Studies on the conformation of α-globulin fromSesamum indicum L. in cationic and nonionic detergents

The circular dichroic spectra of α-globulin fromSesamum indicum L. was recorded in the presence of cetyltrimethyl ammonium bromide, Triton X-100 and Brij-36T. The protein in 0.2 M phosphate buffer pH 7.4 had about 25% Β-structure and 5% α-helix, the rest being aperiodic or irregular structure and a-helix, structure was increased by cationic detergent cetyl trimethyl ammonium bromide. But, the increase in α-helix content was much less than that induced by an anionic detergent, sodium dodecyl sulphate. In non-ionic detergent like Brij-36T and Triton X-100, specific Β-structures like II-Β and I-Β were formed along with changes in α-helical and aperiodic structures. These results suggest that the protein has a fairly labile quaternary structure. Part of this work was presented at the Second FAOB Congress and Golden Jubilee Meeting of the Society of Biological Chemists (India) held during December 14–18, 1980 at Bangalore, India (Indian J. Biochem. Biophys.) and the work was done at the Biochemistry Department, Brandeis University, Waltham, Massachusetts 02254 USA.     

Association-dissociation of glycinin in urea,guanidine hydrochloride and sodium dodecyl sulphate solutions

The effect of urea, guanidine hydrochloride and sodium dodecyl sulphate on glycinin, the high molecular weight protein fraction from soybean has been investigated by analytical ultracentrifugation. Urea and guanidine hydrochloride dissociate the protein to a ‘2S’ protein through the intermediary 7S and 4S proteins. Howeαer, in sodium dodecyl sulphate the protein directly dissociates to a 2S protein. Analysis of the data by calculation of per cent fraction and S_20,w value indicates that dissociation and denaturation of glycinin occur simultaneously in the presence of the aboαe reagents but to different extents.     

Nucleoside diphosphate kinase A as a controller of AMP-kinase in airway epithelia

This review integrates recent understanding of a novel role for NDPK-A in two related directions: Firstly, its role in an airway epithelial cell when bound to the luminal (apical) membrane and secondly in the cytosol of many different cells (epithelial and non-epithelial) where an isoform-specific interaction occurs with a regulatory partner, AMPKα1. Thus NDPK-A is present in both a membrane and cytosolic environment but in the apical membrane, its roles are not understood in detail; preliminary data suggest that it co-localises with the cystic fibrosis protein (CFTR). In cytosol, we find that NDPK-A is coupled to the catalytic alpha1 isoform of the AMP-activated protein kinase (AMPKα subunit), which is part of a heterotrimeric protein complex that responds to cellular energy status by switching off ATP-consuming pathways and switching on ATP-generating pathways when ATP is limiting. We find that ATP is located within this complex and ‘fed’ from NDPK to AMPK without ever ‘seeing’ bulk solution. Importantly, the reverse can also happen such that AMPK activity can be made to decline when NDPK-A ‘steals’ ATP from AMPK. Thus we propose a novel paradigm in NDPK-A function by suggesting that AMP-kinase can be regulated by NDPK-A, independently of AMP.     

Mapping and proteomic analysis of albumin and globulin proteins in hexaploid wheat kernels (Triticum aestivum L.)

Albumins and globulins of wheat endosperm represent 20% of total kernel protein. They are soluble proteins, mainly enzymes and proteins involved in cell functions. Two-dimensional gel immobiline electrophoresis (2DE) (pH 4-7) × SDS-Page revealed around 2,250 spots. Ninety percent of the spots were common between the very distantly related cultivars ‘Opata 85’ and ‘Synthetic W7984’, the two parents of the International Triticeae Mapping Initiative (ITMI) progeny. ‘Opata’ had 130 specific spots while ‘Synthetic’ had 96. 2DE and image analysis of the soluble proteins present in 112 recombinant inbred lines of the F9-mapped ITMI progeny enabled 120 unbiased segregating spots to be mapped on 21 wheat (Triticum aestivum L. em. Thell) chromosomes. After trypsic digestion, mapped spots were subjected to MALDI-Tof or tandem mass spectrometry for protein identification by database mining. Among the ‘Opata’ and ‘Synthetic’ spots identified, many enzymes have already been mapped in the barley and rice genomes. Multigene families of Heat Shock Proteins, beta-amylases, UDP-glucose pyrophosphorylases, peroxydases and thioredoxins were successfully identified. Although other proteins remain to be identified, some differences were found in the number of segregating proteins involved in response to stress: 11 proteins found in the modern selected cultivar ‘Opata 85’ as compared to 4 in the new hexaploid `Synthetic W7984’. In addition, ‘Opata’ and ‘Synthetic’ differed in the number of proteins involved in protein folding (2 and 10, respectively). The usefulness of the mapped enzymes for future research on seed composition and characteristics is discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Influence of endogenous and exogenous RNases on the variation of pollen cytosolic-free Ca2+ in Pyrus serotina Rehd

