New strategy for selective protein cleavage

Hoyer, Daniel; Cho, Ho; Schultz, Peter G.

doi:10.1021/ja00164a076

Cited by 136 publications

(74 citation statements)

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“…In order to circumvent this problem, as well as to achieve higher resolution footprinting, we also used the iron-EDTA cleavage method (35)(36)(37) on these complexes (data not shown). In this technique, a diffusible hydroxyl free radical is generated in solution from the iron-EDTA-peroxide reagent, and this radical rapidly and non-sequence-specifically cleaves the exposed peptide bonds of the folded protein.…”

Section: Quantitative Tryptic Footprinting Of the H-gp61-k Construct-mentioning

confidence: 99%

Interactions of Bacteriophage T4-coded Primase (gp61) with the T4 Replication Helicase (gp41) and DNA in Primosome Formation

Jing

Dong²,

Latham³

et al. 1999

Journal of Biological Chemistry

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One primase (gp61) and six helicase (gp41) subunits interact to form the bacteriophage T4-coded primosome at the DNA replication fork. In order to map some of the detailed interactions of the primase within the primosome, we have constructed and characterized variants of the gp61 primase that carry kinase tags at either the N or the C terminus of the polypeptide chain. These tagged gp61 constructs have been probed using several analytical methods. Proteolytic digestion and protein kinase protection experiments show that specific interactions with single-stranded DNA and the T4 helicase hexamer significantly protect both the N-and the Cterminal regions of the T4 primase polypeptide chain against modification by these procedures and that this protection becomes more pronounced when the primase is assembled within the complete ternary primosome complex. Additional discrete sites of both protection and apparent hypersensitivity along the gp61 polypeptide chain have also been mapped by proteolytic footprinting reactions for the binary helicase-primase complex and in the three component primosome. These studies provide a detailed map of a number of gp61 contact positions within the primosome and reveal interactions that may be important in the structure and function of this central component of the T4 DNA replication complex.Most known DNA replication complexes, including the bacteriophage T4-coded system, are organized into subassemblies of proteins that perform equivalent functions within the moving replication fork. Since the T4 complex is probably the simplest replication system that includes separate processivity clamp and clamp loader and primosome subassemblies, the T4 system has often served as a useful paradigm to elucidate and model some of the mechanistically important interactions that also occur within the replication complexes of higher organisms.The central replication machinery of bacteriophage T4 consists of seven phage-encoded proteins that work together with the DNA of the replication fork to form the minimal complex that permits efficient leading and lagging DNA synthesis and replication fork movement (1). These protein components are organized into three functional subassemblies within the replication complex. These are (i) the DNA-dependent DNA replication polymerase of T4 (gp43), which contains both synthesis and exonuclease (3Ј 3 5Ј) editing domains and works together with the T4-coded single-stranded DNA binding protein (gp32) to catalyze DNA synthesis on both strands of the replication fork; (ii) the T4-coded polymerase accessory proteins (gp44/62 and gp45), which form the processivity clamp (gp45) and ATPdependent clamp-loading (gp44/62) machinery of the T4 system; and (iii) the T4 coded helicase (gp41) and primase (gp61) components, which interact to form a primosome subassembly that contains an ATP-dependent helicase (gp41) that "opens" the template DNA ahead of the replication fork and a primase (gp61) that catalyzes the template-directed polymerization of the RNA primers that initiate th...

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Section: Quantitative Tryptic Footprinting Of the H-gp61-k Construct-mentioning

confidence: 99%

Interactions of Bacteriophage T4-coded Primase (gp61) with the T4 Replication Helicase (gp41) and DNA in Primosome Formation

Jing

Dong²,

Latham³

et al. 1999

Journal of Biological Chemistry

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“…The chemistry of selective Fe-mediated cleavage of peptide bonds is uncertain. Both oxidative and hydrolytic mechanisms have been proposed (16)(17)(18)29). In the presence of ascorbate, H 2 O 2 , and Fe 2ϩ ions, OH⅐ radicals are generated by the Fenton reaction, but OH⅐ radicals in the medium are not involved in cleavage of Na͞K-ATPase.…”

