Membrane proteins: from sequence to structure

The prediction of protein structure from sequence has been along-standing goal of molecular biology. Integral membrane proteins,once abhorred by protein chemists and crystallographers becauseof their insolubility and stubborn refusal to yield good crystals,now appear to hold great promises for efficient structure predictionand engineering. This is mainly due to the constraints on permissiblestructures imposed by the lipid environment, and to the apparentuncoupling between an initial membrane targeting and insertionprocess which determines the overall topological arrangementof the transmembrane segments and a subsequent –condensation—of these segments into a unique folded state. Recent work suggeststhat the membrane insertion process is controlled by simplesequence elements composed of different combinations of longhydrophobic regions and flanking charged residues. In this reviewwe sketch the most unportant structural rules relating aminoacid sequence to membrane insertion to fully folded molecule,and their use for prediction and protein-engineering purposes.     

HOMOGENEITY OF THE FRIABLE FLOUR OF MALTING BARLEY

A detailed comparison was made of the properties of the friable flours and non-friable residues of two samples of malted barley of different nitrogen contents. The friable flours were sieved and fractionated to give a range of particle sizes, and the intact malt, whole friable flour, non-friable residues and fractionated friable flours were subjected to a range of analyses. Endosperm fractionation studies showed that the pattern of enzymic degradation of proteins in the modified friable flour of low nitrogen malt was more uniform than the corresponding pattern of protein breakdown in the friable flour of high nitrogen malt. Examination of the non friable residues showed that cell wall breakdown in the high nitrogen malt was less extensive than the low nitrogen malt. It is proposed that the high protein levels in the endosperm caused starch/protein compacting which limited endosperm hydration and enzymic modification during malting. The friability scores of high nitrogen malts may given an over estimate of endosperm modification.     

The terminal protein of the linear DNA plasmid pGKL2 shares an N-terminal domain of the plasmid-encoded DNA polymerase

The 36K protein attached at the 5′ end of the linear DNA plasmid pGKL2 from the yeast Kluyveromyces lactis was first purified and characterized. The terminal protein was purified from cells (1 kg wet weight) by ammonium sulphate precipitation and two rounds of centrifugation to equilibrium in CsCl gradients. The pGKL2 was present only in the post-microsomal supernatant. Approximately 10 mg of the purified pGKL2 was recovered and digested with DNase I. The terminal protein (final ca. 0·8 mg) was homogeneous by electrophoresis and we determined the N-terminal amino acid sequence up to ten residues, showing that it existed in the cryptic N-terminal domain of pGKL2-ORF2 (DNA polymerase) sequence.     

SOME BARLEY GRAIN AND GREEN MALT PROPERTIES AND THEIR INFLUENCE ON MALT HOT-WATER EXTRACT: II. PROTEIN,PROTEINASE AND MOISTURE

This paper continues the study of variation between varieties in some properties of barley and malt and examines protein-related characters and moisture uptake. Initial rate of moisture uptake was more closely related to enzyme activities than maximum moisture content. Significant varietal variation was found in all characters and this variation was greater than environmental variation with the exception of grain nitrogen concentration. In the endosperm endopeptidase activity was the character most highly correlated with malt hot water extract (HWE). The combined influence of protein- and β-glucan-related characters was investigated and the relative influence of each group of characters on HWE varied in different sections of the grain. In the endosperm the protein- and β-glucan-related characters accounted for 30% and 60% of the variation in HWE respectively.     

Yeast KEX2 protease and mannosyltransferase I are localized to distinct compartments of the secretory pathway

The KEX2 protease (product of the KEX2 gene) functions late in the secretory pathway of Saccharomyces cerevisiae by cleaving the polypeptide chains of prepro-killer toxin and prepro-alpha-factor at paired basic amino acid residues. The intracellular vesicles containing KEX2 protease sedimented in density gradients to a position distinct from those containing mannosyltransferase I (product of the MNN1 gene), a marker enzyme for the Golgi complex. The recovery of intact compartments containing these enzymes approached 80% after sedimentation. We propose that the KEX2 protease and mannosyltransferase I reside within distinct compartments.     

Utilisation of free and protein-bound methionine sulphoxide by the chicken

The biological availability of free and protein-bound methionine sulphoxide (MSO) was examined in nine growth experiments with chickens. The source of protein-bound MSO was fish meals oxidised with hydrogen peroxide. Bound MSO in oxidised fish meals was equally well utilised by the chicken as was bound methionine in unoxidised fish meals when the meals were the sole protein sources in the diet. When used as supplements to a low-methionine diet, oxidised fish meals were slightly less well utilised than L -methionine and unoxidised meals at the highest supplementation levels. At a low level there was no difference. Free L -MSO was less well utilised than free L -methionine when added to a soya bean meal diet and when used in purified diets. In the latter cystine was found to improve the utilisation of MSO. High levels of MSO were found in muscle extracts of chickens even when no MSO was present in their diet.     

