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PubMed1999

Hydrogen exchange/electrospray ionization mass spectrometry studies of structural features of proteins and protein/protein interactions.

The rate at which amide hydrogens located at the peptide backbone in protein/protein complexes undergo hydrogen/deuterium exchange is highly dependent on whether the amide groups participate in binding. Here, a new mass spectrometric method is presented in which this effect is utilized for the characterization of protein/ligand binding sites. The information obtained is which region within the protein participates in binding. The method includes hydrogen/deuterium exchange of receptor and ligand protein amide protons, binding, and back exchange. After this procedure those backbone amide groups that participate in protein binding are protected from back exchange and therefore still deuterated. These regions were then identified by peptic proteolysis, fast microbore high-performance liquid chromatography separation, and electrospray ionization mass spectrometry. The approach has been applied to the investigation of structural features of insulin-like growth factor I (IGF-I) and the interaction of insulin-like growth factor I with IGF-I binding protein 1. The data show that the approach can provide information on the location of the hydrophobic core of IGF-1 and on two regions that are mainly involved in binding to IGF-I binding protein 1. The data are consistent with results obtained with other approaches. The amount of sample required for one experiment is in the subnanomolar range.

Amino Acid Sequence↗

PubMed2001

Rat glutathione S-transferase M4-4: an isoenzyme with unique structural features including a redox-reactive cysteine-115 residue that forms mixed disulphides with glutathione.

Although the existence of the rat glutathione S-transferase (GST) M4 (rGSTM4) gene has been known for some time, the corresponding protein has not as yet been purified from tissue. A recombinant rGSTM4-4 was thus expressed in Escherichia coli from a chemically synthesized rGSTM4 gene. The catalytic efficiency (k(cat)/K(m)) of rGSTM4-4 for the 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction was 50-180-fold less than that of the well-characterized homologous rGSTM1-1, and the pH optimum for the same reaction was 8.5 for rGSTM4-4 as opposed to 6.5 for rGSTM1-1. Molecular-modelling studies predict that key substitutions in the helix alpha4 region of rGSTM4-4 account for this pK(a) difference. A notable structural feature of rGSTM4-4 is the Cys-115 residue in place of the Tyr-115 of other Mu-class GSTs. The thiol group of Cys-115 is redox-reactive and readily forms a mixed disulphide even with GSH; the S-glutathiolated form of the enzyme is catalytically active. A mutated rGSTM4-4 (C115Y) had 6-10-fold greater catalytic efficiency than the wild-type rGSTM4-4. Trp-45, a conserved residue among Mu-class GSTs, is essential in rGSTM4-4 for both enzyme activity and binding to glutathione affinity matrices. Antibodies directed against either the unique C-terminal undecapeptide or tridecapeptide of rGSTM4 reacted with rat and mouse liver GSTs to reveal an orthologous mouse GSTM4-4 present at low basal levels but which is inducible in mouse liver. This subclass of rodent Mu GSTs with redox-active Cys-115 residues could have specialized physiological functions in response to oxidative stress.

Amino Acid Sequence↗

PubMed1986

Proto-oncogene c-ros codes for a molecule with structural features common to those of growth factor receptors and displays tissue specific and developmentally regulated expression.

A recombinant DNA clone containing cellular sequences homologous to the transforming sequence, v-ros, of avian sarcoma virus UR2 was isolated from a chicken genomic DNA library. Heteroduplex mapping and nucleotide sequencing reveal that the v-ros sequences are distributed in nine exons ranging from 65 to 204 nucleotides on cellular ros (c-ros) DNA over a range of 11 kilobases. Comparison of the deduced amino acid sequences of c-ros and v-ros shows two differences: v-ros contains a three-amino-acid insertion within the hydrophobic domain presumed to be involved in membrane association, and (ii) the carboxyl 12 amino acids of v-ros are completely different from those of the deduced c-ros sequence. The deduced amino acid sequence of c-ros bears striking structural features similar to those of insulin and epidermal growth factor receptors, including the presumed hydrophobic membrane binding domain, amino acids flanking the domain, and the distance between the domain and the catalytic region of the kinase activity. The expression of c-ros appears to be under a very stringent control. When tissues at various stages of chicken development were analyzed, only kidney was found to contain a significant level of c-ros RNA. The level of c-ros RNA in kidney tissue is most abundant in 7- to 14-day-old chickens. Finally, nucleotide sequences of c-ros DNA and UR2-associated helper viral genome at regions corresponding to the gag ros recombination site suggest that the junction has been formed by RNA splicing.

