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• W2034383690 abstract "Small, acid-soluble spore proteins (SASP) of the α/β-type from several Bacillus species were cross-linked into homodimers, heterodimers and homooligomers with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of linear plasmid DNA. Significant protein cross-linking was not detected in the absence of DNA. In all four α/β-type SASP examined, the amino donor in the EDC induced amide cross-links was the α-amino group of the protein. However, the carboxylate containing amino acid residues involved in cross-linking varied. In SASP-A and SASP-C ofBacillus megaterium two conserved glutamate residues, which form part of the germination protease recognition sequence, were involved in cross-link formation. In SspC from Bacillus subtilis and Bce1 from Bacillus cereus the acidic residues involved in cross-link formation were not in the protease recognition sequence, but at a site closer to the N terminus of the proteins. These data indicate that, although there are likely to be subtle structural differences between different α/β-type SASP, the N-terminal regions of these proteins are involved in protein-protein interactions while in the DNA bound state. Small, acid-soluble spore proteins (SASP) of the α/β-type from several Bacillus species were cross-linked into homodimers, heterodimers and homooligomers with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of linear plasmid DNA. Significant protein cross-linking was not detected in the absence of DNA. In all four α/β-type SASP examined, the amino donor in the EDC induced amide cross-links was the α-amino group of the protein. However, the carboxylate containing amino acid residues involved in cross-linking varied. In SASP-A and SASP-C ofBacillus megaterium two conserved glutamate residues, which form part of the germination protease recognition sequence, were involved in cross-link formation. In SspC from Bacillus subtilis and Bce1 from Bacillus cereus the acidic residues involved in cross-link formation were not in the protease recognition sequence, but at a site closer to the N terminus of the proteins. These data indicate that, although there are likely to be subtle structural differences between different α/β-type SASP, the N-terminal regions of these proteins are involved in protein-protein interactions while in the DNA bound state. Between 5 and 10% of the total protein in spores of theBacillus and Clostridium species of bacteria is α/β-type small, acid-soluble spore protein (α/β-type SASP) 1The abbreviations used are: SASP, small, acid-soluble spore proteins; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis; GPR, germination protease. (1Setlow P. Annu. Rev. Microbiol. 1988; 42: 319-338Crossref PubMed Scopus (176) Google Scholar, 2Setlow P. Annu. Rev. Microbiol. 1995; 49: 29-54Crossref PubMed Scopus (331) Google Scholar). These proteins are encoded by four to seven monocistronic genes in each species, and their amino acid sequences are highly conserved both within and betweenBacillus species (1Setlow P. Annu. Rev. Microbiol. 1988; 42: 319-338Crossref PubMed Scopus (176) Google Scholar, 2Setlow P. Annu. Rev. Microbiol. 1995; 49: 29-54Crossref PubMed Scopus (331) Google Scholar). The α/β-type SASP are nonspecific DNA-binding proteins which are synthesized only within the forespore compartment during sporulation (3Mason J.M. Hackett R.H. Setlow P. J. Bacteriol. 1988; 170: 239-244Crossref PubMed Scopus (88) Google Scholar, 4Sun D. Stragier P. Setlow P. Genes Dev. 1989; 3: 141-149Crossref PubMed Scopus (107) Google Scholar). Typically, two major α/β-type SASP accumulate to high levels within the spore, while the minor α/β-type SASP are found at much lower levels. The level of total α/β-type SASP in spores is sufficient to saturate the spore chromosome, and the binding of these proteins to spore DNA is the major determinant of spore resistance to UV radiation and a significant determinant of spore heat resistance (1Setlow P. Annu. Rev. Microbiol. 1988; 42: 319-338Crossref PubMed Scopus (176) Google Scholar, 2Setlow P. Annu. Rev. Microbiol. 1995; 49: 29-54Crossref PubMed Scopus (331) Google Scholar). Bacillus subtilis spores which lack the two major α/β-type SASP (α and β) are much more sensitive to UV radiation and heat than are wild type spores (5Mason J.M. Setlow P. J. Bacteriol. 