Section: Figurementioning

confidence: 99%

“…The use of complexed transition metals, such as EDTA-Fe or phenanthroline-Cu, for selective oxidative cleavage of nucleic acids is well established (15). Although specific metal-catalyzed cleavage of proteins is less characterized, there are several examples of applications to soluble enzymes using complexed or free Fe 2ϩ and Cu 2ϩ ions, directed or naturally bound to specific sites (16)(17)(18)(19)(20). The term ''affinity cleavage reagent'' describes substrates or other molecules that complex the heavy metals and direct them to sites where cleavage occurs (16).…”

Section: Introductionmentioning

confidence: 99%

“…Although specific metal-catalyzed cleavage of proteins is less characterized, there are several examples of applications to soluble enzymes using complexed or free Fe 2ϩ and Cu 2ϩ ions, directed or naturally bound to specific sites (16)(17)(18)(19)(20). The term ''affinity cleavage reagent'' describes substrates or other molecules that complex the heavy metals and direct them to sites where cleavage occurs (16). In principle, analysis of the cleavage pattern should provide spatial information on peptide bonds in proximity to the metal.…”

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confidence: 99%

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Fe-catalyzed cleavage of the α subunit of Na/K-ATPase: Evidence for conformation-sensitive interactions between cytoplasmic domains

Goldshleger

Karlish

1997

Proc. Natl. Acad. Sci. U.S.A.

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Incubation of Na͞K-ATPase with ascorbate plus H 2 O 2 produces specific cleavage of the ␣ subunit. Five fragments with intact C termini and complementary fragments with intact N termini were observed. The ) ion. Thus, cleavage patterns can provide information on spatial organization of the polypeptide chain. We propose that highly conserved regions of the ␣ subunit, within the minor and major cytoplasmic loops, interact in the E 2 or E 2 (Rb) conformations but move apart in the E 1 or E 1 Na conformations. We discuss implications of domain interactions for the energy transduction mechanism. Fe-catalyzed cleavages may be applicable to other P-type pumps or membrane proteins.Recent studies of structure-function relations of P-type cation pumps, such as Na͞K-, H͞K-, and Ca-ATPases, have focused on identification of residues of the catalytic subunits involved in cation or ATP binding or on their topological organization, comprising 10 transmembrane segments (M1-M10) (1-3). Cation occlusion sites are located within trans-membrane segments, and site-directed mutagenesis results suggest that side chains within M4, M5, M6, and M8 ligate cations (2, 4-6). The ATP binding site is located within the major cytoplasmic loop, and probable ligating residues are being identified by chemical modification or mutagenesis (1). By comparison with progress in these areas, our understanding of the energy transduction mechanism lags behind. Earlier studies provided a wealth of information on kinetics of cation transport, phosphorylation-dephosphorylation, transport-linked E 1 -E 2 conformational transitions, and cation occlusion (7,8). Transmission of force between ATP and cation binding sites and their structural reorganization in E 1 and E 2 conformations must underlie the energy transduction process. However, the structural basis of these events is poorly understood. Some evidence has been obtained using proteolysis or site-directed mutations. Specific proteolytic cleavages of Na͞K-or Ca-ATPase in the first cytoplasmic loop between trans-membrane segments M2 and M3, characteristically inhibit ATPase activity and stabilize E 1 conformations (9-11). In addition, there are several highly conserved sequences in the cytoplasmic domains of all P-type pumps, including TGES in the loop between M2 and M3 and MVTGD and TGDGVNDSPALKK in the major cytoplasmic loop before M5. Mutations in these sequences, in Ca-or H-ATPase, inhibit activity and stabilize E 1 forms (and in some cases also inhibit phosphorylation) (2,(12)(13)(14). Thus, the conserved sequences serve an important function, perhaps mediating conformational interactions between ATP and cation sites. However, their specific roles are unknown.This paper describes metal-catalyzed cleavage for study of structure-function relations and conformational transitions of Na͞K-ATPase. The use of complexed transition metals, such as EDTA-Fe or phenanthroline-Cu, for selective oxidative cleavage of nucleic acids is well established (15). Although specific metal-catalyzed cleavage of prot...