Amino Acid Sequence↗

PubMed2000

Efficient generation of a hepatitis B virus cytotoxic T lymphocyte epitope requires the structural features of immunoproteasomes.

Interferon (IFN)-gamma-induced cells express the proteasome subunits low molecular weight protein (LMP)2, LMP7, and MECL-1 (multicatalytic endopeptidase complex-like 1), leading to the formation of immunoproteasomes. Although these subunits are thought to optimize MHC class I antigen processing, the extent of their role and the mechanistic aspects involved remain unclear. Herein, we study the proteolytic generation of an human histocompatibility leukocyte antigen (HLA)-Aw68-restricted hepatitis B virus core antigen (HBcAg) cytotoxic T lymphocyte (CTL) epitope that is recognized by peripheral blood lymphocytes from patients with acute self-limited but not chronic hepatitis B virus (HBV). Immunological data suggest that IFN-gamma-induced rather than uninduced HeLa cells process and present the HBV CTL epitope upon infection with HBcAg-expressing vaccinia viruses. Analyses of 20S proteasome digests of synthetic polypeptides covering the antigenic HBcAg peptide demonstrate that only immunoproteasomes efficiently perform the cleavages needed for the liberation of this HBV CTL epitope. Although the concerted presence of the three immunosubunits appears essential, we find that both catalytically active LMP7 and inactive LMP7 T1A support CTL epitope generation. We conclude that LMP7 influences the structural features of 20S proteasomes, thereby enhancing the activity of the LMP2 and MECL-1 catalytic sites, which provide cleavage specificity. Thus, LMP7 incorporation is of greater functional importance for the generation of an HBV CTL epitope than cleavage specificity.

Amino Acid Sequence↗

PubMed1993

Structural features of the trans-activation response RNA element of equine infectious anemia virus.

A 25-nucleotide RNA with the sequence of the trans-activation response (TAR) element of equine infectious anemia virus (EIAV) was analyzed by biochemical methods and by one- and two-dimensional NMR spectroscopy. NMR, nuclease probing, and polyacrylamide gel migration rates show that the RNA consists of an A-helical stem capped by two non-Watson-Crick U-G base pairs and a compact four-nucleotide loop. The loop is stabilized by base stacking, with loop nucleotides C12 and C15 stacked upon U11 and G16, respectively. Near the 5' end of the molecule, the stem contains a bulge at nucleotide C2, most likely a result of base pairing between G1 and C25. A method for distinguishing RNA stem-loops from palindromic dimers is described and was used to confirm that the EIAV TAR RNA has a stem-loop structure at conditions used for NMR spectroscopy. RNase A and RNase T1 cleavage patterns are consistent with the structural features derived from the NMR data.

Base Composition↗

PubMed1991

Sequence analysis of the S RNA of the African arenavirus Mopeia: an unusual secondary structure feature in the intergenic region.

