Protein content and amino acid composition of some varieties of grain sorghum

Determination of the protein content and lysine levels of a number of nonhybrid varieties of grain sorghum indicates large variations in the protein content. Statistical analysis of data on amounts of lysine shows that a negative correlation exists between per cent lysine in the protein and per cent protein in the seed. The proportion of various protein fractions in endosperm of five varieties of grain sorghum of both low- and high-protein type has been determined. Results show that prolamine and glutelin are the principal protein fractions, and increased protein levels in sorghum varieties are correlated with an increase mainly in the prolamine fraction. Nine high- and low-protein varieties of grain sorghum have been analyzed for their amino acid composition by ion exchange procedures. One of the high-protein genetic varieties of sorghum has a high concentration of lysine in the seed. Amino acid composition of the protein fractions of two varieties is also reported. These data permit an evaluation of the nutritional quality of sorghum protein and factors that influence the quality of the protein.

Novel Pentameric Structure of the Diarrhea-Inducing Region of the Rotavirus Enterotoxigenic Protein NSP4

A novel pentameric structure which differs from the previously reported tetrameric form of the diarrhea-inducing region of the rotavirus enterotoxin NSP4 is reported here. A significant feature of this pentameric form is the absence of the calcium ion located in the core region of the tetrameric structures. The lysis of cells, the crystallization of the region spanning residues 95 to 146 of NSP4 (NSP4(95-146)) of strain ST3 (ST3: NSP4(95-146)) at acidic pH, and comparative studies of the recombinant purified peptide under different conditions by size-exclusion chromatography (SEC) and of the crystal structures suggested pH-, Ca(2+)-, and protein concentration-dependent oligomeric transitions in the peptide. Since the NSP4(95-146) mutant lacks the N-terminal amphipathic domain (AD) and most of the C-terminal flexible region (FR), to demonstrate that the pentameric transition is not a consequence of the lack of the N- and C-terminal regions, glutaraldehyde cross-linking of the Delta N72 and Delta N94 mutant proteins, which contain or lack the AD, respectively, but possess the complete C-terminal FR, was carried out. The results indicate the presence of pentamers in preparations of these longer mutants. Detailed SEC analyses of Delta N94 prepared under different conditions, however, revealed protein concentration-dependent but metal ion-and pH-independent pentamer accumulation at high concentrations which dissociated into tetramers and lower oligomers at low protein concentrations. While calcium appeared to stabilize the tetramer, magnesium in particular stabilized the dimer. Delta N72 existed primarily in the multimeric form under all conditions. These findings of a calcium-free NSP4 pentamer and its concentration-dependent and largely calcium-independent oligomeric transitions open up a new dimension in an understanding of the structural basis of its multitude of functions.

Early protein malnutrition disrupts cerebellar development and impairs motor coordination

Maternal malnutrition affects every aspect of fetal development. The present study asked the question whether a low-protein diet of the mother could result in motor deficits in the offspring. Further, to examine whether cerebellar pathology was correlated with motor deficits, several parameters of the postnatal development of the cerebellum were assayed. This is especially important because the development of the cerebellum is unique in that the time scale of development is protracted compared with that of the cortex or hippocampus. The most important result of the study is that animals born to protein-deficient mothers showed significant delays in motor development as assessed by rotarod and gait analysis. These animals also showed reduced cell proliferation and reduced thickness in the external granular layer. There was a reduction in the number of calbindin-positive Purkinje cells (PC) and granular cells in the internal granular layer. However, glial fibrillary acidic protein-positive population including Bergmann glia remained unaffected. We therefore conclude that the development of the granular cell layer and the PC is specifically prone to the effects of protein malnutrition potentially due to their protracted developmental period from approximately embryonic day 11 to 13 until about the third postnatal week.