1986; 167: 174-178Crossref PubMed Google Scholar). During the first few minutes of spore germination, α/β-type SASP are quickly degraded by a sequence-specific protease termed germination protease (GPR) (1Setlow P. Annu. Rev. Microbiol. 1988; 42: 319-338Crossref PubMed Scopus (176) Google Scholar, 2Setlow P. Annu. Rev. Microbiol. 1995; 49: 29-54Crossref PubMed Scopus (331) Google Scholar). Structural studies of purified α/β-type SASP and α/β-type SASP·DNA complexes have shown that significant changes in these proteins' structure occur upon binding to DNA, as α/β-type SASP are predominantly unfolded in solution but acquire significant α-helical content upon binding to DNA 2S. C. Mohr and P. Setlow, unpublished results. (6Rao H. Mohr S.C. Fairhead H. Setlow P. FEBS Lett. 1992; 305: 115-120Crossref PubMed Scopus (9) Google Scholar). The α/β-type SASP cover 4–6 base pairs of DNA, and binding of these proteins to DNA is highly cooperative, particularly to DNAs bound with low affinity. (7Nicholson W.L. Setlow B. Setlow P. J. Bacteriol. 1990; 172: 6900-6906Crossref PubMed Google Scholar). Electron micrographs of α/β-type SASP·DNA complexes indicate that the protein forms a helical coat along the DNA (8Griffith J. Makhov A. Santiago-Lara L. Setlow P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8224-8228Crossref PubMed Scopus (36) Google Scholar), suggesting that there are extensive interactions between α/β-type SASP when bound to DNA, although these proteins are monomers in solution 3B. Setlow and P. Setlow, unpublished results. (9Setlow P. J. Biol. Chem. 1975; 250: 8168-8173Abstract Full Text PDF PubMed Google Scholar). Consequently, it is possible that interactions between adjacent α/β-type SASP along the DNA backbone may be important for the α/β-type SASP/DNA binding interaction. To determine which regions of the proteins are involved in interactions between α/β-type SASP bound to DNA, we have performed protein cross-linking studies with the zero-length cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). We have identified EDC-catalyzed protein cross-links in four different α/β-type SASP from Bacillus species, and the identification of these cross-links has yielded new insights into the interaction of α/β-type SASP on DNA. TheEscherichia coli strains used include: JM107 (F′traD36 proA+ proB+ lacIq lacZ ΔM15/endA1 gyrA96(Nalr) thi hsdR17 supE44 relA1Δ(lac-proAB) mcrA) (Life Technologies, Inc.), JM83 (ara Δ(lac-proAB) rpsL φ80lacZΔM15) (10Yanisch-Perron C. Vieira J. Messing J. Gene ( Amst. ). 1985; 33: 103-119Crossref PubMed Scopus (11462) Google Scholar), BL21(DE3) (T7 RNA polymerase under control of the lac promoter) (11Studier F.W. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4831) Google Scholar), and BMH 71-18 (F′ proAB lacIq lacZΔM15 thi supEΔ(lac-proAB) mutS::Tn10) (CLONTECH Laboratories, Inc.). TheBacillus strains used were Bacillus cereus T (originally obtained from H. O. Halvorson) and Bacillus megaterium QMB1551, ATCC no. 12872 (originally obtained from H. Levinson). E. coli strains were routinely grown in 2× YT medium (16 g of tryptone, 10 g of yeast extract, 5 g of NaCl per liter) at 37 °C with shaking. For the overexpression of cloned genes encoding α/β-type SASP, the medium was supplemented with 100 μg/ml ampicillin (JM107) or 200 μg/ml ampicillin and 0.5% glucose (BL21).B. megaterium was sporulated at 30 °C in supplemented nutrient broth, and spores were harvested and cleaned as described previously (12Setlow P. Kornberg A. J. Bacteriol. 1969; 100: 1155-1160Crossref PubMed Google Scholar). Oligonucleotides were designed to polymerase chain reaction amplify a 512-base pair fragment containing the gene encoding Bce1 (13Loshon C.A. Fliss E.R. Setlow B. Foerster H.F. Setlow P. J. Bacteriol. 1986; 167: 168-173Crossref PubMed Google Scholar) from B. cereus genomic DNA; the amplified fragment contained the gene's ribosome binding site and transcription terminator (13Loshon C.A. Fliss E.R. Setlow B. Foerster H.F. Setlow P. J. Bacteriol. 1986; 167: 168-173Crossref PubMed Google Scholar). The upstream primer, BCE1–1 (5′-AAAGGATCCTTATTATTTCATAATTTGTAGC; complementary to nucleotides 119–140) (13Loshon C.A. Fliss E.R. Setlow B. Foerster H.F. Setlow P. J. Bacteriol. 