Mopeia virus is an apparently nonpathogenic African arenavirus which can protect animals from subsequent challenge by the closely related Lassa virus. As a step toward understanding these differences in pathogenicity and the means by which Mopeia virus infection can protect against subsequent Lassa virus infection, cDNA clones corresponding to 3419 nucleotides of Mopeia virus S RNA were isolated and sequenced. Two open reading frames, encoding the glycoprotein precursor (GPC) and nucleocapsid (N) proteins, were located in the ambisense arrangement characteristic of the arenaviruses. Comparison of the amino acid sequences of the translation products with those of two Lassa virus strains showed considerable conservation, with 74 and 80% identity for the two glycoproteins G1 and G2, and 74% identity for the N protein. The putative dibasic site of GPC cleavage (R-R) was conserved, as were the potential N-linked glycosylation sites. A striking difference between Mopeia virus and Lassa virus was identified in the noncoding intergenic region. Instead of the single hairpin structure formed by base-pairing of complementary sequences which is usually found, the Mopeia virus S RNA has the potential to form two hairpins. These hairpins were similar in sequence and may have been formed in a duplication event during RNA replication. The possible contribution of this secondary structure feature to differences in pathogenicity between Mopeia and Lassa viruses is discussed.

Amino Acid Sequence↗

PubMed2001

Photoaffinity labeling of mutant neurokinin-1 receptors reveals additional structural features of the substance P/NK-1 receptor complex.

Photoaffinity labeling, receptor site-directed mutagenesis, and high-resolution NMR spectroscopy have been combined to further define the molecular details of the binding of substance P (SP) to the rat neurokinin-1 (NK-1) receptor. Mutant NK-1 receptors were constructed by substituting Ala for Met174 and/or Met181: residues previously identified as the sites of covalent attachment of radioiodinated, photoreactive derivatives of SP containing p-benzoyl-L-phenylalanine (Bpa) in positions 4 and 8, respectively. Photoaffinity labeling of the M181A mutant using radioiodinated Bpa8-SP resulted in a marked reduction in photoincorporation efficiency compared to the wild-type receptor. In contrast, photoaffinity labeling of the M174A mutant using radioiodinated Bpa4-SP gave the unexpected result of an increase in the efficiency of photoincorporation compared to the wild-type receptor. Enzymatic and chemical fragmentation analysis of the photolabeled receptor mutants established that the sites of covalent attachment were not the substituted alanine, but rather the other methionine on the second extracellular (E2) loop sequence, that is not the primary site of attachment in the wild-type receptor. The results thus suggest a close spatial relationship between Met174 and Met181 on the NK-1 receptor. To evaluate this structural disposition, NMR analyses were performed on a synthetic peptide with a sequence corresponding to the entire E2 loop and segments of the adjoining transmembrane helices to anchor the peptide in the lipids used to mimic a membrane. The structural features of the E2 loop include a centrally located alpha-helix, extending from Pro175 to Glu183, as well as smaller alpha-helices at the termini, corresponding to the transmembrane regions. The two methionine residues are located on the same face of the central alpha-helix, approximately 11 A apart from each other, and are therefore consistent with the conclusions of the photoaffinity labeling results.

Amino Acid Sequence↗

PubMed1993

Isolation and sequencing of Escherichia coli gene proP reveals unusual structural features of the osmoregulatory proline/betaine transporter, ProP.

Transporters encoded in genetic loci putP, proP and proU mediate proline and/or betaine accumulation by Escherichia coli K-12. The ProP and ProU systems are osmoregulatory. Activation of ProP in response to hyperosmotic stress has been demonstrated both in vivo and in vitro. It therefore serves as a model experimental system for the analysis of osmosensory and osmoregulatory mechanisms. We developed methodologies which will facilitate the identification of proline transporter genes by functional complementation of putP proP proU bacteria. E. coli gene proP was isolated and located within a chromosomal DNA fragment. Deletion, complementation and sequence analysis revealed putative promoter and transcription termination signals flanking a 1500 base-pair open reading frame. The predicted 55 kDa ProP protein was hydrophobic. In vitro expression of proP yielded a protein whose apparent molecular mass was determined to be 42 kDa by polyacrylamide gel electrophoresis under denaturing conditions. Database searches and cluster analysis defined relationships among the ProP sequence and those of integral membrane proteins that comprise a transporter superfamily. Members of the superfamily catalyze facilitated diffusion or ion linked transport of organic solutes in prokaryotes and eukaryotes. Multiple alignment revealed particularly close correspondence among the ProP protein, citrate transporters from E. coli and Klebsiella pneumoniae and an alpha-ketoglutarate transporter from E. coli. The predicted ProP sequence differed from those closely similar sequences in possessing an extended central hydrophilic loop and a carboxyl terminal extension. Unlike other protein sequences within the transporter superfamily, the carboxyl terminal extension of ProP was strongly predicted to participate in formation of an alpha-helical coiled coil. These data suggest that the ProP protein catalyzes solute-ion cotransport. Its unusual structural features may be related to osmoregulation of its activity.

