N Terminus (n + terminus)

Distribution by Scientific Domains

Selected Abstracts

N-terminal destruction signals lead to rapid degradation of the major histocompatibility complex class II transactivator CIITA

Felix Schnappauf
Abstract Major histocompatibility complex (MHC) class II molecules play an essential role for the cellular immune response by presenting peptide antigens to CD4+ T cells. MHC class II molecules and genes show a highly complex expression pattern, which is orchestrated through a master regulatory factor, called CIITA (class II transactivator). CIITA controls MHC class II expression not only qualitatively, but also quantitatively, and has therefore a direct influence on the CD4 T cell-dependent immune response. CIITA is itself tightly regulated not only on the transcriptional level, but as we show here also on the protein level. CIITA is subjected to a very rapid protein turnover and shows a half-life of about 30,min. Inhibition of degradation by proteasome inhibitors and the identification of ubiquitylated CIITA intermediates indicate that the degradation of CIITA is mediated by the ubiquitin-proteasome system. We identified two regions mediating degradation within the N-terminal domain of CIITA. N-terminal fusions or deletions stabilized CIITA, indicating that the N termini contribute to degradation. Several non-functional CIITA mutants are partially stabilized, but we provide evidence that transcriptional activity of CIITA is not directly linked to degradation. [source]

Asymmetric amino acid compositions of transmembrane ,-strands

Aaron K. Chamberlain
Abstract In contrast to water-soluble proteins, membrane proteins reside in a heterogeneous environment, and their surfaces must interact with both polar and apolar membrane regions. As a consequence, the composition of membrane proteins' residues varies substantially between the membrane core and the interfacial regions. The amino acid compositions of helical membrane proteins are also known to be different on the cytoplasmic and extracellular sides of the membrane. Here we report that in the 16 transmembrane ,-barrel structures, the amino acid compositions of lipid-facing residues are different near the N and C termini of the individual strands. Polar amino acids are more prevalent near the C termini than near the N termini, and hydrophobic amino acids show the opposite trend. We suggest that this difference arises because it is easier for polar atoms to escape from the apolar regions of the bilayer at the C terminus of a ,-strand. This new characteristic of ,-barrel membrane proteins enhances our understanding of how a sequence encodes a membrane protein structure and should prove useful in identifying and predicting the structures of trans-membrane ,-barrels. [source]

Binding of Helix-Threading Peptides to E. coli 16S Ribosomal RNA and Inhibition of the S15,16S Complex

CHEMBIOCHEM, Issue 12 2005
Barry D. Gooch
Abstract Helix-threading peptides (HTPs) constitute a new class of small molecules that bind selectively to duplex RNA structures adjacent to helix defects and project peptide functionality into the dissimilar duplex grooves. To further explore and develop the capabilities of the HTP design for binding RNA selectively, we identified helix 22 of the prokaryotic ribosomal RNA 16S as a target. This helix is a component of the binding site for the ribosomal protein S15. In addition, the S15,16S RNA interaction is important for the ordered assembly of the bacterial ribosome. Here we present the synthesis and characterization of helix-threading peptides that bind selectively to helix 22 of E. coli 16S RNA. These compounds bind helix 22 by threading intercalation placing the N termini in the minor groove and the C termini in the major groove. Binding is dependent on the presence of a highly conserved purine-rich internal loop in the RNA, whereas removal of the loop minimally affects binding of the classical intercalators ethidium bromide and methidiumpropyl,EDTA,Fe (MPE,Fe). Moreover, binding selectivity translates into selective inhibition of formation of the S15,16S complex. [source]

Catalytically Active Tetramodular 6-Deoxyerythonolide B Synthase Fusion Proteins

CHEMBIOCHEM, Issue 11 2003
Corinne M. Squire Dr.
Easy as 1, 2, 3? Erythromycin (see scheme) is biosynthesised on a polyketide synthase consisting of three discrete bimodular protein subunits which, in the natural system, must dock together to form the active system. This paper details an experiment in which either two or all three of these proteins are translationally fused through their C and N termini. The tetramodular fusions are shown to be competent in biosynthesis. [source]

