Probing the unfolding pathway of alpha1-antitrypsin.
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Protein misfolding plays a role in the pathogenesis of many diseases. alpha1-Antitrypsin misfolding leads to the accumulation of long chain polymers within the hepatocyte, reducing its plasma concentration and predisposing the patient to emphysema and liver disease. In order to understand the misfolding process, it is necessary to examine the folding of alpha1-antitrypsin through the different structures involved in this process. In this study we have used a novel technique in which unique cysteine residues were introduced at various positions into alpha1-antitrypsin and fluorescently labeled with N, N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)ethylenediamine. The fluorescence properties of each protein were studied in the native state and as a function of guanidine hydrochloride-mediated unfolding. The studies found that alpha1-antitrypsin unfolded through a series of intermediate structures. From the position of the fluorescence probes, the fluorescence quenching data, and the molecular modeling, we show that unfolding of alpha1-antitrypsin occurs via disruption of the A and C beta-sheets followed by the B beta-sheet. The implications of these data on both alpha1-antitrypsin function and polymerization are discussed. (+info)
A kinetic mechanism for the polymerization of alpha1-antitrypsin.
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The mutation in the Z deficiency variant of alpha1-antitrypsin perturbs the structure of the protein to allow a unique intermolecular linkage. These loop-sheet polymers are retained within the endoplasmic reticulum of hepatocytes to form inclusions that are associated with neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma. The process of polymer formation has been investigated here by intrinsic tryptophan fluorescence, fluorescence polarization, circular dichroic spectra and extrinsic fluorescence with 8-anilino-1-naphthalenesulfonic acid and tetramethylrhodamine-5-iodoacetamide. These biophysical techniques have demonstrated that alpha1-antitrypsin polymerization is a two-stage process and have allowed the calculation of rates for both of these steps. The initial fast phase is unimolecular and likely to represent temperature-induced protein unfolding, while the slow phase is bimolecular and associated with loop-sheet interaction and polymer formation. The naturally occurring Z, S, and I variants and recombinant site-directed reactive loop and shutter domain mutants of alpha1-antitrypsin were used to demonstrate the close association between protein stability and rate of alpha1-antitrypsin polymerization. Taken together, these data allow us to propose a kinetic mechanism for alpha1-antitrypsin polymer formation that involves the generation of an unstable intermediate, which can form polymers or generate latent protein. (+info)
Role of the second extracellular loop of human C3a receptor in agonist binding and receptor function.
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The C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor with an unusually large second extracellular loop (e2 loop, approximately 172 amino acids). To determine the function of this unique structure, chimeric and deletion mutants were prepared and analyzed in transfected RBL-2H3 cells. Whereas replacement of the C3aR N-terminal segment with that from the human C5a receptor had minimal effect on C3a binding, substitution of the e2 loop with a smaller e2 loop from the C5a receptor (C5aR) abolished binding of 125I-C3a and C3a-stimulated calcium mobilization. However, as much as 65% of the e2 loop sequence (amino acids 198-308) may be removed without affecting C3a binding or calcium responses. The e2 loop sequences adjacent to the transmembrane domains contain multiple aspartate residues and are found to play an important role in C3a binding based on deletion mutagenesis. Replacement of five aspartate residues in the e2 loop with lysyl residues significantly compromised both the binding and functional capabilities of the C3a receptor mediated by intact C3a or by two C3a analog peptides. These data suggest a two-site C3a-C3aR interaction model similar to that established for C5a/C5aR. The anionic residues near the N and C termini of the C3aR e2 loop constitute a non-effector secondary interaction site with cationic residues in the C-terminal helical region of C3a, whereas the C3a C-terminal sequence LGLAR engages the primary effector site in C3aR. (+info)
Homologous plasminogen N-terminal and plasminogen-related gene A and B peptides. Characterization of cDNAs and recombinant fusion proteins.
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The cDNA corresponding to exons 2-4 of the processed human plasminogen (Pgn) gene, encoding the N-terminal peptide domain (NTP), has been cloned, expressed in Escherichia coli as a recombinant protein (r-NTP) containing a hexahistidine tag, and refolded to the native structure that contains two internal cystine bridges. RNA expression of the two Pgn-related genes, PRG A and PRG B, that potentially encode 9-kDa polypeptides having extensive similarity to the NTP has been investigated. Using RNA-based PCR with liver RNA as template, we demonstrate that PRG A encodes a detectable mRNA species. PRG A and PRG B have been found to be transcribed in the liver and yield virtually identical mRNAs. Neither of the PRGs are expressed in a variety of other normal tissues, as determined by Northern blot analysis. Factor-Xa digestion of the tagged r-NTP yields cleavage products which indicates that the expressed r-NTP domain of Pgn is endowed with a flexible conformation. Recombinant PRG B protein (r-PRG B) fused to a hexahistidine tag was purified and analyzed for structural integrity. Preliminary 1H-NMR spectroscopic data for r-NTP and r-PRG B indicate relatively fast amide 1H-2H exchange in 2H2O and close conformational characteristics for the two homologous polypeptides. Far ultraviolet-CD spectra for r-NTP and r-PRG B at pH 7.0 indicate similar defined secondary structure content for both domains, with 13-17% alpha-helix and 24-27% antiparallel beta-sheet. The fact that two transcriptionally active genes encode almost identical polypeptides supports the hypothesis that the Pgn NTP, together with the putative polypeptides encoded by the PRGs, may serve an important function, such as controlling the conformation of Pgn and thus its susceptibility to tissue activators. (+info)
Solution structure of a lipid transfer protein extracted from rice seeds. Comparison with homologous proteins.