Amino Acid Sequence↗

PubMed1980

Physicochemical characterization of the ribosomal RNA species of the Mollusca. Molecular weight, integrity and secondary-structure features of the RNA of the large and small ribosomal subunits.

1. The rRNA species of the Cephalopoda Octopus vulgaris and Loligo vulgaris were found to have unexpectedly high sedimentation coefficients and molecular weights. In 0.1 M-NaCl the L-rRNA (RNA from large ribosomal subunit) has the same s20 value as the L-rRNA of the mammals (30.7S), whereas the S-rRNA (RNA from small ribosomal subunit) sediments at a faster rate (20.1S) than the S-rRNA of both the mammals and the fungi (Neurospora crassa) (17.5S). The molecular weights of the L-rRNA were determined by gel electrophoresis in formamide and found to be 1.66 X 10(6) (Octupus) and 1.89 X 10(6) (Loligo); the mol.wt. of the S-rRNA of both species is 0.96 X 10(6), i.e. much larger than that of the mammals (0.65 X 10(6)) and almost coincident with that of the '23S' RNA of the prokaryotes. 2. By contrast, the less evolved Gastropoda and Lamellibranchiata (Murex trunculus and Macrocallista chione) have S-rRNA and L-rRNA species with mol.wts. of 0.65 X 10(6) and approx. 1.40 X 10(6).3. All the mature L-rRNA molecules of the cephalopoda are composed of two unequal fragments held together by regions of hydrogen-bonding having a similar, low, thermal stability in the two species; the molecular weights of the two fragments composing the L-rRNA are estimated to be 0.96 X 10(6) and 0.88 X 10(6) (Loligo) and 0.96 X 10(6) and 0.65 X 10(6) (Octupus). THe S-rRNA of both species is a continuous chain with exactly the same molecular weight (0.96 X 10(6)) as the heavier of the two fragments of the L-rRNA. 4. The secondary-structure features of the L-rRNA and S-rRNA species of the Caphalopoda were investigated by thermal 'melting' analysis in 4.0 M-guanidinium chloride; 60-70% of the residues are estimated to form short, independently 'melting' bihelical segments not more than 10 base-pairs in length. 5. Bases are unevenly distributed between non-helical and bihelical portions of the rRNA molecules, G and C residues being preferentially concentrated in bihelical comains. 6. The secondary-structure regions of the L-rRNA species of Octopus and Loligo are heterogenous, including two discrete fractions of independently 'melting' species that give rise to biphasic 'melting' profiles: a fraction consisting of shorter (G + C)-poorer segments (60-68% G + C, not more than 5 base-pairs in length) and a fraction consisting of longer (G + C)-richer segments (80-88% G + C, 5-10 base-pairs in length). No evidence for heterogeneity has been detected in the S-rRNa.

Animals↗

PubMed2001

Structural features of peptide analogs of human histocompatibility leukocyte antigen class I epitopes that are more potent and immunogenic than wild-type peptide.