The objective of this study was to examine whether S-RNase plays a specific role in the pre-germinated Pyrus pollen. Effects of exogenous RNase and endogenous S-RNase on concentration of cytosolic-free calcium ([Ca²⁺]_i) variation of pre-germinated Pyrus pollen were studied. [Ca²⁺]_i variation caused by different RNases were complex. In 1 h after being cultured, exogenous RNase, RNase T₁ and RNase A, and endogenous incompatible ‘Hohsui’ RNase promoted the [Ca²⁺]_i of ‘Hohsui’ pollen. Acid proteins of ‘Hohsui’ had no remarkable influence on the [Ca²⁺]_i of self-pollen. Endogenous compatible ‘Kohsui’ RNase reduced the [Ca²⁺]_i of ‘Hohsui’ pollen, but compatible ‘Hohsui’ RNase can stimulate the [Ca²⁺]_i of ‘Kohsui’ pollen. RNase T₁, RNase A and incompatible ‘Kohsui’ S-RNase can also make ‘Kohsui’ pollen [Ca²⁺]_i increase. Different from ‘Hohsui’ pollen, acid proteins of ‘Hohsui’ pull down the ‘Kohsui’ pollen [Ca²⁺]_i remarkably. Conclusion can be made that during the prophase of pollen germination, endogenous S-RNase has no specific effect on pollen [Ca²⁺]_i changes.     

Steroidal glycoalkaloid content of potato,tomato and their somatic hybrids

Summary Analyses of leaves and ‘tubers’ from somatic hybrids of potato and tomato (‘pomato’ with plastids of potato, ‘topato’ with plastids of tomato) produced by fusion of protoplasts from liquid cultures of dihaploid potato and mesophyll of tomato revealed the presence of the two major potato glycoalkaloids (α-solanine and α-chaconine) as well as the tomato glycoalkaloid (αtomatine). The total alkaloid content of leaves was greater than that of ‘tubers’ and similar to levels in the foliage of parent plants. However, glycoalkaloids were more abundant in hybrid ‘tubers’ than in normal potato tubers by a factor of 5–15. In hybrid foliage, approximately 98% of the alkaloid present was of potato origin whereas in ‘tubers’ the reverse was the case, with tomatine comprising 60–70% of the total alkaloid. The similarities in alkaloid content and ratios between the pomato and the topato lines indicate that plastomes do not influence the biosynthesis and distribution of these alkaloids. The results indicate that major secondary metabolites may prove useful for assessing the hybrid nature of such plants.     

Identification of Isoforms of G Proteins and PKC that Colocalize with Tight Junctions

Recent evidence suggests that the formation and permeability of tight junctions are actively regulated by second-messenger-generating systems involving G proteins and protein kinase C (PKC). A possible specific target for these regulatory proteins is the tight junction protein ZO-1. An extensive immunocytochemical study was performed in cultured epithelial monolayers of MDCK and Caco-2 cells to identify which isoforms of G proteins and PKC are present at or near the zonula occludens complex. Antibodies against α-subunits of each one of the four major subfamilies were used for the localization of the G proteins. For the PKC localization, antibodies against eight different isoforms were used. In confluent monolayers, Gα₁₂ and PKC ζ, were the only isoforms of these proteins present at the cell borders. In subconfluent monolayers, Gα₁₂ and PKC ζ were found at the plasma membrane only along the areas of lateral cell-cell contact. These isoforms formed a pattern of distribution very similar to the ZO-1 protein. The present findings indicate that Gα₁₂ and PKC ζ may be part of the zonula occludens complex and may locally regulate formation and permeability of tight junctions. Received: 29 July 1995/Revised: 13 October 1995     

Germination kinetics and seed reserve mobilization in two flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.) cultivars under moderate salt stress