Structural effects of a dimer interface mutation on catalytic activity of triosephosphate isomerase.The role of conserved residues and complementary mutations

The active site of triosephosphate isomerase (TIM, EC: 5.3.1.1), a dimeric enzyme, lies very close to the subunit interface. Attempts to engineer monomeric enzymes have yielded well-folded proteins with dramatically reduced activity. The role of dimer interface residues in the stability and activity of the Plasmodium falciparum enzyme, PfTIM, has been probed by analysis of mutational effects at residue 74. The PfTIM triple mutant W11F/W168F/Y74W (Y74W*) has been shown to dissociate at low protein concentrations, and exhibits considerably reduced stability in the presence of denaturants, urea and guanidinium chloride. The Y74W* mutant exhibits concentration-dependent activity, with an approximately 22-fold enhancement of kcat over a concentration range of 2.5–40 μm, suggesting that dimerization is obligatory for enzyme activity. The Y74W* mutant shows an approximately 20-fold reduction in activity compared to the control enzyme (PfTIM WT*, W11F/W168F). Careful inspection of the available crystal structures of the enzyme, together with 412 unique protein sequences, revealed the importance of conserved residues in the vicinity of the active site that serve to position the functional K12 residue. The network of key interactions spans the interacting subunits. The Y74W* mutation can perturb orientations of the active site residues, due to steric clashes with proximal aromatic residues in PfTIM. The available crystal structures of the enzyme from Giardia lamblia, which contains a Trp residue at the structurally equivalent position, establishes the need for complementary mutations and maintenance of weak interactions in order to accommodate the bulky side chain and preserve active site integrity.

When good neighbors don't need fences: temporal landscape partitioning among baboon social groups

Intraspecific competition is a key factor shaping space-use strategies and movement decisions in many species, yet how and when neighbors utilize shared areas while exhibiting active avoidance of one another is largely unknown. Here, we investigated temporal landscape partitioning in a population of wild baboons (Papio cynocephalus). We used global positioning system (GPS) collars to synchronously record the hourly locations of five baboon social groups for similar to 900 days, and we used behavioral, demographic, and life history data to measure factors affecting use of overlap areas. Annual home ranges of neighboring groups overlapped substantially, as predicted (baboons are considered non-territorial), but home ranges overlapped less when space use was assessed over shorter time scales. Moreover, neighboring groups were in close spatial proximity to one another on fewer days than predicted by a null model, suggesting an avoidance-based spacing pattern. At all time scales examined (monthly, biweekly, and weekly), time spent in overlap areas was greater during time periods when groups fed on evenly dispersed, low-quality foods. The percent of fertile females in social groups was negatively correlated with time spent in overlap areas only during weekly time intervals. This suggests that broad temporal changes in ecological resources are a major predictor of how intensively overlap areas are used, and groups modify these ecologically driven spacing patterns at short time scales based on female reproductive status. Together, these findings offer insight into the economics of territoriality by highlighting the dynamics of spacing patterns at differing time scales.

Recognition of Interaction Interface Residues in Low-Resolution Structures of Protein Assemblies Solely from the Positions of C alpha Atoms

Background: The number of available structures of large multi-protein assemblies is quite small. Such structures provide phenomenal insights on the organization, mechanism of formation and functional properties of the assembly. Hence detailed analysis of such structures is highly rewarding. However, the common problem in such analyses is the low resolution of these structures. In the recent times a number of attempts that combine low resolution cryo-EM data with higher resolution structures determined using X-ray analysis or NMR or generated using comparative modeling have been reported. Even in such attempts the best result one arrives at is the very course idea about the assembly structure in terms of trace of the C alpha atoms which are modeled with modest accuracy. Methodology/Principal Findings: In this paper first we present an objective approach to identify potentially solvent exposed and buried residues solely from the position of C alpha atoms and amino acid sequence using residue type-dependent thresholds for accessible surface areas of C alpha. We extend the method further to recognize potential protein-protein interface residues. Conclusion/Significance: Our approach to identify buried and exposed residues solely from the positions of C alpha atoms resulted in an accuracy of 84%, sensitivity of 83-89% and specificity of 67-94% while recognition of interfacial residues corresponded to an accuracy of 94%, sensitivity of 70-96% and specificity of 58-94%. Interestingly, detailed analysis of cases of mismatch between recognition of interface residues from C alpha positions and all-atom models suggested that, recognition of interfacial residues using C alpha atoms only correspond better with intuitive notion of what is an interfacial residue. Our method should be useful in the objective analysis of structures of protein assemblies when positions of only C alpha positions are available as, for example, in the cases of integration of cryo-EM data and high resolution structures of the components of the assembly.

