn(II)-induced shift of the thermal transition of a-LA, exposing some portion of thermally denatured protein. The results suggest that the binding of Zn(II) to the first Zn(II)-(or Cu(lI))-specific site does not cause any drastic changes in the overall structure of a-LA. The acidic form of a-LA (at pH 2.2 and 37°C) was digested by pepsin at rates similar to that for the apo-or Cu(II), Ca(II)-loaded forms by trypsin or a-chymotrypsin at neutralpH. Complexation of a-LA with bis-ANS affords protection against pepsin cleavage. It is suggested that the protective effects of similar small lipophilic compounds to a-LA may have physiological significance (eog., for nutritional transport).

The giant, ~3.6-MDa hexagonal bilayer hemoglobin (Hb) of Lumbricus terrestris consist of twelve 213-kDa globin subassemblies, each comprised of three disulfide-bonded trimers and three monomer globin chains, tethered to a central scaffolding of 36-42 linkers L1-L4 (24-32 kDa). It is known to contain 50-80 Ca and 2-4 Cu and Zn; the latter are thought to be responsible for the superoxide dismutase activity of the Hb. Total reflection X-ray fluorescence spectrometry was used to determine the Ca, Cu, and Zn contents of the Hb dissociated at pH ~2.2, the globin dodecamer subassembly, and linker subunits L2 and L4. Although the dissociated Hb retained 20 Ca 2+ and all the Cu and Zn, the globin subassembly had 0.4 to ~3 Ca 2+ , depending on the method of isolation, and only traces of Cu and Zn. The linkers L2 and L4, isolated by reversed-phase high-pressure liquid chromatography at pH ~2.2, had 1 Ca per mole and very little Cu and Zn. Electrospray ionization mass spectrometry of linker L3 at pH ~2.2 and at neutral pH demonstrated avid binding of 1 Ca 2+ and additional weaker binding of 7 Ca 2+ in the presence of added Ca 2+ . Based on these and previous results which document the heterogeneous nature of the Ca 2+ -binding sites in Lumbricus Hb, we propose three classes of Ca 2+ -binding sites with affinities increasing in the following order: (i) a large number of sites (>100) with affinities lower than EDTA associated with linker L3 and dodecamer subassembly, (ii) ~30 sites with affinities higher than EDTA occurring within the cysteine-rich domains of linker L3 and dodecamer subassembly, and (iii) ~25 very high affinity sites associated with the linker subunits L1, L2, and L4. It is likely that the low-affinity type (i) sites are the ones involved in the effects of 1-100 mM Group IIA cations on Lumbricus Hb structure and function, namely increased stability of its quaternary structure and increased affinity and cooperativity of its oxygen binding.

The giant, ~3.6-MDa hexagonal bilayer hemoglobin (Hb) of Lumbricus terrestris consist of twelve 213-kDa globin subassemblies, each comprised of three disulfide-bonded trimers and three monomer globin chains, tethered to a central scaffolding of 36-42 linkers L1-L4 (24-32 kDa). It is known to contain 50-80 Ca and 2-4 Cu and Zn; the latter are thought to be responsible for the superoxide dismutase activity of the Hb. Total reflection X-ray fluorescence spectrometry was used to determine the Ca, Cu, and Zn contents of the Hb dissociated at pH ~2.2, the globin dodecamer subassembly, and linker subunits L2 and L4. Although the dissociated Hb retained 20 Ca 2+ and all the Cu and Zn, the globin subassembly had 0.4 to ~3 Ca 2+ , depending on the method of isolation, and only traces of Cu and Zn. The linkers L2 and L4, isolated by reversed-phase high-pressure liquid chromatography at pH ~2.2, had 1 Ca per mole and very little Cu and Zn. Electrospray ionization mass spectrometry of linker L3 at pH ~2.2 and at neutral pH demonstrated avid binding of 1 Ca 2+ and additional weaker binding of 7 Ca 2+ in the presence of added Ca 2+ . Based on these and previous results which document the heterogeneous nature of the Ca 2+ -binding sites in Lumbricus Hb, we propose three classes of Ca 2+ -binding sites with affinities increasing in the following order: (i) a large number of sites (>100) with affinities lower than EDTA associated with linker L3 and dodecamer subassembly, (ii) ~30 sites with affinities higher than EDTA occurring within the cysteine-rich domains of linker L3 and dodecamer subassembly, and (iii) ~25 very high affinity sites associated with the linker subunits L1, L2, and L4. It is likely that the low-affinity type (i) sites are the ones involved in the effects of 1-100 mM Group IIA cations on Lumbricus Hb structure and function, namely increased stability of its quaternary structure and increased affinity and cooperativity of its oxygen binding.