1986; 167: 168-173Crossref PubMed Google Scholar) and downstream primer, BCE1–2 (5′-AAAGGATCCTTTTAAGTATGCTTTTTCCTGC; complementary to nucleotides 592–613) (13Loshon C.A. Fliss E.R. Setlow B. Foerster H.F. Setlow P. J. Bacteriol. 1986; 167: 168-173Crossref PubMed Google Scholar), each contained BamHI restriction sites and 5′-flanking sequences (underlined residues) for cloning purposes. The BamHI-digested polymerase chain reaction product was agarose gel-purified and ligated intoBamHIdigested plasmid pET3 (11Studier F.W. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4831) Google Scholar), generating plasmid pPS2532 in which BclI digestion confirmed that the gene encoding Bce1 was under the control of the T7 promoter. Plasmid pPS2532 was used to transform E. coli strain DE3(BL21) to ampicillin resistance. The E10K mutant form of Bce1 was generated with the TransformerTM site-directed mutagenesis kit fromCLONTECH according to manufacturer's instructions. Phosphorylated primers complementary, except for designed mismatches (underlined bases), to the unique AlwNI restriction site of pET3 (5′-CCTGTTACTAGTGGATGCTGC) and the Gly6–Gly15 coding region of the gene encoding Bce1 (5′-GGAAGTCGTAATAAAGTATTAGTTCGAGGC) (13Loshon C.A. Fliss E.R. Setlow B. Foerster H.F. Setlow P. J. Bacteriol. 1986; 167: 168-173Crossref PubMed Google Scholar) were used with plasmid pPS2532 as a template to synthesize a mutagenized plasmid lacking the AlwNI site, and with a lysine codon replacing the codon for glutamate 10 of bce1. The mutagenized plasmid was digested with AlwNI prior to transformation into the mismatch repair deficient E. coli strain (mutS::Tn10, Tetr) supplied with the mutagenesis kit. Mutagenized plasmid was enriched by plasmid isolation, digestion with AlwNI and retransformation intoE. coli strain JM83. One clone was isolated and the identity of the mutagenized plasmid, termed pPS2734, was confirmed by DNA sequencing. SspC and SASP-C were overexpressed in E. coli strain JM107 from pDG148 derived plasmids containing the α/β-type SASP genes under control of an isopropyl β-d-thiogalactopyranoside inducible promoter as described previously (14Setlow B. Hand A.R. Setlow P. J. Bacteriol. 1991; 173: 1642-1653Crossref PubMed Google Scholar, 15Stragier P. Bonamy C. Karmazyn-Campelli C. Cell. 1988; 52: 697-704Abstract Full Text PDF PubMed Scopus (306) Google Scholar). Bce1 and Bce1E10K were overexpressed in E. coli strain DE3(BL21) from plasmids pPS2532 and pPS2734, respectively. SspC, Bce1, and Bce1E10Kwere extracted from dry ruptured E. coli cells with 3% acetic acid/30 mm HCl as described previously (16Hayes C.S. Setlow P. J. Bacteriol. 1997; 178: 6020-6027Crossref Google Scholar). SASP-C and SASP-A were extracted with 3% acetic acid (9Setlow P. J. Biol. Chem. 1975; 250: 8168-8173Abstract Full Text PDF PubMed Google Scholar) from dry rupturedE. coli cells and dry ruptured spores of B. megaterium strain QMB1551, respectively. All α/β-type SASP were purified as described previously (9Setlow P. J. Biol. Chem. 1975; 250: 8168-8173Abstract Full Text PDF PubMed Google Scholar). α/β-Type SASP (0.5 mg/ml) with or without cesium chloride gradient-purified,EcoRI-linearized pUC19 plasmid DNA (100 μg/ml) were incubated in 1 ml of 5 mm sodium phosphate (pH 7.5) at 24 °C for 20 min prior to addition of EDC to 25 mm. The 5:1 (w/w) ratio of protein to DNA is sufficient to saturate the DNA with α/β-type SASP, although SASP-A binds more weakly than do the other α/β-type SASP tested (17Setlow B. Sun D. Setlow P. J. Bacteriol. 1992; 174: 2312-2322Crossref PubMed Google Scholar). Under these conditions, approximately 50% of the α/β-type SASP are bound to the DNA. The cross-linking reactions were incubated for 30 min at 24 °C, followed by dialysis in Spectrapor 3 tubing against 1 liter of 10 mmsodium phosphate (pH 7.5) at 4 °C for 18 h. Dialyzed cross-linking reactions were frozen, lyophilized, dissolved in sample buffer and run on Tris-Tricine SDS-PAGE (18Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10477) Google Scholar). Gels were stained with Coomassie Blue, destained, and monomeric and cross-linked proteins were excised with a clean razor blade. Proteins were electroeluted