Certain peptide analogs that carry substitutions at residues other than the main major histocompatibility complex anchors and are surprisingly much more antigenic than wild-type peptide (heteroclitic analogs). To date, it was unknown how frequently wild-type epitopes could be modified to obtain heteroclitic activity. In this study, we analyzed a large panel of analogs of two different human histocompatibility leukocyte antigen (HLA)-A2.1-restricted epitopes and found that heteroclitic analogs were associated with higher magnitude responses and increased (up to 10(7)-fold) sensitivity to antigen, and corresponded to conservative or semiconservative substitutions at odd-numbered positions in the middle of the peptide (positions 3, 5, or 7). These findings were validated by performing additional immunogenicity studies in murine and human systems with four additional epitopes. The biological relevance of heteroclitic analogs was underlined when predicted analogs of the p53.261 epitope was shown to induce cytotoxic T lymphocytes (CTLs) that recognize low concentrations of peptide (high avidity) in vivo and demonstrate in vitro antitumor recognition. Finally, in vitro immunization of human peripheral blood mononuclear cells with two heteroclitic analogs resulted in recruitment of more numerous CTLs which were associated with increased antigen sensitivity. In conclusion, heteroclitic analogs were identified in each of the six cases studied and structural features were defined which allow identification of such analogs. The strong CTL immunity elicited by heteroclitic epitopes suggest that they could be of significant value in vaccination against tolerant or weakly immunogenic tumor-associated and viral antigens.

Animals↗

PubMed1992

The sequence HGLGHGHEQQHGLGHGH in the light chain of high molecular weight kininogen serves as a primary structural feature for zinc-dependent binding to an anionic surface.

The histidine-glycine-rich region of the light chain of cleaved high molecular weight kininogen (HK) is thought to be responsible for binding to negatively charged surfaces and initiation of the intrinsic coagulation, fibrinolytic, and kinin-forming systems. However, the specifically required amino acid sequences have not been delineated. An IgG fraction of a monoclonal antibody (MAb) C11C1 to the HK light chain was shown to inhibit by 66% the coagulant activity and by 57% the binding of HK to the anionic surface of kaolin at a concentration of 1.5 microM and 27 microM, respectively. Proteolytic fragments of HK were produced by successive digestion with human plasma kallikrein and factor XIa (FXIa). Those polypeptides that bound tightly (Kd = 0.77 nM) to a C11C1 affinity column were eluted at pH 3.0 and purified by membrane filtration. On 15% SDS polyacrylamide electrophoresis, the approximate M(r) was 7.3 kDa (range 6.2-8.1 kDa). Based on N-terminal sequencing, this polypeptide (1(2)), which extends from the histidine residue 459 to a lysine at position 505, 509, 511, 512, 515, or 520, inhibits by 50% the coagulant activity expressed by HK at a concentration of 22 microM. The synthetic peptide HGLGHGH representing the N-terminal of the 1(2)) fragment was synthesized, tested, and found at 4 mM to inhibit the procoagulant activity of HK 50%. A synthetic heptadecapeptide, HGLGHGHEQQHGLGHGH (residues 459-475) included within the 1(2) fragment, and with the ability to bind zinc, inhibited 50% of the HK coagulant activity at a concentration of 325 microM in the absence and presence of added Zn2+ (30 microM). The specific binding of 125I-HK to a negatively charged surface (kaolin) was inhibited 50% by unlabeled HK (5 microM). HGLGHGH, at a concentration of 7.0 mM, inhibited the binding to kaolin by 50%. The heptadecapeptide inhibited the specific binding of 125I-HK to kaolin by 50%, at a concentration of 2.3 mM, in the absence of Zn2+. In contrast, when Zn2+ was added, the concentration to achieve 50% inhibition decreased to 630 microM, indicating that Zn2+ was required to attain a favorable conformation for binding. Moreover, the 1(2) fragment was found to inhibit 50% of the 125I-HK binding to kaolin at a concentration of 380 microM. These results suggest that residues contained within the 1(2) fragment, notably HGLGHGHEQQHGLGHGH, serves as a primary structural feature for binding to a negatively charged surface.