p38a is a significant target for drug designing against cancer. The overproduction of p38a MAPK promotes tumorigenesis in head and neck squamous cell carcinoma (HNSCC). The ATP binding and an allosteric site referred as DFG are the key sites of the p38a mitogen activated protein kinase (MAPK) exploited for the design of inhibitors. This study demonstrated design of peptide inhibitor on the basis of allosteric site using Glide molecular docking software and the biochemical analysis of the best modeled peptide. The best fitted tetrapeptide (FWCS) in the allosteric site inhibited the pure recombinant and serum p38a of HNSCC patients by 74 and 72%, respectively. The potency of the peptide was demonstrated by its IC 50 (4.6 nM) and K D (3.41610 210 M) values, determined by ELISA and by surface plasmon resonance (SPR) technology, respectively. The cell viability of oral cancer i.e. KB cell line was reduced in dose dependent manner by 60 and 97% by the treatment of peptide and the IC 50 was 600 and 210 mM after 24 and 72 h incubation, respectively. Our result provides an insight for the development of a proficient small peptide as a promising anticancer agent targeting DFG site of p38a kinase.

Predicting the three-dimensional (3D) functional structures of proteins remains an important computational milestone in molecular biology to be achieved. This feat is hinged on a clear understanding of the mechanism which proteins use to fold into their native structures. Since Levinthal's paradox, there has been a lot of progress in understanding this mechanism. Most of the earlier attempts were caught between assigning either hydrophobic interactions or hydrogen bonding as the dominant folding force. However, a consensus now seems to be emerging about hydrogen bonding being a stronger force. Interestingly, a view from chaperone action may further throw some light on the nature of the folding mechanism. Thus the very mechanisms which prevent protein aggregation and misfolding, could help us have a better understanding of the folding mechanism itself.

In Antheraea mylitta (D) (Lepidoptera, Saturniidae), the effect of photoperiod on total DNA, RNA, and protein concentration in the middle silk gland (MSG) and posterior silk gland (PSG) was studied. The photoperiod caused enhancement in the secretory activity and stimulates the sericogenesis process in MSG and PSG. The total nucleic acids and protein concentration of MSG and PSG under 24L:00D condition reached maximum level and then it decreased. Maximum nucleic acids and protein concentration were observed during long-day period and the minimum during short-day period. The photoperiod activated or inhibited the biological clock which acted upon the respective endocrine glands accordingly and control the process of sericogenesis. The larvae underwent significant changes with high DNA, RNA and protein concentration under 24L:00D, which is highly suitable for the silk protein synthesis.

A comparison of both amino acid composition and sequence of the rabbit uteroglobin (UG) subunit and the rat seminal vesicle sperm-binding protein (rSBP) by computer analysis indicates homology between the two polypeptide chains. These findmgs are supported by immunological studies showing the occurrence of similar antigenic determinants. In addition, our data indicate the glutamine-9 of the rat seminal vesicle sperm-binding protein and glutamine-40 of UG as the possible glutamine residues involved when the proteins act as transglutaminase (TGase) substrates. The latter results represent an interesting approach to determining the general structural features of the acyl donor site in the TGase-catalyzed reaction.

A comparison of both amino acid composition and sequence of the rabbit uteroglobin (UG) subunit and the rat seminal vesicle sperm-binding protein (rSBP) by computer analysis indicates homology between the two polypeptide chains. These findmgs are supported by immunological studies showing the occurrence of similar antigenic determinants. In addition, our data indicate the glutamine-9 of the rat seminal vesicle sperm-binding protein and glutamine-40 of UG as the possible glutamine residues involved when the proteins act as transglutaminase (TGase) substrates. The latter results represent an interesting approach to determining the general structural features of the acyl donor site in the TGase-catalyzed reaction.