from polyacrylamide gel slices into 50 mmNH4HCO3, 0.1% SDS using ElutrapTMseparation chambers (Schleicher & Schuell). Gel purified proteins were frozen, lyophilized, dissolved in 100 μl of MilliQ-H2O, and precipitated with 800 μl of cold acetone. Precipitated proteins were washed with 500 μl of cold acetone and dissolved in freshly prepared 8 m urea prior to trypsin digestion. EDC-treated proteins (∼20–40 μg) were digested with trypsin (Worthington, 5 μg) in 100 μl of 0.2 m NH4HCO3, 10 mm CaCl2, 1.2 m urea at 37 °C for 15–18 h. Tryptic digests were run on reverse-phase high performance liquid chromatography (HPLC) using a Waters 680 gradient controller, two Waters 501 pumps, a Waters U6K injector, and a Vydac protein C4 column (3.9 × 150 mm). Tryptic digests were loaded onto the reverse phase column in 100% buffer A (0.06% trifluoroacetic acid) followed by 5 min of washing with 100% buffer A. Peptides were eluted at a flow rate of 1 ml/min with a discontinuous linear gradient as follows: 5–30 min, 0–30% buffer B (0.052% trifluoroacetic acid in 80% acetonitrile); 30–50 min, 30–40% buffer B; 50–70 min, 40–100% buffer B. Peptides were detected by their UV absorption at 214 nm with a Waters 481 spectrophotometer, and fractions containing peptides were collected with an Isco 2150 peak separator and an Isco Foxy fraction collector. Molecular masses of HPLC-purified peptides were determined by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry using a Perseptive Biosystems Linear MALDI-TOF instrument. External calibration consisting of two standards (angiotensin, 1297.5 Da; and ACTH(18–39), 2466.7 Da) was used for all determinations, resulting in ±0.15% mass accuracy. Peptides from HPLC fractions (1 μl, ∼1–10 pmol/μl) were mixed and dried on the instrument stage with an equal volume of α-cyano-4-hydroxycinnamic acid (10 mg/ml) in 50% acetonitrile. Amino acid analysis was conducted as described previously (9Setlow P. J. Biol. Chem. 1975; 250: 8168-8173Abstract Full Text PDF PubMed Google Scholar). Peptide sequences were determined with an ABI model 492A Procise automated protein sequencer. In an effort to identify the interacting regions of α/β-type SASP bound to DNA, we decided to use protein cross-linking to trap interacting amino acid residues for subsequent biochemical analysis. Most α/β-type SASP do not contain cysteine residues (1Setlow P. Annu. Rev. Microbiol. 1988; 42: 319-338Crossref PubMed Scopus (176) Google Scholar) and therefore several protein cross-linking reagents which rely upon thiol chemistry could not be used for this study. The α/β-type SASP are small proteins (6–7.6 kDa) which are monomeric in solution3 (9Setlow P. J. Biol. Chem. 1975; 250: 8168-8173Abstract Full Text PDF PubMed Google Scholar) and appear to interact with one another only when bound to DNA. We were interested in regions of close contact between α/β-type SASP and therefore decided to use cross-linking reagents with short- or zero-length linker arms. EDC, a water-soluble carbodiimide, gave efficient cross-linking of α/β-type SASP only in the presence of DNA (see below). Consequently, we chose this reagent for further work. Protein cross-linking with EDC usually involves the formation of an amide bond between either an N-terminal α-amino or lysine ε-amino group and the carboxyl side chain of aspartate/glutamate residues. Therefore, in contrast to cross-linkers that contain flexible linker arms several angstroms in length, EDC induced cross-links should occur only between residues that are in very close proximity to one another. The proteins chosen for this study were SASP-A and SASP-C from B. megaterium, SspC from B. subtilis, and Bce1 fromB. cereus (Fig. 1). Most of the variability between these proteins occurs near the N termini which vary both in length and amino acid sequence (Fig. 1) (1Setlow P. Annu. Rev. Microbiol. 1988; 42: 319-338Crossref PubMed Scopus (176) Google Scholar). All α/β-type SASP lack the N-terminal methionine residue which is the only residue that is removed post-translationally (Fig. 1) (1Setlow P. Annu. Rev. Microbiol. 