Mechanism of modulation of T cell responses by N-palmitoylated peptides

Clara Bueno
Abstract Small structural changes in the antigenic peptides recognized by TCR can alter the biological properties of those peptides and convert them into weak agonists, partial agonists, or antagonists of these receptors. These altered peptide ligands (APL) are usually generated by conservative amino acid substitutions at TCR contact residues. Here, we show that APL with therapeutic properties can also be generated by attachment of palmitic acid at the N terminus of the peptide without the need to modify the peptide's primary sequence. Using N-palmitoylated pigeon cytochrome-c peptide 81,104 (PALPCC81,104), we were able to induce T cell hyporesponsiveness to the wild-type peptide in vitro. More importantly, administration of the PALPCC81,104 to mice reduced the responsiveness to the native peptide when tested ex vivo. Biochemical and functional experiments indicated that the action of N-palmitoylated peptides was due to the conversion of the native peptide into a weak agonist that could then induce T cell anergy. Our results demonstrate that N-palmitoylation of antigenic peptides is a feasible strategy to generate APL, as it avoids the need to screen multiple amino acid variants of each specific antigen to identify those with therapeutic properties. [source]

Studies into factors contributing to substrate specificity of membrane-bound 3-ketoacyl-CoA synthases

FEBS JOURNAL, Issue 19 2002
Brenda J. Blacklock
We are interested in constructing a model for the substrate-binding site of fatty acid elongase-1 3-ketoacyl CoA synthase (FAE1 KCS), the enzyme responsible for production of very long chain fatty acids of plant seed oils. Arabidopsis thaliana and Brassica napus FAE1 KCS enzymes are highly homologous but the seed oil content of these plants suggests that their substrate specificities differ with respect to acyl chain length. We used in vivo and in vitro assays of Saccharomyces cerevisiae -expressed FAE1 KCSs to demonstrate that the B. napus FAE1 KCS enzyme favors longer chain acyl substrates than the A. thaliana enzyme. Domains/residues responsible for substrate specificity were investigated by determining catalytic activity and substrate specificity of chimeric enzymes of A. thaliana and B. napus FAE1 KCS. The N-terminal region, excluding the transmembrane domain, was shown to be involved in substrate specificity. One chimeric enzyme that included A. thaliana sequence from the N terminus to residue 114 and B. napus sequence from residue 115 to the C terminus had substrate specificity similar to that of A. thaliana FAE1 KCS. However, a K92R substitution in this chimeric enzyme changed the specificity to that of the B. napus enzyme without loss of catalytic activity. Thus, this study was successful in identifying a domain involved in determining substrate specificity in FAE1 KCS and in engineering an enzyme with novel activity. [source]

Intracellular site of ,-secretase cleavage for A,42 generation in Neuro 2a cells harbouring a presenilin 1 mutation

FEBS JOURNAL, Issue 7 2000
Shinji Sudoh
Previously, we reported that mutations in presenilin 1 (PS1) increased the intracellular levels of amyloid ,-protein (A,)42. However, it is still not known at which cellular site or how PS1 mutations exert their effect of enhancing A,42,,-secretase cleavage. In this study, to clarify the molecular mechanisms underlying this enhancement of A,42,,-secretase cleavage, we focused on determining the intracellular site of the cleavage. To address this issue, we used APP,C100 encoding the C-terminal ,-amyloid precursor protein (APP) fragment truncated at the N terminus of A, (C100); C100 requires only ,-secretase cleavage to yield A,. Mutated PS1 (M146L)-induced Neuro 2a cells showed enhanced A,1,42 generation from transiently expressed C100 as well as from full-length APP, whereas the generation of A,1,40 was not increased. The intracellular generation of A,1,42 from transiently expressed C100 in both mutated PS1 -induced and wild-type Neuro 2a cells was inhibited by brefeldin A. Moreover, the generation of A,1,42 and A,1,40 from a C100 mutant containing a di-lysine endoplasmic reticulum retention signal was greatly decreased, indicating that the major intracellular site of ,-secretase cleavage is not the endoplasmic reticulum. The intracellular generation of A,1,42/40 from C100 was not influenced by monensin treatment, and the level of A,1,42/40 generated from C100 carrying a sorting signal for the trans -Golgi network was higher than that generated from wild-type C100. These results using PS1 -mutation-harbouring and wild-type Neuro 2a cells suggest that A,42/40,,-secretase cleavages occur in the Golgi compartment and the trans -Golgi network, and that the PS1 mutation does not alter the intracelluar site of A,42,,-secretase cleavage in the normal APP proteolytic processing pathway. [source]