Amino Acid Sequence↗

PubMed1990

Structural features of the cytoplasmic region of CD4 required for internalization.

CD4, the T cell surface antigen, is phosphorylated and internalized when T cells are activated or treated with a phorbol ester, PMA. The actual phosphorylation sites have been identified and the role of phosphorylation of each on CD4 internalization investigated. Seven different mutants, in each of which one, two or all three of the serine residues of the cytoplasmic region was modified to alanine(s) (CD4.SA mutants) and one mutant in which the whole amino acid sequence from Gln421 to the C-terminal Ile433 was changed (CD4.EP mutant) were constructed and used to determine the effect of phosphorylation on CD4 internalization. Ser408 was the most efficiently phosphorylated by PMA treatment, Ser415 next and Ser431 to a minor extent. The effect of mutation on internalization was well matched with the effect on extent of phosphorylation, i.e. Ser408 was the residue most important for internalization. However, complete inhibition of CD4 internalization was achieved only by mutating all three serine residues. Interestingly, the mutant CD4.EP in which Ser408 was present and phosphorylated was not measurably internalized, suggesting that phosphorylation of Ser408 induces CD4 internalization only when other structural features of the cytoplasmic domain remain intact. In addition, the data suggest the existence of an additional minor pathway for CD4 internalization which is phosphorylation independent.

Amino Acid Sequence↗

PubMed1981

Structural features of glutamine substrates for transglutaminases. Specificities of human plasma factor XIIIa and the guinea pig liver enzyme toward synthetic peptides.

Studies on the glutamine substrate specificities of human plasma factor XIIIa and guinea pig liver transglutaminase have been made using variants of the synthetic peptide substrate, Ser-Val-Leu-Ser-Leu-Ser-Gln-Ser-Lys-Val-Leu-Pro-Val-Pro-Glu. The sequence of this effective peptide substrate corresponds to the primary site of factor XIIIa-catalyzed amine incorporation into beta-casein, the most sensitive known macromolecular substrate for this enzyme (Gorman, J.J., and Folk, J.E. (1980) J. Biol. Chem. 255, 419-427). Variations in specificity observed with factor XIIIa for peptides containing single substitutions and multiple substitutions in this sequence are indications that several important determinants for enzyme recognition are contained therein. Among these are several of the hydrophobic amino acid residues and the lysine residue. Less pronounced changes in specificity occur with the liver enzyme and the differences in effects of the various substitutions reveal important differences in specificity requirements of factor XIIIa and the liver enzyme. Comparisons of the activities of the enzymes toward the synthetic peptides to their activities toward macromolecular substrates suggest that higher order macromolecular structural features contribute to specificity.

Amino Acid Sequence↗

PubMed1996

Structural features of thyroid hormone response elements that increase susceptibility to inhibition by an RTH mutant thyroid hormone receptor.

The chicken lysozyme silencer F2 (F2) thyroid hormone response element (TRE) contains an unusual everted palindromic arrangement, has a high affinity for thyroid hormone receptor (TR) homodimers, and is especially sensitive to dominant negative inhibition by, the T3 resistance (RTH) mutant TR beta P453H. We used various TREs and TR mutations to determine the mechanisms for this sensitivity. Changing the F2 orientation from an everted palindrome to a direct repeat with a 4-bp gap (DR+4) (F2-DR) decreased the sensitivity to inhibition at high T3 concentrations, while a loss of this sensitivity occurred with a palindromic arrangement of these same half-sites. F2 contains the dinucleotide TG 5' to each consensus half-site conforming to the optimal TR-binding octamer, YRRGGTCA. A T to A change in position 1 of both F2 half-sites markedly reduced T3-induction, yet only slightly reduced TR homodimer or TR-retinoid X receptor (RXR) heterodimer binding. The TR beta ninth heptad mutation, L428R, prevents TR heterodimerization with RXR and eliminates the inhibitory effect of the P453H mutant TR on the F2-DR, but not the F2 element. Structural features of a TRE that favor strong TR binding of both TR homodimers and TR-RXR heterodimers containing the mutant TR, such as the everted palindromic conformation or the optimal TR-binding consensus octamer, enhance the sensitivity of a TRE to inhibition by the mutant TR. Thus, both half-site orientation and sequence contribute to the sensitivity of a given TRE to dominant negative inhibition by a mutant TR.