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Nuclear magnetic resonance (NMR) spectroscopy was used to determine the three dimensional structure of rice nonspecific lipid transfer protein (ns-LTP), a 91 amino acid residue protein belonging to the broad family of plant ns-LTP. Sequence specific assignment was obtained for all but three HN backbone 1H resonances and for more than 95% of the 1H side-chain resonances using a combination of 1H 2D NOESY; TOCSY and COSY experiments at 293 K. The structure was calculated on the basis of four disulfide bridge restraints, 1259 distance constraints derived from 1H-1H Overhauser effects, 72 phi angle restraints and 32 hydrogen-bond restraints. The final solution structure involves four helices (H1: Cys3-Arg18, H2: Ala25-Ala37, H3: Thr41-Ala54 and H4: Ala66-Cys73) followed by a long C-terminal tail (T) with no observable regular structure. N-capping residues (Thr2, Ser24, Thr40), whose side-chain oxygen atoms are involved in hydrogen bonds with i + 3 amide proton additionally stabilize the N termini of the first three helices. The fourth helix involving Pro residues display a mixture of alpha and 3(10) conformation. The rms deviation of 14 final structures with respect to the average structure is 1.14 +/- 0.16 A for all heavy atoms (C, N, O and S) and 0.72 +/- 0.01 A for the backbone atoms. The global fold of rice ns-LTP is close to the previously published structures of wheat, barley and maize ns-LTPs exhibiting nearly identical pattern of the numerous sequence specific interactions. As reported previously for different four-helix topology proteins, hydrophobic, hydrogen bonding and electrostatic mechanisms of fold stabilization were found for the rice ns-LTP. The sequential alignment of 36 ns-LTP primary structures strongly suggests that there is a uniform pattern of specific long-range interactions (in terms of sequence), which stabilize the fold of all plant ns-LTPs. (+info)
Denatured states of human carbonic anhydrase II: an NMR study of hydrogen/deuterium exchange at tryptophan-indole-H(N) sites.
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Hydrogen/deuterium (H/D) exchange measurements in low and moderate concentrations of GuHCI were conducted on the side chain H(N) atoms of the seven tryptophans of pseudo wild-type human carbonic anhydrase II. Tryptophans 5, 16 and 245, situated in or close to the N-terminal domain were found to have little protection against exchange. The H/D exchange results for Trp-123, Trp-192 and Trp-209 showed that a previously identified molten globule and the native state gave a similar protection against exchange. Global unfolding of the protein is necessary for the efficient exchange at Trp-97, which is located in the central part of the beta-sheet. (+info)
Ligand recognition and domain structure of Vps10p, a vacuolar protein sorting receptor in Saccharomyces cerevisiae.
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Vp10p is a receptor that sorts several different vacuolar proteins by cycling between a late Golgi compartment and the endosome. The cytoplasmic tail of Vps10p is necessary for the recycling, whereas the lumenal domain is predicted to interact with the soluble ligands. We have studied ligand binding to Vps10p by introducing deletions in the lumenal region. This region contains two domains with homology to each other. Domain 2 binds carboxypeptidase Y (CPY), proteinase A (PrA) and hybrids of these proteases with invertase. Moreover, we show that aminopeptidase Y (APY) is a ligand of Vps10p. The native proteases compete for binding to domain 2. Binding of CPY(156)-invertase or PrA(137)-invertase, on the other hand, do not interfere with binding of CPY to Vps10p. Furthermore, the Q24RPL27 sequence known to be important for vacuolar sorting of CPY, is of little importance in the Vps10p-dependent sorting of CPY-invertase. Apparently, domain 2 contains two different binding sites; one for APY, CPY and PrA, and one for CPY-invertase and PrA-invertase. The latter interaction seems not to be sequence specific, and we suggest that an unfolded structure in these ligands is recognized by Vps10p. (+info)
Folding stability of the kinetoplastid membrane protein-11 (KMP-11) from Leishmania infantum.
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Kinetoplastid membrane protein-11 (KMP-11) is a major component of the cell surface of kinetoplastids, and acts as a potent B- and T-cell immunogen during Leishmania infection. Here we report that the Leishmania infantum KMP-11 secondary structure adopts mainly an alpha-helical conformation at pH 7.5 and that its urea- and thermally-induced unfolding constitute a fully reversible two-step process. This allows estimation of a half-denaturation temperature of approximately 65 degrees C, a delta GDH2O at 20 degrees C of approximately 14.63 kJ.mol-1, and an increment of the reaction heat of approximately 183.92 kJ.mol-1 and an entropy of approximately 543.4 J.mol-1.deg-1, respectively, for the native-denatured equilibrium of the KMP-11 in solution. We also report that the KPM-11 protein is induced to adopt a molten globule state at a pH range between pH 4 and pH 6. As a whole, the stability and the specific features of the denaturing effect induced by changes in pH are similar in KMP-11 to various other lipoproteins. (+info)