Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Alterations in the structure of the DNA-binding protein specified by gene 32 of bacteriophage T4 have been detected using partial trypsin digestion as a conformational probe. Limited tryptic hydrolysis of the gene 32 protein removes a fragment ("B" region), of 21 amino acids from the NH2 terminus and a 6,200-dalton fragment ("A" region) from the COOH terminus. Poly(dT), poly(dC), and single-stranded DNA increase the rate of tryptic hydrolysis of the "A" region but decrease the rate of tryptic hydrolysis of the "B" region. Oligonucleotides, which are too short to permit cooperative binding of the gene 32 protein, do not alter the rate of tryptic hydrolysis of either the "A" or "B" regions. A model which accounts for these findings requires that the "B" region be involved in gene 32 protein:gene 32 protein interactions when the gene 32 protein: DNA complex is formed. As a consequence of the gene 32 protein:DNA interaction, the "A" region should be able to participate more effectively in vivo and in vitro with other proteins involved in T4 DNA metabolism.
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PMID:Structural changes in the T4 gene 32 protein induced by DNA polynucleotides. 63 79

Varicella-zoster virus (VZV) open reading frame (ORF) 62 potentially encodes a protein with considerable amino acid homology to the herpes simplex virus (HSV) immediate-early regulatory polypeptide ICP4 (or IE3). To identify and characterize its protein product(s) (IE62), we used a rabbit antiserum prepared against a synthetic peptide corresponding to the C-terminal 13 amino acids of the predicted protein. This antiserum reacted with phosphorylated polypeptides of 175 to 180 kDa that were made in VZV-infected cells and in cells infected with a vaccinia virus recombinant expressing IE62, but not in control-infected cells, confirming its specificity and reactivity to the IE62 protein. The antiserum recognized a 175-kDa polypeptide in purified virions that comigrated with a major structural protein. Comparison of this reactivity with that of an antipeptide antiserum directed against the VZV ORF 10 product (homologous to the HSV major structural protein VP16) indicates similar levels of ORF 62 and ORF 10 polypeptides in VZV virions. In contrast, antipeptide antiserum directed against the VZV ORF 29 product, the homolog of the HSV major DNA-binding protein, failed to recognize any protein in our virion preparations. Treatment of virions with detergents that disrupt the virion envelope did not dissociate IE62 from the nucleocapsid-tegument structure of the virion. Differential sensitivity of VZV virion IE62 to trypsin digestion in the presence or absence of Triton X-100 indicates that IE62 is protected from trypsin degradation by the virus envelope; since it is not a nucleocapsid protein, we conclude that it is part of the tegument. Finally, we show that the virion 175-kDa protein either can autophosphorylate or is a major substrate in vitro for virion-associated protein kinase activity.
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PMID:The varicella-zoster virus immediate-early protein IE62 is a major component of virus particles. 130 52

The nucleic acid binding domain of the adenovirus type 2 (or type 5) DNA-binding protein (DBP) was characterized by using limited proteolysis and photochemical cross-linking. Three proteases were used to generate fragments of DBP which retained the ability to bind to single-stranded DNA. One fragment, a 35-kDa tryptic product, was partially sequenced and found to contain amino acid residues 153 to approximately 470. This fragment further defines the minimum region of the protein which is required for nucleic acid binding. The DNA binding pocket of DBP was defined by using ultraviolet irradiation to cross-link covalently the carboxyl-terminal portion of the protein to the oligonucleotide p(dT)14. Cross-linked complexes were digested with trypsin, and peptides which were associated with the oligonucleotide were isolated by anion-exchange and reverse-phase ion-pairing high performance liquid chromatography. Two DBP peptides comprised of residues 294-308 and 415-434 were isolated by this approach. Sequence analysis indicated that methionine 299 and phenylalanine 418 were probable sites of cross-linking between their respective peptides and the oligonucleotide; hence these residues may represent contact points between DBP and single-stranded DNA. Both residues are highly conserved and are near, but not identical to, regions of the protein implicated previously in DNA binding.
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PMID:Characterization of the nucleic acid binding region of adenovirus DNA binding protein by partial proteolysis and photochemical cross-linking. 151 23