Amino Acid Sequence↗

PubMedDate not supplied

Structural features of cultured epithelial cells from the adult rat epididymis.

Epithelial cells isolated from the caput epididymidis of adult rats were placed in primary culture and examined daily for ten days for changes in external anatomy, reorganization of cytoskeletal components, maintenance of characteristic cytoplasmic features, and response to media formulated to minimize nonepithelial cell proliferation. Significant cell attachment to the substrate began after the first 24 hours of culture. After attachment, the cells underwent a progressive flattening and became closely applied to the substrate. This was accompanied by a redistribution of microvilli on the cell surface and a reorganization of cytoskeletal elements within the cell. After flattening, the cultured cells displayed an extensive array of 10-nm filaments which were associated with the desmosomes attaching adjacent cells. Immunofluorescence studies demonstrated that these were keratin-containing intermediate filaments and 2-D gel electrophoresis of intact cells and cell cytoskeletons revealed that a family of "keratin-like" polypeptides were major components of the cells. Epithelial cell attachment, morphology, and maintenance in the primary culture were unaffected by D-valine, cytosine arabinoside, or both; however, these agents, either individually or in combination, reduced significantly the number of cells incorporating 3H-thymidine. These data show that isolated epithelial cells retain some differentiated structural features that characterize the intact cell and that enriched cultures of epithelial cells can be maintained under conditions where fibroblast proliferation is inhibited.

Animals↗

PubMed2000

DNA breakage detection-FISH (DBD-FISH) in human spermatozoa: technical variants evidence different structural features.

Non-irradiated and X-irradiated (80 Gy) human spermatozoa were processed for in situ DNA breakage detection-FISH (DBD-FISH) of the whole genome, following two alternative variations of the basic technique. In the first, cells were initially incubated in the alkaline unwinding solution for transformation of DNA breaks into single-stranded DNA (ssDNA) to be hybridized, followed by the lysing solutions for protein removal. In the second, incubation in the lysing solutions was carried out before the denaturation step. The first approach yielded two subpopulations. While most sperm nuclei were faintly labeled and had chromocenters, a small subpopulation was strongly and homogeneously labeled, due to extensive DNA breakage. X-ray exposure increased the surface and mean fluorescence intensity. Otherwise, when the denaturation step was performed after protein extraction, all sperm nuclei yielded strong and dispersed FISH signals. Protein removal allows access of the unwinding solution to the DNA, which has abundant alkali-labile sites, and thus gives rise to large areas of ssDNA that are labeled by FISH. X-ray exposure increased the dispersion of FISH signals but decreased their mean fluorescence intensity. A linear dose-response was generated using the second experimental variant, being 30 Gy the lowest dose for detecting induction of damage by X-rays in mature sperm chromatin. These results indicate that DBD-FISH is not only useful for in situ detection of DNA breakage but also for revealing structural features of chromatin.

DNA↗

PubMed1999

Structural features contributing to complex formation between glycogen phosphorylase and phosphorylase kinase.