Epitope-Imprinted Nanoparticles as Transforming Growth Factor-β3 Sequestering Ligands to Modulate Stem Cell Fate

Growth factors (GFs) are biomolecules with potent biological effects but inherent limitations hinder their potential as therapeutic agents and cell culture supplements in tissue engineering and regenerative medicine (TERM). Biomaterials that sequester endogenous GFs by affinity binding might circumvent such limitations and thus are being increasingly investigated. Here, molecularly imprinted nanoparticles (MINPs) are proposed as specific abiotic ligands for GFs. As a proof of concept, a conformational epitope of transforming growth factor-β3 (TGF-β3) is designed and surface imprinted onto polyacrylamide-based nanoparticles by inverse microemulsion polymerization. It is found that, depending on the polymerization mixture composition, MINPs can recognize and preferentially bind TGF-β3, either in noncompetitive assays or from a complex human fluid (platelet lysate). Substrates functionalized with MINPs are then used for 2D culture of adipose-derived stem cells. Remarkably, gene and protein expression profiles show a marked upregulation of SOX-9, suggesting activation of TGF-β3 signaling pathways without requiring supplementation with exogenous GF. Likewise, culturing these cells in pellets incorporating MINPs previously incubated with platelet lysate results in higher collagen II-rich matrix deposition, compared to pellets incorporating non-imprinted nanoparticles. In summary, results suggest MINPs can be used as cost-effective, stable, and scalable alternative abiotic GF ligands to guide cell fate in TERM applications.     

Effects of cultivar and soil fertility on grain protein yield,grain protein content,flour yield and breadmaking quality of wheat

Grain protein content affects the flour yield and breadmaking characteristics of wheat (Triticum aestivum L). In this study, grain protein yield, grain protein content, flour yield and loaf volume were quantified for four wheat cultivars (Inia, Carina, Kariega and SST 86) grown under six different soil fertility regimes in a long-term fertilisation and irrigation experiment at the University of Pretoria. The experimental design was a randomised complete block replicated four times, with fertility as the main plots and cultivars as the subplot treatments. Grain protein yield, flour yield, loaf volume and mixograph dough peak mixing time varied among cultivars and soil fertility situations. Grain protein content differed among cultivars, but mixograph water absorption and dough characteristics did not differ. The highest grain protein yield was 873 kg ha⁻¹ for Carina and the lowest 527 kg ha⁻¹ for SST 86. Grain protein content averaged 131 g kg⁻¹ for Carina and 122 g kg⁻¹ for Kariega. Breadmaking performance showed that in a well-balanced soil fertility situation, Kariega produced 1025 cm³ of loaf volume while Inia averaged 950 cm³. Grain protein yield increased with increasing soil fertility, but grain protein content, flour yield, loaf volume, water absorption and mixograph peak mixing time varied with soil fertility. The interaction between cultivar and soil fertility was significant for grain protein yield, grain protein content, flour yield, loaf volume and water absorption but not dough peak mixing time. The results indicate cultivar differences in breadmaking quality characteristics and that soil fertility status affects grain protein yield, grain protein content, flour yield, loaf volume potential and water absorption but not mixograph peak mixing time and dough characteristics. © 1999 Society of Chemical Industry     

Analysis of the peroxisomal acyl‐CoA oxidase gene product from Pichia pastoris and determination of its targeting signal

Acyl‐CoA oxidase (Pox1p) is involved in the β‐oxidation of fatty acids and is targeted to the peroxisomal matrix via the use of different signals in various organisms. In rat, mouse and human, Pox1p contains a canonical peroxisomal targeting signal 1 (PTS1), whereas in the yeasts Candida tropicalis, Saccharomyces cerevisiae, C. maltosa and Yarrowia lipolytica neither a PTS1 nor a PTS2 sequence is present, suggesting that Pox1p might be targeted to the peroxisomes via a third unknown pathway. Alternatively, since proteins lacking a PTS sequence can enter peroxisomes in association with other polypeptides containing a PTS, Pox1p might ‘piggy‐back’ its way into the peroxisomal matrix together with other proteins. To understand the mechanism of peroxisomal targeting of a yeast Pox1p, we cloned the Pichia pastoris POX1 gene to study the pathway of import of PpPox1p into peroxisomes. The gene was cloned by PCR, hybridization and plasmid rescue. The 2157 bp gene encodes a protein with a predicted molecular weight of 80 kDa. Antisera against PpPox1p detected a protein specifically induced on oleate with an apparent molecular weight of 72 kDa. Immunolocalization studies confirmed the peroxisomal localization of PpPox1p. The carboxy‐terminus of PpPox1p ends with a PTS1‐like sequence, APKI. The sequence PKI was necessary for transport of PpPox1p into peroxisomes and interacted with the PTS1 receptor, Pex5p. Furthermore, addition of the sequence APKI to the C‐terminus of the green fluorescent protein directed this fusion protein to the peroxisome. Therefore, PpPox1p uses the PTS1 pathway for its import into peroxisomes. Copyright © 1999 John Wiley & Sons, Ltd.     

Water Vapor Permeability of Whey Protein Emulsion Films as Affected by pH

The water vapor permeability (WVP) of whey protein isolate-beeswax emulsion films was investigated as related to pH. Lower WVP was observed for films cast from solutions at pH 7.0. When pH of the film-forming solution was lowered, resulting film WVP increased. At the isoelectric point, WVP was the highest. As pH of the emulsion approached pI, a sharp change in viscosity occurred due to an increase in protein aggregation. This increase in viscosity probably lowered lipid mobility and reduced interconnectivity among lipid droplets, resulting in the higher WVP. For minimum WVP, such films should be applied at pH different from pI.