Prediction of protein-protein interactions in dengue virus coat proteins guided by low resolution cryoEM structures

Background: Dengue virus along with the other members of the flaviviridae family has reemerged as deadly human pathogens. Understanding the mechanistic details of these infections can be highly rewarding in developing effective antivirals. During maturation of the virus inside the host cell, the coat proteins E and M undergo conformational changes, altering the morphology of the viral coat. However, due to low resolution nature of the available 3-D structures of viral assemblies, the atomic details of these changes are still elusive. Results: In the present analysis, starting from C alpha positions of low resolution cryo electron microscopic structures the residue level details of protein-protein interaction interfaces of dengue virus coat proteins have been predicted. By comparing the preexisting structures of virus in different phases of life cycle, the changes taking place in these predicted protein-protein interaction interfaces were followed as a function of maturation process of the virus. Besides changing the current notion about the presence of only homodimers in the mature viral coat, the present analysis indicated presence of a proline-rich motif at the protein-protein interaction interface of the coat protein. Investigating the conservation status of these seemingly functionally crucial residues across other members of flaviviridae family enabled dissecting common mechanisms used for infections by these viruses. Conclusions: Thus, using computational approach the present analysis has provided better insights into the preexisting low resolution structures of virus assemblies, the findings of which can be made use of in designing effective antivirals against these deadly human pathogens.

Protein Hydration and Water Structure: X-ray Analysis of a Closely Packed Protein Crystal with Very Low Solvent Content

Low-humidity monoclinic lysozyme, resulting from a water-mediated transformation, has one of the lowest solvent contents (22% by volume) observed in a protein crystal. Its structure has been solved by the molecular replacement method and refined to an R value of 0.175 for 7684 observed reflections in the 10–1.75 Å resolution shell. 90% of the solvent in the well ordered crystals could be located. Favourable sites of hydration on the protein surface include side chains with multiple hydrogen-bonding centres, and regions between short hydrophilic side chains and the main-chain CO or NH groups of the same or nearby residues. Major secondary structural features are not disrupted by hydration. However, the free CO groups at the C terminii and, to a lesser extent, the NH groups at the N terminii of helices provide favourable sites for water interactions, as do reverse turns and regions which connect β-structure and helices. The hydration shell consists of discontinuous networks of water molecules, the maximum number of molecules in a network being ten. The substrate-binding cleft is heavily hydrated, as is the main loop region which is stabilized by water interactions. The protein molecules are close packed in the crystals with a molecular coordination number of 14. Arginyl residues are extensively involved in intermolecular hydrogen bonds and water bridges. The water molecules in the crystal are organized into discrete clusters. A distinctive feature of the clusters is the frequent occurrence of three-membered rings. The protein molecules undergo substantial rearrangement during the transformation from the native to the low-humidity form. The main-chain conformations in the two forms are nearly the same, but differences exist in the side-chain conformation. The differences are particularly pronounced in relation to Trp 62 and Trp 63. The shift in Trp 62 is especially interesting as it is also known to move during inhibitor binding.

Withania somnifera reverses Alzheimer's disease pathology by enhancing low-density lipoprotein receptor-related protein in liver

A 30-d course of oral administration of a semipurified extract of the root of Withania somnifera consisting predominantly of withanolides and withanosides reversed behavioral deficits, plaque pathology, accumulation of beta-amyloid peptides (A beta) and oligomers in the brains of middle-aged and old APP/PS1 Alzheimer's disease transgenic mice. It was similarly effective in reversing behavioral deficits and plaque load in APPSwInd mice (line J20). The temporal sequence involved an increase in plasma A beta and a decrease in brain A beta monomer after 7 d, indicating increased transport of A beta from the brain to the periphery. Enhanced expression of low-density lipoprotein receptor-related protein (LRP) in brain microvessels and the A beta-degrading protease neprilysin (NEP) occurred 14-21 d after a substantial decrease in brain A beta levels. However, significant increase in liver LRP and NEP occurred much earlier, at 7 d, and were accompanied by a rise in plasma sLRP, a peripheral sink for brain A beta. In WT mice, the extract induced liver, but not brain, LRP and NEP and decreased plasma and brain A beta, indicating that increase in liver LRP and sLRP occurring independent of A beta concentration could result in clearance of A beta. Selective down-regulation of liver LRP, but not NEP, abrogated the therapeutic effects of the extract. The remarkable therapeutic effect of W. somnifera mediated through up-regulation of liver LRP indicates that targeting the periphery offers a unique mechanism for A beta clearance and reverses the behavioral deficits and pathology seen in Alzheimer's disease models.