Antigenic characterization of Trypanosoma evansi using sera from experimentally and naturally infected bovines, equines, dogs, and coatis Caracterização antigênica do Trypanosoma evansi usando soros de bovinos, equinos, cães e quatis experimentalmente e naturalmente infectados

The application of computational design of protein is mainly seen in the mutations of its active sites for increasing the reliability of that particular protein. Two essential examples from our groups are the stability improvement of riboflavin synthase and lipase enzyme. Specific mutations were introduced to their active and binding sites. Thus, by running molecular dynamics simulations, it is confirmed that the mutations improve the protein stability significantly. However, as the future of green chemistry is eventually aligned with nanotechnology, the atomic and even the subatomic features of the protein improvement will be put into the discussion. Nanotechnology is nowadays could not separate by the computational procedures. Besides molecular dynamics, finding the binding and active sites, as well as their chemical interactions, are important aspects of the nano-based computational design. In this respect, it could be related that the bioinformatics tools in Green Chemistry should be aligned with nanotechnology to produce finegrained protein design that environmentally friendly.

Structural and functional data on 2S albumins and particularly rape seed napins are reviewed and, based on the coordinates of the three-dimensional structure of napin-like albumin BnIb, are used to model different rape napins. Surprisingly, the modeled napins, despite great sequence homology, differ in tertiary arrangements of the polypeptide chains. It is proposed that these differences in 3D structures of the analyzed rape napins may reflect their functions, which may cover many other potential beneficial purposes besides simple storage.

Histone-poly(A) hybrid molecules were used for transport experiments with resealed nuclear envelopes and after attachment of a cleavable cross-linker (SASD) to identify nuclear proteins. In contrast to histories, the hybrid molecules cannot be accumulated in resealed nuclear envelopes, and in contrast to poly(A), the export of hybrids from preloaded nuclear envelopes is completely impaired. The experiments strongly confirm the existence of poly(A) as an export signal in mRNA which counteracts the nuclear location signals (NLS) in histones. The contradicting transport signals in the hybrid molecules impair translocation through the nuclear pore complex. The failure to accumulate hybrid molecules into resealed nuclear envelopes results from the covalent attachment of polyadenylic acid to histones in a strict 1:1 molar ratio. This was demonstrated in control transport experiments where radiolabeled histones were simply mixed with nonlabeled poly(A) or radiolabeled poly(A) mixed with nonlabeled histones. In comparison, control uptake experiments with histories covalently linked to a single UMP-mononucleotide are strongly enhanced. Such controls exclude the conceivable possibility of a simple masking of the nuclear location signal in the histones by the covalent attached poly(A) moiety. Photoreactive histone-poly(A) hybrid analogs serve to identify nuclear envelope proteins--presumably in the nuclear pore--with molecular weights of 110, 80, and 71.4 kDa.

As a prerequisite for the synthesis of affinity labels, we describe methods to couple histories to ribonucleic acids. For the synthesis of these covalent hybrid molecules, we used a population of histones HI, H2A, H2B, H3, and H4 from calf thymus and polyadenylic acid with an average chain length of up to 260-280 bases, representing the size of poly(A)-tails from mature mRNAs. Three methods were investigated. (a) Poly(A) containing an 8-N3-A residue was cross-linked to histones by ultraviolet irradiation. (b) The 3'-end of the polynucleotide was connected to a mononucleotide containing an aliphatic amino group, and the resulting poly(A)-derivative was coupled to histones via derivation with a bromoacetyl group. (c) The Y-end of the polynucleotide was oxidized with sodium periodate and bound covalently to an amino group of the polypeptide. To demonstrate the RNA content of the hybrid molecule, the poly(A) was removed with RNase T2.