Protease-Activated Receptors: A Means of Converting Extracellular Proteolysis into Intracellular Signals

IUBMB LIFE, Issue 6 2002
E. J. Mackie
Abstract Protease-activated receptors (PARs) mediate cellular responses to a variety of extracellular proteases. The four known PARs constitute a subgroup of the family of seven-transmembrane domain G protein-coupled receptors and activate intracellular signalling pathways typical for this family of receptors. Activation of PARs involves proteolytic cleavage of the extracellular domain, resulting in formation of a new N terminus, which acts as a tethered ligand. PAR-1, -3, and -4 are relatively selective for activation by thrombin whereas PAR-2 is activated by a variety of proteases, including trypsin and tryptase. Recent studies in mice genetically incapable of expressing specific PARs have defined roles for PAR-1 in vascular development, and for PAR-3 and -4 in platelet activation, which plays a fundamental role in blood coagulation. PAR-1 has also been implicated in a variety of other biological processes including inflammation, and brain and muscle development. Responses mediated by PAR-2 include contraction of intestinal smooth muscle, epithelium-dependent smooth muscle relaxation in the airways and vasculature, and potentiation of inflammatory responses. The area of PAR research is rapidly expanding our understanding of how cells communicate and control biological functions, in turn increasing our knowledge of disease processes and providing potential targets for therapeutic intervention. [source]

Confined dynamics of a ribosome-bound nascent globin: Cone angle analysis of fluorescence depolarization decays in the presence of two local motions

PROTEIN SCIENCE, Issue 10 2009
Jamie P. Ellis
Abstract We still know very little about how proteins achieve their native three-dimensional structure in vitro and in the cell. Folding studies as proteins emerge from the mega Dalton-sized ribosome pose special challenges due to the large size and complicated nature of the ribosome-nascent chain complex. This work introduces a combination of three-component analysis of fluorescence depolarization decays (including the presence of two local motions) and in-cone analysis of diffusive local dynamics to investigate the spatial constraints experienced by a protein emerging from the ribosomal tunnel. We focus on E. coli ribosomes and an all-,-helical nascent globin in the presence and absence of the cotranslationally active chaperones DnaK and trigger factor. The data provide insights on the dynamic nature and structural plasticity of ribosome-nascent chain complexes. We find that the sub-ns motions of the N-terminal fluorophore, reporting on the globin dynamics in the vicinity of the N terminus, are highly constrained both inside and outside the ribosomal tunnel, resulting in high-order parameters (>0.85) and small cone semiangles (<30°). The shorter globin chains buried inside the tunnel are less spatially constrained than those of a reference sequence from a natively unfolded protein, suggesting either that the two nascent chain sequences have a different secondary structure and therefore sample different regions of the tunnel or that the tunnel undergoes local structural adjustments to accommodate the globin sequence. Longer globins emerging out of the ribosomal tunnel are also found to have highly spatially constrained slow (ns) motions. There are no observable spectroscopic changes in the absence of bound chaperones. [source]

The FliK protein and flagellar hook-length control

Richard C. Waters
Abstract The bacterial flagellum is a highly complex prokaryotic organelle. It is the motor that drives bacterial motility, and despite the large amount of energy required to make and operate flagella, motile organisms have a strong adaptive advantage. Flagellar biogenesis is both complex and highly coordinated and it typically involves at least three two-component systems. Part of the flagellum is a type III secretion system, and it is via this structure that flagellar components are exported. The assembly of a flagellum occurs in a number of stages, and the "checkpoint control" protein FliK functions in this process by detecting when the flagellar hook substructure has reached its optimal length. FliK then terminates hook export and assembly and transmits a signal to begin filament export, the final stage in flagellar biosynthesis. As yet the exact mechanism of how FliK achieves this is not known. Here we review what is known of the FliK protein and discuss the evidence for and against the various hypotheses that have been proposed in recent years to explain how FliK controls hook length, FliK as a molecular ruler, the measuring cup theory, the role of the FliK N terminus, the infrequent molecular ruler theory, and the molecular clock theory. [source]

Position dependence of the 13C chemical shifts of ,-helical model peptides.