Detection of the protein dimers, multiple monomeric states and hydrated forms of Plasmodium falciparum triosephosphate isomerase in the gas phase

Dimeric and monomeric forms of the enzyme triosephosphate isomerase (TIM) from Plasmodium falciparum (Pf) have been detected under conditions of nanoflow by electrospray mass spectrometry. The dimer (M = 55 663 Da) exhibits a narrow charge state distribution with intense peaks limited to values of 18(+) to 21(+), maximal intensity being observed for charge states 19(+) and 20(+). A monomeric species with a charge state distribution ranging from 11(+) to 16(+) is also observed, which may be assigned to folded dissociated subunits. Complete dimer dissociation results under normal electrospray condition. The effects of solution pH and source temperature have been investigated. The observation of four distinct charge state distributions which may be assigned to a dimer, folded monomer, partially folded monomer and unfolded monomer is reported. Circular dichromism and fluorescence studies of Pf TIM at low pH support the retention of substantial secondary and tertiary structures. Satellite peaks in mass spectra corresponding to hydrated species are also observed and isotope shift upon deuteration is demonstrated. The analysis of all available independent crystal structures of Pf TIM and TIMs from other organisms permits identification of structurally conserved water molecules. Hydration observed in the dimer and folded monomeric forms in the gas phase may correspond to these conserved sites.

Future switching technology: bacterial protein-based programmable all-optical switch for integrated optics applications?

We report on the bacterial protein-based all-optical switches which operate at low laser power, high speed and fulfil most of the requirements to be an ideal all-optical switch without any moving parts involved. This consists of conventional optical waveguides coated with bacteriorhodopsin films at switching locations. The principle of operation of the switch is based on the light-induced refractive index change of bacteriorhodopsin. This approach opens the possibility of realizing proteinbased all-optical switches for communication network, integrated optics and optical computers.

Protein Synthesis in Mycobacterium tuberculosis H37Rv and the Effect of Streptomycin in Streptomycin-Susceptible and -Resistant Strains

An efficient in vitro amino acid-incorporating system from Mycobacterium tuberculosis H37Rv was standardized. Ribonucleic acid (RNA) isolated from phage-infected M. smegmatis cells served as natural messenger RNA and directed the incorporation of 14C-amino acids into protein. The effects of various antitubercular drugs and “known inhibitors” of protein synthesis on amino acid incorporation were studied. Antibiotics like chloramphenicol and tetracycline inhibited mycobacterial protein synthesis, though they failed to prevent the growth of the organism. This failure was shown to be due to the impermeability of mycobacteria to these drugs by use of “membrane-active” agents along with the antibiotics in growth inhibition studies. Several independent streptomycin-resistant mutants of M. tuberculosis H37Rv were isolated. Streptomycin inhibited the incorporation of 14C-amino acids into proteins by whole cells of a streptomycin-susceptible strain by more than 90%, whereas very little or no inhibition was observed in either high-level or low-level streptomycin-resistant strains.

Thermodynamic characterization of the reversible, two-state unfolding of maltose binding protein, a large two-domain protein

The folding and stability of maltose binding protein (MBP) have been investigated as a function of pH and temperature by intrinsic tryptophan fluorescence, far- and near-UV circular dichroism, and high-sensitivity differential scanning calorimetric measurements. MBP is a monomeric, two-domain protein containing 370 amino acids. The protein is stable in the pH range of 4-10.5 at 25 degrees C. The protein exhibits reversible, two-state, thermal and guanidine hydrochloride-mediated denaturation at neutral pH. The thermostability of MBP is maximal at pH 6, with a Tm of 64.9 degrees C and a deltaHm of 259.7 kcal mol(-1). The linear dependence of deltaHm on Tm was used to estimate a value of deltaCp of 7.9 kcal mol(-1) K(-1) or 21.3 cal (mol of residue)(-1) K(-1). These values are higher than the corresponding deltaCp's for most globular proteins studied to date. However, the extrapolated values of deltaH and deltaS (per mole of residue) at 110 degrees C are similar to those of other globular proteins. These data have been used to show that the temperature at which a protein undergoes cold denaturation depends primarily on the deltaCp (per mol of residue) and that this temperature increases with an increase in deltaCp. The predicted decrease in stability of MBP at low temperatures was experimentally confirmed by carrying out denaturant-mediated unfolding studies at neutral pH at 2 and 28 degrees C.