1988; 42: 319-338Crossref PubMed Scopus (176) Google Scholar). SASP-A and SASP-C are major α/β-type SASP in spores, whereas SspC and Bce1 are minor proteins. For all four proteins little or no protein-protein cross-linking was detected in the absence of added DNA, while significant protein-protein cross-linking was detected in reactions containing α/β-type SASP and DNA (Fig. 2, and data not shown). The extent of protein-protein cross-link formation and the number of higher order oligomers formed varied for each protein tested (Fig. 2). By overloading polyacrylamide gels, decamers could be easily detected in cross-linking reactions with Bce1 and DNA, whereas only small amounts of trimer were detected in reactions with SASP-A and DNA (Fig. 2 and data not shown). The efficiency of protein cross-linking corresponded roughly to the affinity of each protein for linear plasmid DNA (Bce1 > SspC ≈ SASP-C > SASP-A) as determined by DNase protection assays (17Setlow B. Sun D. Setlow P. J. Bacteriol. 1992; 174: 2312-2322Crossref PubMed Google Scholar) (data not shown), although the observed cross-linking efficiency of SASP-A was lower than expected.Figure 2EDC cross-linking of α/β-type SASP is DNA-dependent. Protein (0.5 mg/ml) with or without linear plasmid DNA (100 μg/ml) was reacted with 25 mm EDC for 30 min at 22 °C, followed by dialysis against 10 mm sodium phosphate (pH 7.5) at 4 °C as described under “Experimental Procedures.” Samples were lyophilized, dissolved in sample buffer, and run on Tris-Tricine SDS-PAGE (16.7% acrylamide) and stained with Coomassie Brilliant Blue R-250. Numbers on the left marginindicate the positions of molecular mass markers in kDa.View Large Image Figure ViewerDownload (PPT) The DNA dependence of cross-link formation between α/β-type SASP suggested that the EDC-generated protein-protein cross-links are formed only between α/β-type SASP that are adjacent to one another on the DNA backbone. These in vitro experiments used only a single α/β-type SASP. However, there are multiple α/β-type SASP in spores, with two proteins present at high levels. Consequently, an obvious question is whether the different α/β-type SASP interact when bound to DNA. To obtain data pertinent to this question we analyzed protein-protein cross-link formation in reactions with two different α/β-type SASP bound to DNA. SASP-A and SASP-C fromB. megaterium were chosen for the initial hetero-cross-linking experiments because they are the two major α/β-type SASP found in spores of B. megaterium (9Setlow P. J. Biol. Chem. 1975; 250: 8168-8173Abstract Full Text PDF PubMed Google Scholar). These proteins also differ sufficiently in molecular mass (SASP-A = 6,260.1 Da and SASP-C = 7,423.3 Da) to allow resolution of the three possible dimeric forms by Tris-Tricine SDS-PAGE. Electrophoretic analysis of cross-linking reactions containing SASP-A, SASP-C, and DNA revealed the presence of a new predominant band that migrated at the position expected for a SASP-A/SASP-C heterodimer (Fig. 3, lane A + C), and this band is indeed a SASP-A/SASP-C heterodimer (see below). Titration experiments demonstrated that the ratio of SASP-A to SASP-C that produces the most heterodimer is ∼3:1 (w/w) (data not shown). This latter ratio approximates the relative levels of these two proteins inB. megaterium spores (9Setlow P. J. Biol. Chem. 1975; 250: 8168-8173Abstract Full Text PDF PubMed Google Scholar). Heterodimers were also formed between SASP-A and SspC from B. subtilis (data not shown). However, in contrast to the SASP-A/SASP-C cross-linking reaction in which the SASP-A/SASP-C heterodimer was the predominant cross-linked product (Fig. 3), SASP-A/SspC heterodimer formation was much less efficient than formation of the SspC homodimer in these reactions (data not shown). Since SspC and SASP-C have similar affinities for linear plasmid DNA in solution, the difference in their formation of heterodimers with SASP-A is presumably due to differences in the amino acid sequences of SspC and SASP-C. There is presently very little detailed structural information available on α/β-type SASP or the complex they form with DNA. Therefore, identification of the amino acid residues involved in EDC-dependent cross-link formation was undertaken to determine which regions of α/β-type SASP are involved in protein-protein interactions that occur in the DNA bound state. Purified monomeric and oligomeric α/β-type SASP from EDC cross-linking reactions