Covalent cross-linking of proteins without chemical reagents. (1/67)

A facile method for the formation of zero-length covalent cross-links between protein molecules in the lyophilized state without the use of chemical reagents has been developed. The cross-linking process is performed by simply sealing lyophilized protein under vacuum in a glass vessel and heating at 85 degrees C for 24 h. Under these conditions, approximately one-third of the total protein present becomes cross-linked, and dimer is the major product. Chemical and mass spectroscopic evidence obtained shows that zero-length cross-links are formed as a result of the condensation of interacting ammonium and carboxylate groups to form amide bonds between adjacent molecules. For the protein examined in the most detail, RNase A, the cross-linked dimer has only one amide cross-link and retains the enzymatic activity of the monomer. The in vacuo cross-linking procedure appears to be general in its applicability because five different proteins tested gave substantial cross-linking, and co-lyophilization of lysozyme and RNase A also gave a heterogeneous covalently cross-linked dimer.  (+info)

Site-directed mutagenesis of conserved charged amino acid residues in ClpB from Escherichia coli. (2/67)

ClpB is a member of a multichaperone system in Escherichia coli (with DnaK, DnaJ, and GrpE) that reactivates strongly aggregated proteins. The sequence of ClpB contains two ATP-binding domains, each containing Walker consensus motifs. The N- and C-terminal sequence regions of ClpB do not contain known functional motifs. In this study, we performed site-directed mutagenesis of selected charged residues within the Walker A motifs (Lys212 and Lys611) and the C-terminal region of ClpB (Asp797, Arg815, Arg819, and Glu826). We found that the mutations K212T, K611T, D797A, R815A, R819A, and E826A did not significantly affect the secondary structure of ClpB. The mutation of the N-terminal ATP-binding site (K212T), but not of the C-terminal ATP-binding site (K611T), and two mutations within the C-terminal domain (R815A and R819A) inhibited the self-association of ClpB in the absence of nucleotides. The defects in self-association of these mutants were also observed in the presence of ATP and ADP. The four mutants K212T, K611T, R815A, and R819A showed an inhibition of chaperone activity, which correlated with their low ATPase activity in the presence of casein. Our results indicate that positively charged amino acids that are located along the intersubunit interface (this includes Lys212 in the Walker A motif of the N-terminal ATP-binding domain as well as Arg815 and Arg819 in the C-terminal domain) participate in intersubunit salt bridges and stabilize the ClpB oligomer. Interestingly, we have identified a conserved residue within the C-terminal domain (Arg819) which does not participate directly in nucleotide binding but is essential for the chaperone activity of ClpB.  (+info)

Both acidic and basic amino acids in an amphitropic enzyme, CTP:phosphocholine cytidylyltransferase, dictate its selectivity for anionic membranes. (3/67)

Amphitropic proteins are regulated by reversible membrane interaction. Anionic phospholipids generally promote membrane binding of such proteins via electrostatics between the negatively charged lipid headgroups and clusters of basic groups on the proteins. In this study of one amphitropic protein, a cytidylyltransferase (CT) that regulates phosphatidylcholine synthesis, we found that substitution of lysines to glutamine along both interfacial strips of the membrane-binding amphipathic helix eliminated electrostatic binding. Unexpectedly, three glutamates also participate in the selectivity for anionic membrane surfaces. These glutamates become protonated in the low pH milieu at the surface of anionic, but not zwitterionic membranes, increasing protein positive charge and hydrophobicity. The binding and insertion into lipid vesicles of a synthetic peptide containing the three glutamates was pH-dependent with an apparent pK(a) that varied with anionic lipid content. Glutamate to glutamine substitution eliminated the pH dependence of the membrane interaction, and reduced anionic membrane selectivity of both the peptide and the whole CT enzyme examined in cells. Thus anionic lipids, working via surface-localized pH effects, can promote membrane binding by modifying protein charge and hydrophobicity, and this novel mechanism contributes to the membrane selectivity of CT in vivo.  (+info)

The Est1 subunit of Saccharomyces cerevisiae telomerase makes multiple contributions to telomere length maintenance. (4/67)

The telomerase-associated Est1 protein of Saccharomyces cerevisiae mediates enzyme access by bridging the interaction between the catalytic core of telomerase and the telomere-binding protein Cdc13. In addition to recruiting telomerase, Est1 may act as a positive regulator of telomerase once the enzyme has been brought to the telomere, as previously suggested by the inability of a Cdc13-Est2 fusion protein to promote extensive telomere elongation in an est1-Delta strain. We report here three classes of mutant Est1 proteins that retain association with the telomerase enzyme but confer different in vivo consequences. Class 1 mutants display a telomere replication defect but are capable of promoting extensive telomere elongation in the presence of a Cdc13-Est2 fusion protein, consistent with a defect in telomerase recruitment. Class 2 mutants fail to elongate telomeres even in the presence of the Cdc13-Est2 fusion, which is the phenotype predicted for a defect in the proposed second regulatory function of EST1. A third class of mutants impairs an activity of Est1 that is potentially required for the Ku-mediated pathway of telomere length maintenance. The isolation of mutations that perturb separate functions of Est1 demonstrates that a telomerase holoenzyme subunit can contribute multiple regulatory roles to telomere length maintenance.  (+info)