Thermodynamics of metal ion binding and denaturation of a calcium binding protein from Entamoeba histolytica

The thermodynamics of tie binding of calcium and magnesium ions to a calcium binding protein from Entamoeba histolytica was investigated by isothermal titration calorimetry (ITC) in 20 mM MOPS buffer (pH 7.0) at 20 degrees C. Enthalpy titration curves of calcium show the presence of four Ca2+ binding sites, There exist two low-affinity sites for Ca2+, both of which are exothermic in nature and with positive cooperative interaction between them. Two other high affinity sites for Ca2+ exist of which one is endothermic and the other exothermic, again with positive cooperative interaction. The binding constants for Ca2+ at the four sites have been verified by a competitive binding assay, where CaBP competes with a chromophoric chelator 5, 5'-Br-2 BAPTA to bind Ca2+ and a Ca2+ titration employing intrinsic tyrosine fluorescence of the protein, The enthalpy of titration of magnesium in the absence of calcium is single site and endothermic in nature. In the case of the titrations performed using protein presaturated with magnesium, the amount of heat produced is altered. Further, the interaction between the high-affinity sites changes to negative cooperativity. No exchange of heat was observed throughout the addition of magnesium in the presence of 1 mM calcium, Titrations performed on a cleaved peptide comprising the N-terminus and the central linker show the existence of two Ca2+ specific sites, These results indicate that this CaBP has one high-affinity Ca-Mg site, one high-affinity Ca-specific site, and two low-affinity Ca-specific sites. The thermodynamic parameters of the binding of these metal ions were used to elucidate the energetics at the individual site(s) and the interactions involved therein at various concentrations of the denaturant, guanidine hydrochloride, ranging from 0.05 to 6.5 M. Unfolding of the protein was also monitored by titration calorimetry as a function of the concentration of the denaturant. These data show that at a GdnHCl concentration of 0.25 M the binding affinity for the Mg2+ ion is lost and there are only two sites which can bind to Ca2+, with substantial loss cooperativity. At concentrations beyond 2.5 M GdnHCl, at which the unfolding of the tertiary structure of this protein is observed by near UV CD spectroscopy, the binding of Ca2+ ions is lost. We thus show that the domain containing the two low-affinity sites is the first to unfold in the presence of GdnHCl. Control experiments with change in ionic strength by addition of KCI in the range 0.25-1 M show the existence of four sites with altered ion binding parameters.

A Novel Invertase from a Thermophilic FungusThermomyces lanuginosus:Its Requirement of Thiol and Protein for Activation

Unlike the invertases from the mesophilic fungi and yeasts, invertase from a thermophilic fungus,Thermomyces lanuginosus,was unusually unstable bothin vivoandin vitro.The following observations suggested that the unstable nature of the enzyme activity in the cell-free extracts was due to the oxidation of the cysteine residue(s) in the enzyme molecule: (a) the addition of dithiothreitol or reduced glutathione stabilized invertase activity during storage of the extracts and also revived enzyme activity in the extracts which had become inactive with time; (b)N-ethylmaleimide, iodoacetamide, oxidized glutathione, cystine, or oxidized coenzyme A-inactivated invertase; (c) invertase activity was low when the ratio reduced/oxidized glutathione was lower and high when this ratio was higher, suggesting regulation of the enzyme by thiol/disulfide exchange reaction. In contrast to the activation of invertase by the thiol compounds and its inactivation by the disulfides in the cell-free extracts, the purified enzyme did not respond to these compounds. Following its inactivation, the purified enzyme required a helper protein in addition to dithiothreitol for maximal activation. A cellular protein was identified that promoted activation of invertase by dithiothreitol and it was called “PRIA” for theprotein which helps inrestoringinvertaseactivity. The revival of enzyme activity was due to the conversion of the inactive invertase molecules into an active form. A model is presented to explain the modulation of invertase activity by the thiol compounds and the disulfides, both in the crude cell-free extracts and in the purified preparations. The requirement of free sulfhydryl group(s) for the enzyme activity and, furthermore, the reciprocal effects of the thiols and the disulfides on invertase activity have not been reported for invertase from any other source. The finding of a novel invertase which shows a distinct mode of regulation demonstrates the diversity in an enzyme that has figured prominently in the development of biochemistry.