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Porin of Haemophilus influenzae type b (341 amino acids; M(r) 37782) determines the permeability of the outer membrane to low molecular mass compounds. Purified Hib porin was subjected to chemical modification of lysine residues by succinic anhydride. Electrospray ionization mass spectrometry identified up to 12 modifications per porin molecule. Tryptic digestion of modified Hib porin followed by reverse phase chromatography and matrix assisted laser desorption ionization time-of-flight mass spectrometry mapped the succinylation sites. Most modified lysines are positioned in surface-located loops, numbers 1 and 4 to 7. Succinylated porin was reconstituted into planar lipid bilayers, and biophysical properties were analyzed and compared to Hib porin: there was an increased average single channel conductance compared to Hib porin (1.24 +/- 0.41 vs. 0.85 +/- 0.40 nanosiemens). The voltage-gating activity of succinylated porin differed considerably from that of Hib porin. The threshold voltage for gating was decreased from 75 to 40 mV. At 80 mV, steady-state conductance for succinylated porin was 50-55% of the instantaneous conductance. Hib porin at 80 mV showed a decrease to 89-91% of the instantaneous current levels. We propose that surface-located lysine residues are determinants of voltage gating for porin of Haemophilus influenzae type b. [ABSTRACT FROM AUTHOR]

Transporters mediate the movement of compounds across the membranes that separate the cell from its environment and across the inner membranes surrounding cellular compartments. It is estimated that one third of a proteome consists of membrane proteins, and many of these are transport proteins. Given the increase in the number of genomes being sequenced, there is a need for computational tools that predict the substrates that are transported by the transmembrane transport proteins. In this paper, we present TranCEP, a predictor of the type of substrate transported by a transmembrane transport protein. TranCEP combines the traditional use of the amino acid composition of the protein, with evolutionary information captured in a multiple sequence alignment (MSA), and restriction to important positions of the alignment that play a role in determining the specificity of the protein. Our experimental results show that TranCEP significantly outperforms the state-of-the-art predictors. The results quantify the contribution made by each type of information used. [ABSTRACT FROM AUTHOR]

Kinks are a structural feature of alpha-helices and many are known to have functional roles. Kinks have previously tended to be defined in a binary fashion. In this paper we have deliberately moved towards defining them on a continuum, which given the unimodal distribution of kink angles is a better description. From this perspective, we examine the conservation of kinks in proteins. We find that kink angles are not generally a conserved property of homologs, pointing either to their not being functionally critical or to their function being related to conformational flexibility. In the latter case, the different structures of homologs are providing snapshots of different conformations. Sequence identity between homologous helices is informative in terms of kink conservation, but almost equally so is the sequence identity of residues in spatial proximity to the kink. In the specific case of proline, which is known to be prevalent in kinked helices, loss of a proline from a kinked helix often also results in the loss of a kink or reduction in its kink angle. We carried out a study of the seven transmembrane helices in the GPCR family and found that changes in kinks could be related both to subfamilies of GPCRs and also, in a particular subfamily, to the binding of agonists or antagonists. These results suggest conformational change upon receptor activation within the GPCR family. We also found correlation between kink angles in different helices, and the possibility of concerted motion could be investigated further by applying our method to molecular dynamics simulations. These observations reinforce the belief that helix kinks are key, functional, flexible points in structures. [ABSTRACT FROM AUTHOR]