A polyclonal antibody was generated against a peptide corresponding to a region opposite the regulatory face of glycogen phosphorylase b (P-b), providing a probe for detecting and quantifying P-b when it is bound to its activating kinase, phosphorylase kinase (PhK). Using both direct and competition enzyme-linked immunosorbent assays (ELISAs), we have measured the extent of direct binding to PhK of various forms of phosphorylase, including different conformers induced by allosteric effectors as well as forms differing at the N-terminal site phosphorylated by PhK. Strong interactions with PhK were observed for both P-b', a truncated form lacking the site for phosphorylation, and P-a, the phosphorylated form of P-b. Further, the binding of P-b, P-b', and P-a was stimulated a similar amount by Mg(2+), or by Ca(2+) (both being activators of PhK). Our results suggest that the presence and conformation of P-b's N-terminal phosphorylation site do not fully account for the protein's affinity for PhK and that regions distinct from that site may also interact with PhK. Direct ELISAs detected the binding of P-b by a truncated form of the catalytic gamma subunit of PhK, consistent with the necessary interaction of PhK's catalytic subunit with its substrate P-b. In contrast, P-b' bound very poorly to the truncated gamma subunit, suggesting that the N-terminal phosphorylatable region of P-b may be critical in directing P-b to PhK's catalytic subunit and that the binding of P-b' by the PhK holoenzyme may involve more than just its catalytic core. The sum of our results suggests that structural features outside the catalytic domain of PhK and outside the phosphorylatable region of P-b may both be necessary for the maximal interaction of these two proteins.

Adenosine Monophosphate↗

PubMed1998

Reconstitution of core light-harvesting complexes of photosynthetic bacteria using chemically synthesized polypeptides. 2. Determination of structural features that stabilize complex formation and their implications for the structure of the subunit complex.

Chemically synthesized polypeptides have been utilized with a reconstitution assay to determine the role of specific amino acid side chains in stabilizing the core light-harvesting complex (LH1) of photosynthetic bacteria and its subunit complex. In the preceding paper [Meadows, K. A., Parkes-Loach, P. S., Kehoe, J. W., and Loach, P. A. (1998) Biochemistry 37, 3411-3417], it was demonstrated that 31-residue polypeptides (compared to 48 and 54 amino acids in the native polypeptides) having the same sequence as the core region of the beta-polypeptide of Rhodobacter sphaeroides (sph beta 31) or Rhodospirillum rubrum (rr beta 31) could form subunit-type complexes. However, neither polypeptide interacted with the native alpha-polypeptides to form a native LH1 complex. In this paper, it is demonstrated that larger segments of the native Rb. sphaeroides beta-polypeptide possess native behavior in LH1 formation. Polypeptides were synthesized that were six (sph beta 37) and ten amino acids (sph beta 41) longer than sph beta 31. Although sph beta 37 exhibited behavior nearly identical to that of sph beta 31, sph beta 41 behaved more like the native polypeptide. In the case of rr beta 31, a polypeptide with four additional amino acids toward the C terminus was synthesized (rr beta 35). Because LH1-forming behavior was not recovered with this longer polypeptide, one or more of the three remaining amino acids at the C-terminal end of the native beta-polypeptide seem to play an important role in LH1 stabilization in Rs. rubrum. Three analogues of the core region of the Rb. sphaeroides beta-polypeptide were synthesized, in each of which one highly conserved amino acid was changed. Evidence was obtained that the penultimate amino acid, a Trp residue, is especially important for subunit formation. When it was changed to Phe, the lambda Max of the subunit shifted from 823 to 811 nm and the association constant decreased about 500-fold. Changing each of two other amino acids had smaller effects on subunit formation. Changing Trp to Phe at the location six amino acid residues toward the C terminus from the His coordinated to Bchl resulted in an approximately 10-fold decrease in the association constant for subunit formation but did not affect the formation of a LH1-type complex compared to sph beta 31. Finally, changing Arg to Leu at the location seven amino acid residues toward the C terminus from the His coordinated to Bchl decreased the association constant for subunit formation by about 30-fold. In this case, no LH1-type complex could be formed. On the basis of these results, in comparison with the crystal structure of the LH2 beta-polypeptide of Rhodospirillum molischianum, two possible structures for the subunit complex are suggested.

Amino Acid Sequence↗