Because of its high contents of protein, α-linolenic-rich oil, lignans, and fiber, demand is increasing for flax(Linum usitatissi-mum L.) and flax seed oil as a food source. In this comparative survey, we examined germination and the mobilization of seed storage products (lipids and soluble proteins) of 3-d-old seedlings from two flax cultivars (N 51 and H 52) challenged with moderate salinity (up to 200 mM NaCl). At the highest salt concentration, germination appeared to be cultivar-dependent, with that of ‘N 51’ being less impaired and delayed than in ’H 52’. Sodium chloride inhibited germination via osmotic and toxic effects, so that seed viability was altered, especially in ‘H 52’. At 200 mM NaCl, lipid mobilization was delayed in the earliest germination phases. This response was associated with increased proportions of linolenic acid contents in both cultivars and more linolenic acid-rich molecular species of TAGs. Irrespective of the salt level, soluble protein contents in both cultivars decreased over time, although a salt-related precocity of protein degradation occurred at 200 mM NaCl.     

Outer membrane proteins: comparing X-ray and NMR structures by MD simulations in lipid bilayers

The structures of three bacterial outer membrane proteins (OmpA, OmpX and PagP) have been determined by both X-ray diffraction and NMR. We have used multiple (7 × 15 ns) MD simulations to compare the conformational dynamics resulting from the X-ray versus the NMR structures, each protein being simulated in a lipid (DMPC) bilayer. Conformational drift was assessed via calculation of the root mean square deviation as a function of time. On this basis the ‘quality’ of the starting structure seems mainly to influence the simulation stability of the transmembrane β-barrel domain. Root mean square fluctuations were used to compare simulation mobility as a function of residue number. The resultant residue mobility profiles were qualitatively similar for the corresponding X-ray and NMR structure-based simulations. However, all three proteins were generally more mobile in the NMR-based than in the X-ray simulations. Principal components analysis was used to identify the dominant motions within each simulation. The first two eigenvectors (which account for >50% of the protein motion) reveal that such motions are concentrated in the extracellular loops and, in the case of PagP, in the N-terminal α-helix. Residue profiles of the magnitude of motions corresponding to the first two eigenvectors are similar for the corresponding X-ray and NMR simulations, but the directions of these motions correlate poorly reflecting incomplete sampling on a ∼10 ns timescale.     

Seasonal studies on alginate and its composition II: Turbinaria conoides (J.Ag.) Kütz. (Fucales, Phaeophyceae)

The alginates extracted from ‘leaf’, ‘stem’ and entire thallus of Turbinaria conoides (J.Ag.) Kütz. were investigated for their viscosity and biochemical constituents namely, β-D-mannuronic acid (M-block}), α-L-guluronic acid (G-Block) and alternating sequences of β–D-mannuronic acid and α-L-guluronic acid (MG-block). Substantial seasonal variation was recorded with high yield of alginate during premonsoon. The yield of alginate was maximum in ‘leaf’ region. In contrast to this, viscosity and G-block were maximum in the ‘stem’ region. A significant positive correlation was observed between viscosity and G-block}. The ratio of guluronic: mannuronic acid was also assessed. Low levels of M/G ratio were recorded in the ‘stem’ region followed by ‘leaf’ and entire thallus.     

Analysis of 3D structural differences in the IgG-binding domains based on the interresidue average-distance statistics

It is well-known that the IgG-binding domain from staphylococcal protein A folds into a 3α helix bundle structure, while the IgG-binding domain of streptococcal protein G forms an (α + β) structure. Recently, He et al. (Biochemistry 44:14055–14061, 2005) made mutants of these proteins from the wild types of protein A and protein G strains. These mutants are referred to as protein A219 and protein G311, and it was showed that these two mutants have different 3D structures, i.e., the 3α helix bundle structure and the (α + β) structure, respectively, despite the high sequence identity (59%). The purpose of our study was to clarify how such 3D structural differences are coded in the sequences with high homology. To address this problem, we introduce a predicted contact map constructed based on the interresidue average-distance statistics for prediction of folding properties of a protein. We refer to this map as an average distance map (ADM). Furthermore, the statistics of interresidue distances can be converted to an effective interresidue potential. We calculated the contact frequency of each residue of a protein in random conformations with this effective interresidue potential, and then we obtained values similar to ϕ values. We refer to this contact frequency of each residue as a p(μ) value. The comparison of the p(μ) values to the ϕ values for a protein suggests that p(μ) values reveal the information on the folding initiation site. Using these techniques, we try to extract the information on the difference in the 3D structures of protein A219 and protein G311 coded in their amino acid sequences in the present work. The results show that the ADM analyses and the p(μ) value analyses predict the information of folding initiation sites, which can be used to detect the 3D difference in both proteins.     