Background: Tandem repetitions within protein amino acid sequences often correspond to regular secondary structures and form multi-repeat 3D assemblies of varied size and function. Developing internal repetitions is one of the evolutionary mechanisms that proteins employ to adapt their structure and function under evolutionary pressure. While there is keen interest in understanding such phenomena, detection of repeating structures based only on sequence analysis is considered an arduous task, since structure and function is often preserved even under considerable sequence divergence (fuzzy tandem repeats). Results: In this paper we present PTRStalker, a new algorithm for ab-initio detection of fuzzy tandem repeats in protein amino acid sequences. In the reported results we show that by feeding PTRStalker with amino acid sequences from the UniProtKB/Swiss-Prot database we detect novel tandemly repeated structures not captured by other state-of-the-art tools. Experiments with membrane proteins indicate that PTRStalker can detect global symmetries in the primary structure which are then reflected in the tertiary structure. Conclusions: PTRStalker is able to detect fuzzy tandem repeating structures in protein sequences, with performance beyond the current state-of-the art. Such a tool may be a valuable support to investigating protein structural properties when tertiary X-ray data is not available. [ABSTRACT FROM AUTHOR]

Unlike all-helices membrane proteins, β-barrel membrane proteins can not be successfully discriminated from other proteins, especially from all-β soluble proteins. This paper performs an analysis on the amino acid composition in membrane parts of 12 β-barrel membrane proteins versus β-strands of 79 all-β soluble proteins. The average and variance of the amino acid composition in these two classes are calculated. Amino acids such as Gly, Asn, Val that are most likely associated with classification are selected based on Fishers discriminant ratio. A linear classifier built with these selected amino acids composition in observed β-strands achieves 100% classification accuracy for 12 membrane proteins and 79 soluble proteins in a four-fold cross-validation experiment. Since at present the accuracy of secondary structure prediction is quite high, a promising method to identify β-barrel membrane proteins is presented based on the linear classifier coupled with predicted secondary structure. Applied to 241 β-barrel membrane proteins and 3855 soluble proteins with various structures, the method achieves 85.48% (206/241) sensitivity and 92.53% specificity (3567/3855). [Copyright &y& Elsevier]

Thrombopoietin (TPO) and its receptor (MPL) are important regulators of megakaryopoiesis. MPL belongs to a cytokine receptor superfamily. To date, all constitutively active MPL mutants have been artificially constructed with amino acid substitutions in the transmembrane domain or extracellular domain of the protein, and they activate signal transduction pathways in Ba/F3 cells that can also be activated by the normal MPL. In this paper, we report a novel spontaneously occurring mutation of MPL, with an amino acid substitution of Trp(508) to Ser(508) in the intracellular domain of MPL, that induces the factor-independent growth of Ba/F3 cells. Examination of intracellular signaling pathways demonstrated that the mutant MPL protein constitutively activates three distinct signaling pathways, SHC-Ras-Raf-MAPK/JNK, JAK-STAT, and PI3K-Akt-Bad. [ABSTRACT FROM AUTHOR]

1. The abundance of tyrosine sulfate in membrane proteins was quantified in four different cell lines and compared to that in soluble cellular and secreted proteins. 2. Upon metabolic labelling of HepG2, Ltk-, AtT20 and PC12 cells with [35S]sulfate or [³H]tyrosine, a fraction enriched in integral membrane proteins was found to contain small, but significant, amounts of protein-bound tyrosine sulfate (up to 2.5% of the total cellular plus secreted protein-bound tyrosine sulfate). On the other hand, the frequency of sulfation of tyrosine residues of membrane proteins was within the same order of magnitude as that of secreted proteins, indicating that the low abundance of tyrosine sulfate in membrane proteins was largely a reflection of the low abundance of these proteins themselves. Consistent with this conclusion were the results of an analysis showing that 14 out of 32 selected membrane-spanning proteins contain potential tyrosine sulfation sites. 3. In HepG2 cells, three tyrosine-sulfated integral membrane glycoproteins of molecular mass 100, 125 and 150 kDa were identified. Characterization of the 150-kDa tyrosine-sulfated membrane protein revealed that it was protected from proteolysis in intact cells, suggesting a localization in an intracellular organelle. 4. Together with the results reported in the preceding paper in this journal, our data suggest that tyrosine sulfation occurs in various classes of trans-Golgi-derived proteins, soluble as well as membrane, and extracellularly exposed as well as intracellularly retained, proteins. This suggests that tyrosine sulfation may have a variety of physiological functions, depending on the individual tyrosine-sulfated protein or protein class. [ABSTRACT FROM AUTHOR]