PROTEIN SCIENCE, Issue 11 2004
Fingerprint of the 20 naturally occurring amino acids
Abstract The position dependence of the 13C chemical shifts was investigated at the density functional level for ,-helical model peptides represented by the sequence Ac-(Ala)i -X-(Ala)j -NH2, where X represents any of the 20 naturally occurring amino acids, with 0 , i , 8 and i + j = 8. Adoption of the locally dense basis approach for the quantum chemical calculations enabled us to reduce the length of the chemical-shift calculations while maintaining good accuracy of the results. For the 20 naturally occurring amino acids in ,-helices, there is (1) significant variability of the computed 13C shielding as a function of both the guest residue (X) and the position along the sequence; for example, at the N terminus, the 13C, and 13C, shieldings exhibit a uniform pattern of variation with respect to both the central or the C-terminal positions; (2) good agreement between computed and observed 13C, and 13C, chemical shifts in the interior of the helix, with correlation coefficients of 0.98 and 0.99, respectively; for 13C, chemical shifts, computed in the middle of the helix, only five residues, namely Asn, Asp, Ser, Thr, and Leu, exhibit chemical shifts beyond the observed standard deviation; and (3) better agreement for four of these residues (Asn, Asp, Ser, and Thr) only for the computed values of the 13C, chemical shifts at the N terminus. The results indicate that 13C,, but not 13C,, chemical shifts are sensitive enough to reflect the propensities of some amino acids for specific positions within an ,-helix, relative to the N and C termini of peptides and proteins. [source]

Crystal structures of possible lysine decarboxylases from Thermus thermophilus HB8

PROTEIN SCIENCE, Issue 11 2004
Mutsuko Kukimoto-Niino
Abstract TT1887 and TT1465 from Thermus thermophilus HB8 are conserved hypothetical proteins, and are annotated as possible lysine decarboxylases in the Pfam database. Here we report the crystal structures of TT1887 and TT1465 at 1.8 Ĺ and 2.2 Ĺ resolutions, respectively, as determined by the multiwavelength anomalous dispersion (MAD) method. TT1887 is a homotetramer, while TT1465 is a homohexamer in the crystal and in solution. The structures of the TT1887 and TT1465 monomers contain single domains with the Rossmann fold, comprising six , helices and seven , strands, and are quite similar to each other. The major structural differences exist in the N terminus of TT1465, where there are two additional , helices. A comparison of the structures revealed the elements that are responsible for the different oligomerization modes. The distributions of the electrostatic potential on the solvent-accessible surfaces suggested putative active sites. [source]

A CH domain-containing N terminus in NuMA?

PROTEIN SCIENCE, Issue 10 2002
Maria Novatchkova
Abstract Nuclear mitotic apparatus protein (NuMA) is an essential vertebrate component in organizing microtubule ends at spindle poles. The NuMA-dynactin/dynein motor multiprotein complex not only explains the transport of NuMA along spindle fibers but also is linked to the process of microtubule focusing. The interaction sites of NuMA to dynein/dynactin have not been mapped. In the yet functionally uncharacterized N terminus of NuMA, we predict a calponin-homology (CH) domain, a motif with binding activity for actin-like molecules. We substantiate the primary sequence analysis-based prediction with secondary structure and fold recognition analysis, and we propose the N-terminal CH domain of NuMA as a likely interaction site for actin-related protein 1 (Arp1) protein of the dynactin/dynein complex. [source]

N-terminal extension changes the folding mechanism of the FK506-binding protein

Alla Korepanova
Abstract Many of the protein fusion systems used to enhance the yield of recombinant proteins result in the addition of a small number of amino acid residues onto the desired protein. Here, we investigate the effect of short (three amino acid) N-terminal extensions on the equilibrium denaturation and kinetic folding and unfolding reactions of the FK506-binding protein (FKBP) and compare the results obtained with data collected on an FKBP variant lacking this extension. Isothermal equilibrium denaturation experiments demonstrated that the N-terminal extension had a slight destabilizing effect. NMR investigations showed that the N-terminal extension slightly perturbed the protein structure near the site of the extension, with lesser effects being propagated into the single ,-helix of FKBP. These structural perturbations probably account for the differential stability. In contrast to the relatively minor equilibrium effects, the N-terminal extension generated a kinetic-folding intermediate that is not observed in the shorter construct. Kinetic experiments performed on a construct with a different amino acid sequence in the extension showed that the length and the sequence of the extension both contribute to the observed equilibrium and kinetic effects. These results point to an important role for the N terminus in the folding of FKBP and suggest that a biological consequence of N-terminal methionine removal observed in many eukaryotic and prokaryotic proteins is to increase the folding efficiency of the polypeptide chain. [source]