The major capsid protein of polyomavirus, VP1, has been expression cloned in Escherichia coli, and the recombinant VP1 protein has been purified to near homogeneity (A. D. Leavitt, T. M. Roberts, and R. L. Garcea, J. Biol. Chem. 260:12803-12809, 1985). With this recombinant protein, a nitrocellulose filter transfer assay was developed for detecting DNA binding to VP1 (Southwestern assay). In optimizing conditions for this assay, dithiothreitol was found to inhibit DNA binding significantly. With recombinant VP1 proteins deleted at the carboxy and amino termini, a region of the protein affecting DNA binding was identified within the first 7 amino acids (MAPKRKS) of the VP1 amino terminus. Southwestern analysis of virion proteins separated by two-dimensional gel electrophoresis demonstrated equivalent DNA binding among the different VP1 isoelectric focusing subspecies, suggesting that VP1 phosphorylation does not modulate this function. By means of partial proteolysis of purified recombinant VP1 capsomeres for assessing structural features of the protein domain affecting DNA binding, a trypsin-sensitive site at lysine 28 was found to eliminate VP1 binding to DNA. The binding constant of recombinant VP1 to polyomavirus DNA was determined by an immunoprecipitation assay (R. D. G. McKay, J. Mol. Biol. 145:471-488, 1981) to be 1 x 10(-11) to 2 x 10(-11) M, which was not significantly different from its affinity for plasmid DNA. McKay analysis of deleted VP1 proteins and VP1-beta-galactosidase fusion proteins indicated that the amino terminus was both necessary and sufficient for DNA binding. As shown by electron microscopy, DNA inhibited in vitro capsomere self-assembly into capsidlike structures (D. M. Salunke, D. L. D. Caspar, and R. L. Garcea, Cell 46:895-904, 1986). Thus, VP1 is a high-affinity, non-sequence-specific DNA-binding protein with the binding function localized near its trypsin-accessible amino terminus. The inhibitory effects of disulfide reagents on DNA binding and of DNA on capsid assembly suggest possible intermediate steps in virion assembly.
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PMID:Characterization of the DNA-binding properties of the polyomavirus capsid protein VP1. 184 46

We have studied the major DNA-binding protein (ICP8) from herpes simplex virus type 1 to identify its DNA-binding site. Since we obtained our protein from a cell line carrying multiple chromosomally located copies of the ICP8 gene, we first analyzed this protein to assess its similarity to the corresponding viral protein. Our protein resembled the viral protein by molecular weight, response to antibody, preference for binding single-stranded DNA, and ability to lower the melting temperature of poly(dA-dT). To define the DNA-binding domain, we subjected the protein to limited trypsin digestion and separated the peptide products on a sodium dodecyl sulfate-polyacrylamide gel. These fragments were then transferred to a nitrocellulose membrane, renatured in situ, and tested for their ability to bind DNA. From this assay, we identified four fragments which both bound DNA and exhibited the expected binding preference for single-stranded DNA. The sequence of the smallest of these fragments was determined and corresponds to a polypeptide spanning residues 300 to 849 in the intact protein. This peptide contains several regions which may be important for DNA binding based on sequence similarities in single-stranded DNA-binding proteins from other herpesviruses and, in one case, on a conserved sequence found in more distant procaryotic and eucaryotic proteins.
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PMID:Characterization of a major DNA-binding domain in the herpes simplex virus type 1 DNA-binding protein (ICP8). 215 71

SS-B/La is a conserved cellular phosphoprotein of 46 to 48 KD that is the target antigen of autoantibodies in sera of patients with Sjogren's syndrome and systemic lupus erythematosus. SS-B/La is also known to be associated with certain small cellular and viral RNA, including adenovirus VAI and VAII RNA. Two relatively protease-resistant domains (X and Y) were defined in SS-B from HeLa cells by using human autoantibodies as reagents. Domain X, a methionine-containing nonphosphorylated 28 KD polypeptide, was found to be resistant to partial digestion with six different proteases. Similar domains were also found in calf and rabbit SS-B. Domain Y, a 23 KD polypeptide, was detected after limited digestion with S. aureus V8 and trypsin. This domain contained little if any methionine, but all the detectable phosphorylated amino acids. Among 16 anti-SS-B sera tested by immunoblotting, 11 (69%) were reactive with both domains, three (19%) only with domain X, and two (13%) only with domain Y. These results showed that there are at least two distinct antigenic epitopes on the 46 to 48 KD SS-B/La protein, each located on a separate structural domain. The asymmetric distribution of methionine and phosphorylated amino acid residues in SS-B/La show striking similarity to the two reported domains of the adenovirus 72 KD DNA-binding protein, and raises questions concerning functional similarities that await investigation.
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PMID:Epitopes, structural domains, and asymmetry of amino acid residues in SS-B/La nuclear protein. 242 60

The multifunctional DNA-binding protein (DBP) encoded by human adenovirus binds RNA. The association of purified DBP with RNA in vitro was demonstrated by using either a gel filtration or a filter binding assay. This association is sensitive to ionic strength and exhibits no apparent sequence specificity. DBP also interacts with RNA in vivo; it can be crosslinked to polyadenylylated RNA by UV-irradiation of intact cells during the late phase of adenovirus infections. The 46-kDa carboxyl-terminal domain of DBP binds RNA in vitro and was found to be associated with polyadenylylated RNA in vivo. This is the same domain that interacts with DNA. However, the differences in sensitivity of DBP to trypsin when bound to RNA versus DNA suggest that RNA and DNA either bind at different sites within this domain or induce different conformational changes within the protein.
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PMID:Association of the adenovirus DNA-binding protein with RNA both in vitro and in vivo. 346 70