1. The abundance of tyrosine sulfate in secretory proteins and in various classes of cellular proteins has been quantified and compared to protein-bound carbohydrate sulfate. 2. HepG2 cells and fibroblasts, two cell types showing only the constitutive pathway of secretion, and PC12 cells, which show both the constitutive and the regulated pathway of secretion, were subjected to pulse-chase and/ or long-term labelling with [35S]sulfate and [³H]tyrosine, followed by analysis of proteins in the cells and medium. Under both conditions of labelling, 65-92% of the protein-bound tyrosine sulfate and 44-84% of the protein bound carbohydrate sulfate were found to be secretory. In HepG2 cells, the frequency of sulfation of tyrosine residues, which can be determined independently from protein abundance and the rate of protein synthesis, was 8-22 times higher in proteins secreted into the medium than in cellular proteins. 3. All cell lines studied contained significant amounts, not only of carbohydrate sulfate, but also of tyrosine sulfate in specific cellular proteins. As shown for fibroblasts, these tyrosine-sulfated proteins were retained within the cells for at least 100 min of chase following a pulse with [35S]sulfate and were almost completely recovered in a light membrane fraction after subcellular fractionation. 4. Lysosomes were found to contain small, but significant, amounts of protein-bound tyrosine sulfate in addition to protein-bound carbohydrate sulfate. Protein-bound tyrosine sulfate in lysosomes reached a peak at 20 min of chase and rapidly disappeared thereafter, whereas protein-bound carbohydrate sulfate accumulated after 20 min of chase. Examination of the known sequences of eleven lysosomal enzymes revealed the presence of potential tyrosine sulfation sites in five of them. 5. Our results show that secretory proteins are the most abundant, but not exclusive, in vivo substrates for tyrosine sulfation and suggest the presence of soluble tyrosine-sulfated proteins in lysosomes and other, as yet unidentified, organelles of the secretory pathway. In the following paper in this journal we describe the abundance of tyrosine sulfate in integral membrane proteins. [ABSTRACT FROM AUTHOR]

Secretion of haemolysin (HlyA) is secA independent, but depends upon two accessory membrane proteins, HlyB and HlyD. encoded by the hly determinant. A fourth (cytoplasmic) protein, HlyC, is required to activate HlyA post-translationally, but has no role in export. Deletion studies have previously shown that the HlyA molecule contains a targeting signal close to the C-terminus which specifically directs its secretion to the medium. This targeting signal has been variously located within the terminal 27, 53, 60 or 113 amino acids. In this paper, we have sought to confirm the presence of a C-terminal targeting signal and to analyse the specificity of the Hly transport system through fusion of C-terminal fragments of HlyA to heterologous polypeptides. A C-terminal fragment (23 kDa) of HlyA. when fused at the C-terminus, efficiently promoted the secretion of the eukaryotic protein prochymosin (PCM) to the medium via HlyB and HlyD. This result is in contrast to previous findings that prochymosin, preceded by the alkaline phosphatase signal sequence, cannot be translocated across the Escherichia coli inner membrane. The HlyA targeting domain was also used to secrete to the medium varying portions of chloramphenicol acetyltransferase (CAT) and 98 per cent of the β-galactosidase (LacZ) molecule (both E. coli cytoplasmic proteins). In the case of the PCM and CAT fusions the efficiency of secretion was reduced as the proportion of the PCM and CAT molecule increased. This result is consistent with inhibition of secretion through the irreversible folding of the larger passenger protein fragments, or the occlusion of the HlyA targeting signal by upstream sequences. Analysis of the nature of the C-terminal domain promoting secretion of prochymosin, demonstrated that shortening the signal domain from 218 to 113 amino acids significantly reduced the efficiency of secretion. This result may also reflect the importance of maintaining an independent... [ABSTRACT FROM AUTHOR]