Histone-poly(A) hybrid molecules were used for transport experiments with resealed nuclear envelopes and after attachment of a cleavable cross-linker (SASD) to identify nuclear proteins. In contrast to histories, the hybrid molecules cannot be accumulated in resealed nuclear envelopes, and in contrast to poly(A), the export of hybrids from preloaded nuclear envelopes is completely impaired. The experiments strongly confirm the existence of poly(A) as an export signal in mRNA which counteracts the nuclear location signals (NLS) in histones. The contradicting transport signals in the hybrid molecules impair translocation through the nuclear pore complex. The failure to accumulate hybrid molecules into resealed nuclear envelopes results from the covalent attachment of polyadenylic acid to histones in a strict 1:1 molar ratio. This was demonstrated in control transport experiments where radiolabeled histones were simply mixed with nonlabeled poly(A) or radiolabeled poly(A) mixed with nonlabeled histones. In comparison, control uptake experiments with histories covalently linked to a single UMP-mononucleotide are strongly enhanced. Such controls exclude the conceivable possibility of a simple masking of the nuclear location signal in the histones by the covalent attached poly(A) moiety. Photoreactive histone-poly(A) hybrid analogs serve to identify nuclear envelope proteins--presumably in the nuclear pore--with molecular weights of 110, 80, and 71.4 kDa.

The structural stability of metmyoglobin in organic solvents and cosolvents was investigated aiming the choice of a suitable medium to perform its dissolution with maintenance of the native folding. The spectroscopic behavior of metmyoglobin solution in UV-Visible and circular dichroism was used to evaluate the solubility and the secondary structure. The results were dependable of the chemical structure of the organic compounds, their polarity and content, in the case of cosolvents. Protic solvents showed better ability than the aprotic ones for the biomolecule dissolution, since they are able to establish hydrogen bonds. Solvents with high polarity usually damage the secondary structure of the protein. Myoglobin was dissolved in pure methanol, ethylene glycol and glycerol. The secondary structure was retained in some extent. The controlled addition of sodium dodecyl sulfate to myoglobin aqueous solution changed the surface moiety of the protein. The complex was extracted to hexane with efficiency of 77%.

Fluorescence spectroscopy and differential scanning calorimetry were used to follow local and global changes in human serum albumin domains during chemical and thermal denaturation of this protein. Results suggests that thermal and chemical treatments involved an unfolding pathway of at least two steps and that domain IIA is not homogeneous. Unfolding at site I exposes a larger hydrophobic area to the solvent than at site II. The bilirubin-binding site showed atypical behavior: a significant increase in the hydrophobic area was exposed to the solvent when its binding site was denatured by guanidine hydrochloride. This result might be due to the high specificity of the bilirubin-binding site, whose binding makes an extensive conformational change in the environment of this site. KEY WORDS: Unfolding; chemical and thermal denaturation; optical probes; albumin domains. native proteins is very sensitive to conformational changes in their environments produced during protein unfolding. Human serum albumin has only one tryptophan residue, at position 214, which is buried in domain II near binding site II. The presence of only one tryptophan turns this protein into an excellent model for following its denaturation process by measuring the fluorescence emission of this fluorophore. However, the information that is obtained from tryptophan fluorescence studies is often not sufficient to characterize changes in the different domains of this protein. Sudlow et al. (1995) characterized two specific ligandbinding sites in albumin and designated them as site I and II. 5-Dimethtlaminonaphthalene-1-sulfonamide (DSNA) 4 binds to site I and dansylsarcosine (DSS) binds to site II. The fluorescence intensities of these ligands are enhanced when they bind to this protein; they act as fluorescent probes in the study of the structure of these binding sites. Albumin also has a specific site for the