A mouse helix-destabilizing protein (HD protein-1) has been purified and characterized, and controlled tryptic digestion has been used to generate two large fragments of this protein and to study structural changes accompanying DNA binding. HD protein-1, a DNA-binding protein that has higher affinity for single-stranded DNA (ssDNA)-cellulose than for double-stranded DNA (dsDNA)-cellulose and is resistant to a dextran sulfate elution from ssDNA-cellulose, was purified from mouse myeloma by the method described by Herrick and Alberts (Herrick, G., and Alberts, B. M. (1976) J. Biol. Chem. 251, 2124-2132). HD protein-1 was heterogeneous with regard to apparent molecular weight (range of Mr = 24,000 to 33,000), but individual Mr species shared extensive primary structure homology as revealed by tryptic peptide mapping. The predominant species of this protein, Mr = 27,000, was resolved from other species and obtained in nearly homogeneous form by preparative isoelectric focusing. Mouse HD protein-1 was capable of lowering the Tm of poly[d(A-T)] by 25 degrees C, indicating that it is a helix-destabilizing protein. Sedimentation boundary analysis revealed that binding to ssDNA was noncooperative and that the binding site covered about 6 nucleotide residues. There was a 35% increase in the intrinsic tryptophan fluorescence of the protein in the presence of ssDNA, suggesting that structural change accompanies binding. Subcellular localization studies indicated that 75% of mouse HD protein-1 is nuclear, but not chromatin-associated, and primary structure analysis indicated that HD protein-1 is distinct from high mobility group proteins 1 and 2, histones, and P8 protein. Tryptic hydrolysis of HD protein-1 produced discrete, large fragments whose apparent molecular weights ranged from 19,000 to 24,000, and whose relative abundance was changed by the presence of ssDNA during the digestion. Thus, a Mr = 22,000 fragment (22 HDP*) predominated in the absence of ssDNA, and a Mr = 19,000, fragment (19 HDP*) predominated in the presence of ssDNA. Poly(dT) and denatured calf thymus DNA were more effective than were other polynucleotides tested in promoting accumulation of 19 HDP*; (dT)8 was as effective as were longer molecules of (dT)n, but (dT)4 and (dT)6 were much less effective, indicating that the binding site involved in 19 HDP* accumulation covered between 6 and 8 residues of (dT)n. Both 19 HDP* and 22 HDP* have the same COOH-terminal end and the same affinity for ssDNA-cellulose as does the native HD protein-1, indicating that a Mr = 8,000 sequence at the NH2-terminal end of HD protein-1 is not required for binding to ssDNA. Even though 22 HDP* retained the ability to bind to ssDNA, it could not be converted to 19 HDP* by further trypsin digestion.
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PMID:Studies on the structure of mouse helix-destabilizing protein-1. DNA binding and controlled proteolysis with trypsin. 625 73

The DNA-binding protein HU from Escherichia coli is a heterodimer constituted of two polypeptide chains termed HU-1 and HU-2, of 90 residues each. Their primary structures were established from structural data obtained from tryptic peptides of each monomer in addition to the structural data provided by the automated Edman degradation of the dimer and by peptides derived from cleavage of the dimer with trypsin, chymotrypsin, V8 staphylococcal protease and dilute acid. The results presented in this paper confirm the amino-terminal and carboxy-terminal sequences of the dimer HU reported previously [Laine et al. (1978) FEBS Lett. 89, 116--120]. The amino acid sequences of proteins HU-1 and HU-2 are identical to those of proteins NS-1 and NS-2 respectively, determined independently by Mende et al. [FEBS Lett. (1978) 96, 395--398]. The amino acid sequences of proteins HU-1 and HU-2 are closely related but differ by 28 residues. These proteins are characterized by their high content of hydrophobic residues represented mostly by alanine. In both proteins, half of the basic residues are scattered along the polypeptide chain and the remainder is found within two short sequences located in the carboxy-terminal part of the molecule. No sequence homology could be established between the proteins HU-1 and HU-2 and any one of the five histones from different eukaryotes.
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PMID:Complete amino-acid sequences of DNA-binding proteins HU-1 and HU-2 from Escherichia coli. 698 59

Osmoregulated porin gene expression in Escherichia coli is controlled by the two-component regulatory system EnvZ and OmpR. EnvZ, the osmosensor, is an inner membrane protein and a histidine kinase. EnvZ phosphorylates OmpR, a cytoplasmic DNA-binding protein, on an aspartyl residue. Phospho-OmpR binds to the promoters of the porin genes to regulate the expression of ompF and ompC. We describe the use of limited proteolysis by trypsin and ion spray mass spectrometry to characterize phospho-OmpR and the conformational changes that occur upon phosphorylation. Our results are consistent with a two-domain structure for OmpR, an N-terminal phosphorylation domain joined to a C-terminal DNA-binding domain by a flexible linker region. In the presence of acetyl phosphate, OmpR is phosphorylated at only one site. Phosphorylation induces a conformational change that is transmitted to the C-terminal domain via the central linker. Previous genetic analysis identified a region in the C-terminal domain that is required for transcriptional activation. Our results indicate that this region is within a surface-exposed loop. We propose that this loop contacts the alpha subunit of RNA polymerase to activate transcription. Mass spectrometry also reveals an unusual dephosphorylated form of OmpR, the potential significance of which is discussed.
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PMID:Phosphorylation-dependent conformational changes in OmpR, an osmoregulatory DNA-binding protein of Escherichia coli. 756 33


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