were digested with trypsin and the products resolved by reverse phase-HPLC. Two types of differences should be detected between the HPLC tryptic maps of dimeric (or oligomeric) and monomeric (but EDC treated) α/β-type SASP. First, the digests of α/β-type SASP dimers should show decreases (∼50%) in the relative yield of some peptide(s) as compared with the monomer, because amino acid residues within this peptide(s) will be in a cross-linked peptide in the dimer. Second, there should be a new peptide peak(s) in HPLC tryptic maps of α/β-type SASP dimers, which should be the peptide containing the cross-link. Detailed analyses, including mass spectrometry, amino acid analysis and amino acid sequencing of the latter peptides should then allow both the unambiguous identification of the peptides in the cross-link, as well as the specific amino acid residues involved. Intramolecular cross-links could also be formed by EDC, as α/β-type SASP go from an unfolded to a more ordered structure on binding to DNA. Intramolecular cross-links could be found within both monomeric and oligomeric proteins, and this modification could be detected by comparing HPLC tryptic maps of EDC treated monomers and untreated protein. However, we never saw evidence for intramolecular cross-link formation in these analyses (data not shown). HPLC analysis identified two unique, closely eluting peptides in the tryptic digest of dimeric SASP-A (Fig. 4 B, peptides labeled1 and 2) which were not present in the digest of the SASP-A monomer (Fig. 4 A). A substantial reduction in the amount of one peptide was also noted in the digest of dimeric SASP-A when compared with that of monomeric SASP-A (Fig. 4 B, peptide labeled 3). No other significant differences were observed between digests of the monomeric and dimeric species. Because the relative amounts of all other peptides appeared to be approximately the same between digests of monomeric and dimeric SASP-A, these data suggested that the cross-link occurred between an amino acid residue in peptide 3 and a residue within a small peptide which has very little UV absorbance. The tryptic digests of the SASP-C monomer and dimer exhibited differences that were very similar to those seen with SASP-A (data not shown). HPLC analysis of the tryptic digest of the SASP-A/SASP-C heterodimer also identified two unique peptides which were not present in digests of SASP-A or SASP-C monomers (data not shown). Both of these unique peptides from the SASP-A/SASP-C heterodimer had HPLC retention times that differed from those of the putative cross-linked peptides identified from the SASP-A and SASP-C homodimers (data not shown). Only two additional significant tryptic peptides were detected in the Bce1 dimer that were not present in the Bce1 monomer (Fig. 5, A and B). One of these peptides (Fig. 5 B, peptide labeled with anasterisk) was an oxidized form of Bce1 tryptic peptide Lys55–Arg66 which contained a methionine sulfoxide residue (data not shown). The other unique peptide, presumably the cross-linked peptide, eluted early in the HPLC gradient (Fig. 5 B, peptide labeled 1). No obvious reduction in the level of any major peptide peak was observed when the HPLC profile of the tryptic digest of monomeric Bce1 was compared with that of dimeric Bce1, suggesting that the cross-link occurred between amino acid residues from two small tryptic peptides. In contrast to SASP-A, SASP-C, and Bce1, analysis of the tryptic digest of the SspC dimer identified only one unique peptide in comparison to the digest of the SspC monomer (data not shown). However, as was found with SASP-A and SASP-C, the amount of one major peptide was decreased significantly in the digest of the SspC dimer as compared with the digest of the monomer (data not shown). Presumably this large peptide is involved in cross-link formation with a rather small peptide. The relatively high efficiency of SspC and Bce1 cross-linking (Fig. 2) also allowed the purification and analysis of cross-linked trimeric and tetrameric species of these proteins. The HPLC profiles of the tryptic digests of the trimeric and tetrameric species of both SspC and Bce1 were essentially identical to the tryptic map of the dimeric forms, with the exception of greater reductions in the larger