Mutation of charged residues in the TR3 death domain does not perturb interaction with TRADD. (5/67)

Members of the death receptor family play a prominent role in developmental and pathological neuronal cell death. The death signal is transduced via interaction between the death domain of the receptor and an intracellular adapter, TRADD. We performed alanine-scanning mutagenesis of specific charged residues in the TR3 death domain to determine whether they play a crucial role in TR3-TR3 and TR3-TRADD interaction. Mutation of charged residues in the second and third helices of the TR3 death domain failed to perturb self-interaction or interaction with TRADD. These data suggest that despite some similarity between the death domains of TR3 and TNFR1 the nature of the interaction with TRADD differs from that reported for TNFR1.  (+info)

Distinct Rab binding specificity of Rim1, Rim2, rabphilin, and Noc2. Identification of a critical determinant of Rab3A/Rab27A recognition by Rim2. (6/67)

Rabphilin, Rim, and Noc2 have generally been believed to be the Rab3 isoform (Rab3A/B/C/D)-specific effectors that regulate secretory vesicle exocytosis in neurons and in some endocrine cells. The results of recent genetic analysis of rabphilin knock-out animals, however, strongly refute this notion, because there are no obvious genetic interactions between Rab3 and rabphilin in nematoda (Staunton, J., Ganetzky, B., and Nonet, M. L. (2001) J. Neurosci. 21, 9255-9264), suggesting that Rab3 is not a major ligand of rabphilin in vivo. In this study, I tested the interaction of rabphilin, Rim1, Rim2, and Noc2 with 42 different Rab proteins by cotransfection assay and found differences in rabphilin, Rim1, Rim2, and Noc2 binding to several Rab proteins that belong to the Rab functional group III (Rab3A/B/C/D, Rab26, Rab27A/B, and Rab37) and/or VIII (Rab8A and Rab10). Rim1 interacts with Rab3A/B/C/D, Rab10, Rab26, and Rab37; Rim2 interacts with Rab3A/B/C/D and Rab8A; and rabphilin and Noc2 interact with Rab3A/B/C/D, Rab8A, and Rab27A/B. By contrast, the synaptotagmin-like protein homology domain of Slp homologue lacking C2 domains-a (Slac2-a)/melanophilin specifically recognizes Rab27A/B but not other Rabs. I also found that alternative splicing events in the first alpha-helical region (alpha(1)) of the Rab binding domain of Rim1 alter the Rab binding specificity of Rim1. Site-directed mutagenesis and chimeric analyses of Rim2 and Slac2-a indicate that the acidic cluster (Glu-50, Glu-51, and Glu-52) in the alpha(1) region of the Rab binding domain of Rim2, which is not conserved in the synaptotagmin-like pro tein homology domain of Slac2-a, is a critical determinant of Rab3A recognition. Based on these results, I propose that Rim, rabphilin, and Noc2 function differently in concert with functional group III and/or VIII Rab proteins, including Rab3 isoforms.  (+info)

A PAR domain transcription factor is involved in the expression from a hematopoietic-specific promoter for the human LMO2 gene. (7/67)

The transcription factor LMO2 is believed to exert its effect through the formation of protein-protein interactions with other DNA-binding factors such as GATA-1 and TAL1. Although LMO2 has been shown to be critical for the formation of the erythroid cell lineage, the gene is also expressed in a number of nonerythroid tissues. In this report, we demonstrate that the more distal of the 2 promoters for the LMO2 gene is highly restricted in its pattern of expression, directing the hematopoietic-specific expression of this gene. Deletion and mutation analyses have identified a critical cis element in the first untranslated exon of the gene. This element is a consensus-binding site for a small family of basic leucine zipper proteins containing a proline and acidic amino acid-rich (PAR) domain. Although all 3 members of this family are produced in erythroid cells, only 2 of these proteins, thyrotroph embryonic factor and hepatic leukemia factor, can activate transcription from this LMO2 promoter element. These findings represent a novel mechanism in erythroid gene regulation because PAR proteins have not previously been implicated in this process.  (+info)

Close pairs of carboxylates: a possibility of multicenter hydrogen bonds in proteins. (8/67)

Covalent attachment of hydrogen to the donor atom may be not an essential characteristic of stable hydrogen bonds. A positively charged particle (such as a proton), located between the two negatively charged residues, may lead to a stable interaction of the two negative residues. This paper analyzes close Asp-Glu pairs of residues in a large set of protein chains; 840 such pairs of residues were identified, of which 28% were stabilized by a metal ion, 12% by a positive residue nearby and 60% are likely to be stabilized by a proton. The absence of apparent structural constraints, secondary structure preferences, somewhat lower B-factors and a distinct correlation between pH and the minimal O-O distance in carboxylate pairs suggest that most of the abnormally close pairs could indeed be stabilized by a shared proton. Implications for protein stability and modeling are discussed.  (+info)

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