Effect of the N1 residue on the stability of the ,-helix for all 20 amino acids

Duncan A.E. Cochran
Abstract N1 is the first residue in an ,-helix. We have measured the contribution of all 20 amino acids to the stability of a small helical peptide CH3CO-XAAAAQAAAAQAAGY-NH2 at the N1 position. By substituting every residue into the N1 position, we were able to investigate the stabilizing role of each amino acid in an isolated context. The helix content of each of the 20 peptides was measured by circular dichroism (CD) spectroscopy. The data were analyzed by our modified Lifson-Roig helix-coil theory, which includes the n1 parameter, to find free energies for placing a residue into the N1 position. The rank order for free energies is Asp,, Ala > Glu, > Glu0 > Trp, Leu, Ser > Asp0, Thr, Gln, Met, Ile > Val, Pro > Lys+, Arg, His0 > Cys, Gly > Phe > Asn, Tyr, His+. N1 preferences are clearly distinct from preferences for the preceding N-cap and ,-helix interior. pKa values were measured for Asp, Glu, and His, and protonation-free energies were calculated for Asp and Glu. The dissociation of the Asp proton is less favorable than that of Glu, and this reflects its involvement in a stronger stabilizing interaction at the N terminus. Proline is not energetically favored at the ,-helix N terminus despite having a high propensity for this position in crystal structures. The data presented are of value both in rationalizing mutations at N1 ,-helix sites in proteins and in predicting the helix contents of peptides. [source]

ATP sensitivity of ATP-sensitive K+ channels: role of the , phosphate group of ATP and the R50 residue of mouse Kir6.2

Scott A. John
ATP-sensitive K (KATP) channels are composed of Kir6, the pore-forming protein, and the sulphonylurea receptor SUR, a regulatory protein. We and others have previously shown that positively charged residues in the C terminus of Kir6.2, including R201 and K185, interact with the , and , phosphate groups of ATP, respectively, to induce channel closure. A positively charged residue in the N terminus, R50, is also important, and has been proposed to interact with either the , or , phosphate group of ATP. To examine this issue, we systematically mutated R50 to residues of different size, charge and hydropathy, and examined the effects on adenine nucleotide sensitivity in the absence and presence of SUR1. In the absence of SUR1, only the size of residue 50 significantly altered ATP sensitivity, with smaller side chains decreasing ATP sensitivity. In the presence of SUR1, however, hydrophathy and charge also played a role. Hydrophilic residues decreased ATP sensitivity more than hydrophobic residues for small size residues, and, surprisingly, negatively charged residues E and D preserved ATP sensitivity and increased ADP sensitivity relative to the wild-type residue R. These observations suggest that a negative charge near position 50, due to either mutation of R50 or the interaction of the , phosphate group of ATP with R50, facilitates closure of the ATP-dependent gate. Mutation of the nearby positively charged residue R54, known to be involved in stabilizing channel opening via electrostatic interactions with phosphatidylinositol 4,5-bisphosphate (PIP2), also caused increased ADP sensitivity as compared with ATP, suggesting a loss of function of ATP's , phosphate. Based on these results, we propose that a phosphate group or a negative charge at position 50 initiates channel closure by destabilizing the electrostatic interactions between negative phosphate groups of PIP2 and residues such as R54. [source]

Functional association of cell death suppressor, Arabidopsis Bax inhibitor-1, with fatty acid 2-hydroxylation through cytochrome b5