peptide partner in the cross-link in the higher oligomers of SspC (data not shown). These data suggest that identical EDC catalyzed cross-links occur between each protein in higher oligomers of cross-linked α/β-type SASP. Various types of information were used to determine the amino acid residues involved in cross-link formation in the different α/β-type SASP. For SASP-A, SASP-C, and SspC, mass spectrometry and amino acid analysis identified the large peptides whose level was decreased in tryptic digests of the dimeric species as Tyr20–Arg37, Phe29–Arg46, and Ser8–Lys27, respectively. This identified one probable partner in the major cross-link formed in these three proteins. Determination of the mass of each peptide tentatively identified as a cross-linked species from tryptic digests of both homo- and heterodimers (Table I), as well as amino acid analyses (data not shown) allowed assignment of the two tryptic peptides in the various cross-links. In all cases, the site of cross-linking was tentatively identified as between the α-amino group of the protein and an acidic group on a separate tryptic peptide. The peptides in the two new peaks from tryptic digests of cross-linked dimers of either SASP-A or SASP-C had virtually identical observed molecular masses (Table I), suggesting that each peak contained the same two tryptic peptides linked together, but cross-linked at a different site. Analyses of the two unique peptides isolated from the tryptic digest of the SASP-A/SASP-C heterodimer predicted that the cross-links were between the α-amino group of one protein and a tryptic peptide in the other protein (Table I).Table IMass determination of cross-linked peptides by MALDI-MSPeptideObserved MassPredicted peptideCalculated average massaAverage masses were determined using MS Digest on the UCSF Mass Spectrometry Home Page; http://www.rafael.ucsf.edu.SASP-A-1bCross-linked peptides are labeled (1) and (2) according to the order of their elution during reverse-phase HPLC (Figs. 4 B and 5 B, and data not shown).2452.8Ala1–Lys5× Tyr20–Arg372454.7SASP-A-2bCross-linked peptides are labeled (1) and (2) according to the order of their elution during reverse-phase HPLC (Figs. 4 B and 5 B, and data not shown).2453.8Ala1–Lys5 × Tyr20–Arg372454.7SASP-C-1bCross-linked peptides are labeled (1) and (2) according to the order of their elution during reverse-phase HPLC (Figs. 4 B and 5 B, and data not shown).2914.6Ala1–Arg9 × Phe29–Arg462915.2SASP-C-2bCross-linked peptides are labeled (1) and (2) according to the order of their elution during reverse-phase HPLC (Figs. 4 B and 5 B, and data not shown).2914.3Ala1–Arg9 × Phe29–Arg462915.2Bce1–1913.8Gly1–Lys2 × Asn9–Arg14915.2SspC2745.1Ala1–Arg5 × Ser8–Lys272744.0SASP-A/C-1bCross-linked peptides are labeled (1) and (2) according to the order of their elution during reverse-phase HPLC (Figs. 4 B and 5 B, and data not shown).2944.6Ala1–Arg9 × Tyr20–Arg372945.2SASP-A/C-2bCross-linked peptides are labeled (1) and (2) according to the order of their elution during reverse-phase HPLC (Figs. 4 B and 5 B, and data not shown).2423.8Ala1–Lys5 × Phe29–Arg462424.7Mass spectrometry was performed as described under “Experimental Procedures.”a Average masses were determined using MS Digest on the UCSF Mass Spectrometry Home Page; http://www.rafael.ucsf.edu.b Cross-linked peptides are labeled (1Setlow P. Annu. Rev. Microbiol. 1988; 42: 319-338Crossref PubMed Scopus (176) Google Scholar) and (2Setlow P. Annu. Rev. Microbiol. 1995; 49: 29-54Crossref PubMed Scopus (331) Google Scholar) according to the order of their elution during reverse-phase HPLC (Figs. 4 B and 5 B, and data not shown). Open table in a new tab Mass spectrometry was performed as described under “Experimental Procedures.” Amino acid sequence analysis definitively identified the cross-linked pepti" @default.
• W2034383690 created "2016-06-24" @default.
• W2034383690 creator A5024707854 @default.
• W2034383690 creator A5042833634 @default.
• W2034383690 date "1998-07-01" @default.
• W2034383690 modified "2023-10-16" @default.
• W2034383690 title "Identification of Protein-Protein Contacts between α/β-Type Small, Acid-soluble Spore Proteins of Bacillus Species Bound to DNA" @default.
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