Phage displayed 6-mer mimotopes with a consensus proline absent in the minimized linear wild-type epitope

Summary Phage displayed random-6-mer libraries were screened with a monoclonal antibody specific for a minimized ‘linear’ 7-mer epitope of the measles virus hemagglutinin protein. No clone with the wild-type sequence was selected and most clones contained a sequence motif not found in the wild-type sequence. Two mimotopes (LYMPQLS, SEMPQLP) were synthesized which inhibited binding to the measles virus 95–135 times better than a wild-type peptide. Sequence comparison of proteins with known 3D-structure indicates that the epitope corresponds to an α-helix, while the best mimotopes have no predicted helix propensity. The proline is thought to be required for inducing a turn neccesary for mimicking part of the α-helix. The higher intrinsic stability of such a mimotope may explain its improved binding and may be more suitable in immunogenicity experiments.     

D-Amino acids in protein de novo design. II. Protein-diastereomerism versus protein-enantiomerism

Summary With a few exceptions, proteins in our biosphere are based exclusively onl-amino acids. The inversion of configuration of all the stereogenic centers in a protein leads to anall-d compound with ‘mirror image’ properties and ‘mirror image’ structure. We propose to use the termprotein-enantiomerism to describe the relationship between two proteins that have the same sequence but whose amino acids have opposite configuration. We will use the termprotein-diastereomerism to define the relationship between two proteins that have the same sequence in which some amino acids have opposite configurations. A classification of type I, II, III, and IV protein-diastereomerism is proposed. By extension, a diastereoprotein is a protein where some amino acids have the same configuration (l ord) while others have the opposite one (d orl). A particular case of diastereoproteins aremesoproteins, also analyzed in this article. In addition to the goal of making proteins resistant to protease degradation, the use ofd-amino acids in protein de novo design may give rise to proteins with structures, and perhaps properties, very different to those of nativeall-l-proteins.     

Influence of assignment on the prediction of transmembrane helices in protein structures

α-Helical transmembrane proteins (TMP_α) are composed of a series of helices embedded in the lipid bilayer. Due to technical difficulties, few 3D structures are available. Therefore, the design of structural models of TMP_α is of major interest. We study the secondary structures of TMP_α by analyzing the influence of secondary structures assignment methods (SSAMs). For this purpose, a published and updated benchmark databank of TMP_α is used and several SSAMs (9) are evaluated. The analysis of the results points to significant differences in SSA depending on the methods used. Pairwise comparisons between SSAMs led to more than 10% of disagreement. Helical regions corresponding to transmembrane zones are often correctly characterized. The study of the sequence–structure relationship shows very limited differences with regard to the structural disagreement. Secondary structure prediction based on Bayes’ rule and using only a single sequence give correct prediction rates ranging from 78 to 81%. A structural alphabet approach gives a slightly better prediction, i.e., only 2% less than the best equivalent approach, whereas the prediction rate with a very different assignment bypasses 86%. This last result highlights the importance of the correct assignment choice to evaluate the prediction assessment.     

Classical and dynamic morphology: toward a synthesis through the space of forms

In plant morphology, most structures of vascular plants can easily be assigned to pre-established organ categories. However, there are also intermediate structures that do not fit those categories associated with a classical approach to morphology. To integrate the diversity of forms in the same general framework, we constructed a theoretical morphospace based on a variety of modalities where it is possible to calculate the morphological distance between plant organs. This paper gives emphasis on shoot, leaf, leaflet and trichomes while ignoring the root. This will allow us to test the hypothesis that classical morphology (typology) and dynamic morphology occupy the same theoretical morphospace and the relationship between the two approaches remains a question of weighting of criteria. Our approach considers the shoot (i.e. leafy stem) as the basic morphological structural unit. A theoretical data table consisting of as many lines as there are possible combinations between different modalities of characters of a typical shoot was generated. By applying a principal components analysis (PCA) to these data it is possible to define a theoretical morphospace of shoots. Typical morphological elements (shoots, leaves, trichomes) and atypical structures (phylloclades, cladodes) including particular cases representing ‘exotic’ structures such as the epiphyllous appendages of Begonia and ‘water shoot’ and ‘leaf’ of aquatic Utricularia were placed in the morphospace. The more an organ differs from a typical shoot, the further away it will be from the barycentre of shoots. By giving a higher weight to variables used in classical typology, the different organ categories appear to be separate, as expected. If we do not make any particular arbitrary choice in terms of character weighting, as it is the case in the context of dynamic morphology, the clear separation between organs is replaced by a continuum. Contrary to typical structures, “intermediate” structures are only compatible with a dynamic morphology approach whether they are placed in the morphospace based on a ponderation compatible with typology or dynamic morphology. The difference in points of view between typology and continuum leads to a particular mode of weighting. By using an equal weighting of characters, contradictions due to the ponderation of characters are avoided, and the morphological concepts of continuum’ and ‘typology’ appear as sub-classes of ‘process’ or ‘dynamic morphology’.     