Shelf-stable milk is consumed worldwide, and this market is expected to continue growing. One quality challenge for UHT milk is age gelation during shelf life, which is in part caused by bacterial heat-stable proteases (HSP) synthesized during the raw milk storage period before heat processing. Some Pseudomonas spp. are HSP producers, and their ability to grow well at refrigeration temperature make them important spoilage organisms for UHT processors to control. Previous studies have shown that lactose oxidase (LO), a natural and commercially available enzyme that produces hydrogen peroxide and lactobionic acid from lactose, can control bacterial growth in raw milk. In this research, we investigated the ability of LO to control HSP producer outgrowth, and thus delay age gelation in UHT milk. Six strains of Pseudomonas spp. were selected based on their ability to synthesize HSP and used as a cocktail to inoculate both raw and sterile (UHT) milk at a level of 1 × 10 5 cfu/mL. Groups were treated with and without LO, stored for 4 d at 6°C, and monitored for cell count and pH. Additionally, a sample from each was tested for HSP activity via particle size analysis (average effective diameter at 90° angle and 658 nm wavelength) and visual inspection on each day of the storage period. The HSP activity results were contrasted using Tukey's HSD test, which showed that in UHT milk, a LO treatment (0.12 g/L) effectively prevented gelation as compared with the control. In raw milk, however, a concentration of 0.24 g/L of LO was needed to obtain a similar effect. This test was scaled up to 19-L pilot plant batches of raw milk where they were challenged with Pseudomonas cocktail, treated with LO for 3 d, and then UHT processed. Resulting UHT milk bottles were monitored for gelation. Significant differences in particle size between the LO-treated samples and the control were observed as early as 1 mo after processing , and gelation was not detected in the LO-treated samples through 6 mo of storage. These results demonstrated that LO can be used to delay age gelation in UHT milk induced by HSP-producing Pseudomonas spp., representing an opportunity to improve quality and reduce postproduction losses in the shelf-stable milk market sector.

Editorial on the Research Topic Insights in protein biochemistry: protein biophysics 2022 This Research Topic highlights diverse biophysical approaches, methods and tools available for a better understanding of structure-function relationships in proteins. Protein biophysics is crucial for understanding how proteins are formed and function. Proteins are the most complex and versatile molecules known to us. They can be controlled by a broad range of mechanisms, which, in turn can regulate a variety of biological processes (

Novel genetic variants for donkey milk lysozyme and b-lactoglobulins I and II have been identified by the combined use of peptide mass mapping and sequencing by tandem mass spectrometry in association with database searching. The novel donkey lysozyme variant designated as lysozyme B (Mr 14,631 Da) differed in three amino acid exchanges, N 49 ® D, Y 52 ® S, and S 61 ® N, from the previously published sequence. Three novel genetic variants for donkey b-lactoglobulins were identified. One of them is a type b-lactoglobulin I with three amino acid exchanges at E 36 ® S, S 97 ® T, and V 150 ® I (b-lactoglobulin I B, Mr 18,510 Da). The two others are type b-lactoglobulins II with two amino acid exchanges at C 110 ® P and M 118 ® T (b-lactoglobulin II B, Mr 18,227 Da) and with three amino acid exchanges at D 96 ® E, C 110 ® P, and M 118 ® T (b-lactoglobulin II C, Mr 18,241 Da). All these primary structures are closely related to those of homologous proteins in horse milk (percent identity >96%).

The class I and II Clostridium histolyticum collagenases (CHC) have been used to identify hyperreactive sites in rat type I, bovine type II, and human type III collagens. The class I CHC attack both collagens at loci concentrated in the N-terminal half of these collagens starting with the site closest to the N-terminus. The class II CHC initiate collagenolysis by attacking both collagens in the interior to produce a mixture of C-terminal 62~000 and a Nterminal 36,000 fragments. Both fragments are next shortened by removal of a 3000 fragment. These results are very similar to those reported earlier for the hydrolysis of rat type I collagen by these CHC, indicating that the three collagens share many hyperreactive sites. Similar reactions carried out with the respective gelatins show that they are cleaved at many sites at approximately the same rate. Thus, the hyperreactivity of the sites identified must be attributed to their environment in the native collagens. N-terminal sequencing of the fragments produced in these reactions has allowed the identification of 16 cleavage sites in the at(I), a2(l), al(II), and a 1 (III) collagen chains. An analysis of the triple helical stabilities of these cleavage site regions as reflected by their imino acid contents fails to yield a correlation between reactivity and triple helical stability. The existence of these hyperreactive CHC cleavage sites suggests that type I, II, and III collagens contain regions that have specific nontriple helical conformations. The sequence of these sites presented here now makes it possible to investigate these conformations by computational and peptide mimetic techniques.