Minoru Nagano
Summary Bax inhibitor-1 (BI-1) is a widely conserved cytoprotective protein localized in the endoplasmic reticulum (ER) membrane. We identified Arabidopsis cytochrome b5 (AtCb5) as an interactor of Arabidopsis BI-1 (AtBI-1) by screening the Arabidopsis cDNA library with the split-ubiquitin yeast two-hybrid (suY2H) system. Cb5 is an electron transfer protein localized mainly in the ER membrane. In addition, a bimolecular fluorescence complementation (BiFC) assay and fluorescence resonance energy transfer (FRET) analysis confirmed that AtBI-1 interacted with AtCb5 in plants. On the other hand, we found that the AtBI-1-mediated suppression of cell death in yeast requires Saccharomyces cerevisiae fatty acid hydroxylase 1 (ScFAH1), which had a Cb5-like domain at the N terminus and interacted with AtBI-1. ScFAH1 is a sphingolipid fatty acid 2-hydroxylase localized in the ER membrane. In contrast, AtFAH1 and AtFAH2, which are functional ScFAH1 homologues in Arabidopsis, had no Cb5-like domain, and instead interacted with AtCb5 in plants. These results suggest that AtBI-1 interacts with AtFAHs via AtCb5 in plant cells. Furthermore, the overexpression of AtBI-1 increased the level of 2-hydroxy fatty acids in Arabidopsis, indicating that AtBI-1 is involved in fatty acid 2-hydroxylation. [source]

Cover Picture: NMR Structure of the Single QALGGH Zinc Finger Domain from the Arabidopsis thaliana SUPERMAN Protein (ChemBioChem 2-3/2003)

CHEMBIOCHEM, Issue 2-3 2003
Carla Isernia Prof.
Abstract The cover picture shows the NMR structure of the SUPERMAN zinc finger domain, which is the first high-resolution structure of a classical zinc finger domain from a plant protein. The structure consists of a very well-defined ,,, motif, typical of all the other Cys2,His2 zinc fingers so far structurally characterized. As a consequence, the QALGGH sequence, which is highly conserved in plant protein classical zinc finger domains, is located at the N terminus of the , helix. Interestingly, this domain region, in animal protein zinc fingers, is constituted of hypervariable residues deputed to the recognition of the DNA bases. Therefore, a peculiar DNA recognition code for the QALGGH zinc finger domain is proposed in the article by Fattorusso and co-workers on p. 171 ff. [source]

New Insight into the Mode of Action of Nickel Superoxide Dismutase by Investigating Metallopeptide Substrate Models

Daniel Tietze M.
Abstract For the first time, the existence of a substrate adduct of a nickel superoxide dismutase (NiSOD) model, based on the first nine residues from the N terminus of the active form of Streptomyces coelicolor NiSOD, has been proven and the adduct has been isolated. This adduct is based on the cyanide anion (CN,), as a substrate analogue of the superoxide anion (O2.,), and the nickel metallopeptide H-HCDLPCGVY-NH2 -Ni. Spectroscopic studies, including IR, UV/Vis, and liquid- and solid-state NMR spectroscopy, show a single nickel-bound cyanide anion, which is embedded in the metallopeptide structure. This complex sheds new light on the question of whether the mode of action of the NiSOD enzyme is an inner- or outer-sphere mechanism. Whereas discussion was previously biased in favor of an outer-sphere electron-transfer mechanism due to the fact that binding of cyanide or azide moieties to the nickel active site had never been observed, our results are a clear indication in favor of the inner-sphere electron-transfer mechanism for the disproportionation of the O2., ion, whereby the substrate is attached to the Ni atom in the active site of the NiSOD. [source]

Solid-Phase Synthesis of DOTA,Peptides

Luis M. De León-Rodriguez Dr.
Abstract A general synthetic route to two DOTA-linked N -Fmoc amino acids (DOTA-F and DOTA-K) is described that allows insertion of DOTA at any endo -position within a peptide sequence. Three model pentapeptides were prepared to test the general utility of these derivatives in solid-phase peptide synthesis. Both DOTA derivatives reacted smoothly by means of standard HBTU activation chemistry to the point of insertion of the DOTA amino acid, but extension of the peptide chain beyond the DOTA-amino acid insertion required the use of pre-activated C -pentafluorophenyl ester N - , -Fmoc amino acids. Three Gal-80 binding peptides (12-mers) were then prepared by using this methodology with DOTA positioned either at the N terminus or at one of two different internal positions;the binding of the resulting GdDOTA-12-mers to Gal-80 were compared. The methodology described here allows versatile, controlled introduction of DOTA into any location within a peptide sequence. This provides a potential method for the screening of libraries of DOTA-linked peptides for optimal targeting properties. [source]