Aligning protein sequence and analysing substitution pattern using a class-specific matrix

Aligning protein sequences using a score matrix has became a routine but valuable method in modern biological research. However, alignment in the ‘twilight zone’ remains an open issue. It is feasible and necessary to construct a new score matrix as more protein structures are resolved. Three structural class-specific score matrices (all-alpha, allbeta and alpha/beta) were constructed based on the structure alignment of low identity proteins of the corresponding structural classes. The class-specific score matrices were significantly better than a structure-derived matrix (HSDM) and three other generalized matrices (BLOSUM30, BLOSUM60 and Gonnet250) in alignment performance tests. The optimized gap penalties presented here also promote alignment performance. The results indicate that different protein classes have distinct amino acid substitution patterns, and an amino acid score matrix should be constructed based on different structural classes. The class-specific score matrices could also be used in profile construction to improve homology detection.     

Peptide Fragment Approach to Prion Misfolding: The Alpha-2 Domain

The mechanism that underlies a multitude of human disorders, including type II diabetes, Parkinson’s, Huntington’s and Alzheimer’s, and the prion encephalopathies, is β-structure expansion through a pathogenic aggregation-prone monomeric form. β-sheet expansion disorders share intermolecular association as a common determinant, being therefore collectively identified as conformational diseases, but little is known about the underlying mechanism. Transmissible spongiform encephalopathies, also known as prion diseases, are all characterised by progressive neuronal degeneration associated to marked extracellular accumulation of an amyloidogenic conformer of the normal cellular prion protein (PrP^C), referred to as the scrapie isoform (PrP^Sc), which is thought to be responsible for the disease symptoms. PrP^C is a ubiquitous 231-amino acid glycoprotein, whose physiological role is still elusive. It is organised as an N-terminal disordered region and a compact C-terminal domain, where secondary structure elements consist of three α-helices (α1, α2 and α3), with an α2-α3 disulphide bridge, and two short β-strands (β1 and β2). Evidence accumulated so far suggests that the protein possesses one or several ‘spots’ of intrinsic conformational weakness, which may trigger generic folding, leading the whole architecture to adopt aggregation-prone conformations. One of such spots is suspected to be the C-terminal side of the α-helix 2, which has recently gained the attention of several investigations because it gathers several disease-associated point mutations, can be strongly fibrillogenic and toxic to neuronal cells, and possesses chameleon conformational behaviour. This paper briefly reviews recent literature on α-2 domain-derived model peptides.     

Regeneration of peach (<Emphasis Type="Italic">Prunus persica</Emphasis> L. Batsch) cultivars and <Emphasis Type="Italic">Prunus persica</Emphasis> × <Emphasis Type="Italic">Prunus dulcis</Emphasis> rootstocks via organogenesis

Somatic peach plants were regenerated from callus derived from the base of stem explants of the scion cultivars ‘UFO-3’, ‘Maruja’, ‘Flariba’ and ‘Alice Bigi’, and the peach × almond rootstocks ‘Garnem’ and ‘GF677’. A protocol for organogenic plant regeneration was developed using three culture media containing different concentrations of 6-benzyladenine (BA) and indolebutyric acid to produce organogenic calli. Shoots were obtained from sliced calli after their transfer to a differentiation culture medium containing 2 mg l⁻¹ BA and 1 mg l⁻¹ α-naphthalene acetic acid. Using this procedure, up to 29 regenerated plants per callus were obtained. The highest regeneration rate was obtained with the peach × almond rootstocks. This work provides an effective protocol that could be utilized for peach transformation research.