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“…Therefore, the a N-terminal domain must contain determinants for dimeriza- [24][25][26][27]; and (iii) we analyze the cleavage products by denaturing PAGE followed by PhosphorImager analysis. Binding of the ligand decreases polypeptidebackbone solvent accessibility at residues it contacts, protecting against hydroxyl-radical-mediated cleavage at residues it contacts and, therefore, resulting in a gap in the "ladder" of cleavage products.…”

Section: And 5)mentioning

confidence: 99%

Determinants of RNA polymerase alpha subunit for interaction with beta, beta', and sigma subunits: hydroxyl-radical protein footprinting.

Heyduk

Severinov

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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Escherichia coli RNA polymerase (RNAP) a subunit serves as the initiator for RNAP assembly, which proceeds according to the pathway 2a -a a2 -* a2.8 -* a2318' -a21813'or. In this work, we have used hydroxyl-radical protein footprinting to define determinants of a for interaction with 13, 13', and cr. Our results indicate that amino acids 30-75 of a are protected from hydroxyl-radical-mediated proteolysis upon interaction with 13 (i.e., in a43, a2483', and a23,1'ar), and amino acids 175-210 of a are protected from hydroxyl-radical-mediated proteolysis upon interaction with .3' (i.e., in a2488' and a24831'r). The protected regions are conserved in the a homologs of prokaryotic, eukaryotic, archaeal, and chloroplast RNAPs and contain sites of substitutions that affect RNAP assembly. We conclude that the protected regions define determinants of a for direct functional interaction with 13 and 13'. The observed maximal magnitude of protection upon interaction with 18 and the observed maximal magnitude of protection upon interaction with 1' both correspond to the expected value for complete protection of one of the two a protomers of RNAP (i.e., 50%Yv protection). We propose that only one of the two a protomers of RNAP interacts with 13 and that only one of the two a protomers of RNAP interacts with 13'.Escherichia coli RNA polymerase holoenzyme (RNAP) has subunit composition a2/3/3'c (for review, see ref. 1). RNAP a, the smallest subunit (329 amino acids), performs at least three critical functions:(i) RNAP a serves as the initiator for RNAP assembly, which proceeds according to the pathway 2a --a2 --a2/213 a2/31 --* a2//'3cT (for review, see ref.2).(ii) RNAP a participates in promoter recognition, making direct sequence-specific a-DNA interactions with promoter upstream elements (ref. (iii) RNAP a participates in transcriptional activation, repression, and elongation, making direct protein-protein interactions with activators, repressors, and elongation factors (refs. 6-9; for review, see refs. 4 and 5).RNAP a consists of two independently folded domains: an N-terminal domain required for RNAP assembly (amino acids 8-235) and a C-terminal domain required for interactions with protnoter upstream elements, activators, repressors, and elongation factors (amino acids 249-329) (refs. 10 and 11; for review, see refs. 4 and 5).The a N-terminal domain, by itself, is able to dimerize and to be assembled into RNAP (6,(12)(13)(14) (20), suggesting that determinants for RNAP assembly may be conserved. The genetic and sequence-comparison results, by themselves, do not distinguish between amino acids of a involved directly in interactions with ,B and /3' and amino acids of a involved solely in maintaining the proper conformation of a. In this work, we have used a biochemical method-i.e., hydroxyl-radical protein footprinting-to define determinants of a for interaction with ,B, /3', and a.Our procedure for hydroxyl-radical protein footprinting has three steps: (i) we 33P-end-label the protein of interest [using an introduce...

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New strategy for selective protein cleavage

Cited by 136 publications

References 0 publications

Interactions of Bacteriophage T4-coded Primase (gp61) with the T4 Replication Helicase (gp41) and DNA in Primosome Formation

Interactions of Bacteriophage T4-coded Primase (gp61) with the T4 Replication Helicase (gp41) and DNA in Primosome Formation

Fe-catalyzed cleavage of the α subunit of Na/K-ATPase: Evidence for conformation-sensitive interactions between cytoplasmic domains

Determinants of RNA polymerase alpha subunit for interaction with beta, beta', and sigma subunits: hydroxyl-radical protein footprinting.

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