FKBP, an 11.8 kD intracellular protein that binds the immunosuppressants FK506 (Ka= 0.4 nM) and rapamycin (Kd = 0.2 nM) with high affinity, was purified to homogeneity from calf thymus. The complete amino acid sequence has been determined by automated Edman degradation of the intact molecule and overlapping fragments generated by proteolytic and chemical cleavage. The analysis revealed a 107 amino acid peptide chain with the following sequence: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFV-LGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPNATLIFDVELLKLE. The molecular weight, calculated from the amino sequence to be 11,778 D, was confirmed by electrospray ionization mass spectrometry. Thus, naturally isolated bovine FKBP does not appear to have any residues modified by glycosylation, phosphorylation, or other posttranslational derivatization processes. Bovine FKBP has only three amino acid residues that differ from human FKBP, whose sequence was elucidated by cloning and sequencing complementary DNA (Standaert et aL, 1990). The protein has a substantial number of hydrophilic peptide segments with prevalent/~-strand type of chain fold. Understanding the biological function of FKBP and other members of the immunophilin class and their respective complexes with immunosuppressive drugs may provide insights into cytoplasmic signalling mechanisms, protein folding and translocation, and other cellular processes.

Chou-Fasman algorithm is an empirical algorithm developed for the prediction of protein secondary structure. Implementation and interpretation of the secondary structure of protein has been done using C programming and the output of the result has been predicted good results compared with SOPMA, PSI Pred and Chou-Fasman v1.1 servers. The predicted protein confirmed good accuracy to PSSP results from C programming of the query protein compared to PDB.

Antibodies directed against recombinant erythropoietin have been obtained by immunization of rabbits with the hormone in Freund's complete adjuvant. Two sets of antibodies are present in the serum of the immunized rabbits. The results of oxidation of the erythropoietin with periodate, inhibition of the antibodies with the structural monosaccharide residues of the hormone, and reaction of the antibodies with lectins of known carbohydrate specificity have established the antibodies to be anti-carbohydrate antibodies. These antibodies may be of value as tracking agents for some diseases and should be useful for detecting abuses of the hormone in enhancing performance in athletic competitions.

Life on Earth is capable of growing from temperatures well below freezing to above the boiling point of water, with some organisms preferring cooler and others hotter conditions. The growth rate of each organism ultimately depends on its intracellular chemical reactions. Here we show that a thermodynamic model based on a single, rate-limiting, enzymecatalysed reaction accurately describes population growth rates in 230 diverse strains of unicellular and multicellular organisms. Collectively these represent all three domains of life, ranging from psychrophilic to hyperthermophilic, and including the highest temperature so far observed for growth (122 0 C). The results provide credible estimates of thermodynamic properties of proteins and obtain, purely from organism intrinsic growth rate data, relationships between parameters previously identified experimentally, thus bridging a gap between biochemistry and whole organism biology. We find that growth rates of both unicellular and multicellular life forms can be described by the same temperature dependence model. The model results provide strong support for a single highly-conserved reaction present in the last universal common ancestor (LUCA). This is remarkable in that it means that the growth rate dependence on temperature of unicellular and multicellular life forms that evolved over geological time spans can be explained by the same model.

Butterflies constitute approximately 10% of lepidopteran insects, and along with silkworms, they can produce silk; however, this feature is often ignored. In the present study, we observed two primary methods used by butterflies to hang pupae on trees using silk: pupa adheraena (Danaus chrysippus) and pupa contigua (Papilio polytes). Anchoring the abdominal ends of pupae with a silk pad was the most common method used in both cases, whereas wrapping silk around the body using a silk girdle was a method unique to pupa contigua. The connection between the cremaster and silk pad was observed to be similar to that between the hook and loop of a Velcro fastener, except that the cremaster hook is anchor-shaped rather than being a single hook. Such a connection will remain secure, ensuring the safety of the pupae during exposure to wind and rain. Through determining the mechanical properties of silk, the performance of butterfly silk was found to be weaker than that of silkworm silk. Therefore, the P. polytes silk girdle adopts the strategy of merging a dozen silk threads to improve its strength and toughness, thereby making it difficult to break. In addition, we explained how the protein sequence and structure of butterfly silk impact its performance. In conclusion, we discovered that butterfly pupae develop unique body features to establish secure bonds with silk. This enables them to effectively undergo metamorphosis and endure harsh weather conditions and surroundings.