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ABSTRACT: Single molecule studies of protamine-DNA interactions have characterized the kinetics of protamine binding to DNA and the morphology of the toroidal subunits that comprise sperm chromatin. The results provided by these studies are reviewed, the advantage of using single molecule techniques is discussed, and the implications of the results to the structure, kinetics of toroid formation, and stability of the DNA-protamine complex are described. New measurements of DNA condensation forces induced by the binding of protamine to DNA are also presented. These forces induce a significant tension in constrained segments of DNA and may contribute to the reduction in volume and shaping of the maturing spermatid cell nucleus.
Protein and Peptide Letters 03/2011; 18(8):802-10. · 1.94 Impact Factor
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ABSTRACT: A variety of arginine-rich peptide sequences similar to those found in viral proteins have been conjugated to other molecules to facilitate their transport into the cytoplasm and nucleus of targeted cells. The selective high affinity ligand (SHAL) (DvLPBaPPP)2LLDo, which was developed to bind only to cells expressing HLA-DR10, has been conjugated to one of these peptide transduction domains, hexa-arginine, to assess the impact of the peptide on SHAL uptake and internalization by Raji cells, a B-cell lymphoma.
An analog of the SHAL (DvLPBaPPP)2LLDo containing a hexa-arginine peptide was created by adding six D-arginine residues sequentially to a lysine inserted in the SHAL's linker. SHAL binding, internalization and residualization by Raji cells expressing HLA-DR10 were examined using whole cell binding assays and confocal microscopy. Raji cells were observed to bind two fold more 111In-labeled hexa-arginine SHAL analog than Raji cells treated with the parent SHAL. Three fold more hexa-arginine SHAL remained associated with the Raji cells after washing, suggesting that the peptide also enhanced residualization of the 111In transported into cells. Confocal microscopy showed both SHALs localized in the cytoplasm of Raji cells, whereas a fraction of the hexa-arginine SHAL localized in the nucleus.
The incorporation of a hexa-D-arginine peptide into the linker of the SHAL (DvLPBaPPP)2LLDo enhanced both the uptake and residualization of the SHAL analog by Raji cells. In contrast to the abundant cell surface binding observed with Lym-1 antibody, the majority of (DvLPBaPPP)2LArg6AcLLDo and the parent SHAL were internalized. Some of the internalized hexa-arginine SHAL analog was also associated with the nucleus. These results demonstrate that several important SHAL properties, including uptake, internalization, retention and possibly intracellular distribution, can be enhanced or modified by conjugating the SHALs to a short polypeptide.
Molecular Cancer 05/2009; 8:25. · 3.99 Impact Factor
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ABSTRACT: Healthy human males produce sperm cells of which about 25-40% have abnormal head shapes. Increases in the percentage of sperm exhibiting aberrant sperm head morphologies have been correlated with male infertility, and biochemical studies of pooled sperm have suggested that sperm with abnormal shape may contain DNA that has not been properly repackaged by protamine during spermatid development. We have used micro-Raman spectroscopy to obtain Raman spectra from individual human sperm cells and examined how differences in the Raman spectra of sperm chromatin correlate with cell shape. We show that Raman spectra of individual sperm cells contain vibrational marker modes that can be used to assess the efficiency of DNA-packaging for each cell. Raman spectra obtained from sperm cells with normal shape provide evidence that DNA in these sperm is very efficiently packaged. We find, however, that the relative protein content per cell and DNA packaging efficiencies are distributed over a relatively wide range for sperm cells with both normal and abnormal shape. These findings indicate that single cell Raman spectroscopy should be a valuable tool in assessing the quality of sperm cells for in-vitro fertilization.
Journal of Biophotonics 04/2009; 2(5):322-32. · 4.34 Impact Factor
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ABSTRACT: To mimic the molecular specificity and cell selectivity of monoclonal antibody (mAb) binding while decreasing size, nanomolecules (selective high-affinity ligands; SHALs), based on in silico modeling, have been created to bind to human leukocyte antigen-DR (HLA-DR10), a signaling receptor protein upregulated on the malignant B-lymphocytes of non-Hodgkin's lymphoma and chronic lymphocytic leukemia. SHALs were synthesized with a biotin or DOTA chelate (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid), using a solid-phase lysine-polyethyleneglycol backbone to link sets of ligands shown previously to bind to HLA-DR10. Using cell-binding and death assays and confocal microscopy, SHAL uptake, residualization, and cytocidal activity were evaluated in HLA-DR10 expressing and nonexpressing live, human lymphoma cell lines. All of the SHALs tested were selective for, and accumulated in, expressing cells. Reflecting binding to HLA-DR10 inside the cells, SHALs having the Ct ligand (3-(2-([3-chloro-5-trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3-oxopropanionic acid) residualized in expressing cells greater than 179 times more than accountable by cell-surface membrane HLA-DR10. Confocal microscopy confirmed the intracellular residualization of these SHALs. Importantly, SHALs with a Ct ligand had direct cytocidal activity, similar in potency to that of Lym-1 mAb and rituximab, selectively for HLA-DR10 expressing lymphoma cells and xenografts. The results show that SHALs containing the Ct ligand residualize intracellularly and have cytocidal effects mediated by HLA-DR10. These SHALs have extraordinary potential as novel molecules for the selective targeting of lymphoma and leukemia for molecular therapy and imaging. Further, these SHALs can be used to transport and residualize cytotoxic agents near critical sites inside these malignant cells.
Cancer Biotherapy & Radiopharmaceuticals 12/2008; 23(6):783-96. · 1.44 Impact Factor
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ABSTRACT: The cytocidal potency of a molecule can be augmented by conjugating a radionuclide for molecular targeted radionuclide therapy (MTRT) for cancer. Radioimmunotherapy (RIT) should be incorporated into the management of patients with B-cell non-Hodgkin's lymphoma (NHL) soon after the patients have proven incurable. Better drugs, strategies, and combinations with other drugs seem certain to make RIT integral to the management of patients with NHL and likely to lead to a cure of the currently incurable NHL. These improved drugs, strategies, and combinations thereof also offer opportunities for RIT to become part of the management of solid malignancies, including epithelial cancers. Smaller radionuclide carriers, such as those used for pretargeted strategies, provide dose intensification. The potential of pretargeted RIT to improve patient outcomes is striking.
Cancer Biotherapy & Radiopharmaceuticals 09/2008; 23(4):383-97. · 1.44 Impact Factor
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ABSTRACT: Here we present modeling and NMR spectroscopic evidence that the function of a Yersinia pestis pMT1 plasmid protein, designated as orf38, is most likely a glutamine binding protein. The modeling was homology-based at a very low level of sequence identity ( approximately 16%) and involved structural comparison of multiple templates, as well as template-substrate interaction analyses. Transferred nuclear Overhauser and saturation transfer difference experiments were used to characterize the binding of sugars and amino acids to orf38. The identification and characterization of an unknown protein function using the strategy presented here has applicability to a variety of research areas, including functional genomics and proteomics efforts.
Protein and Peptide Letters 02/2008; 15(9):887-94. · 1.94 Impact Factor
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Rod Balhorn,
Saphon Hok,
Patricia A Burke,
Felice C Lightstone,
Monique Cosman,
Adam Zemla,
Gary Mirick,
Julie Perkins,
Arutselvan Natarajan,
Michele Corzett,
Sally J DeNardo,
Huguette Albrecht,
Jeff P Gregg,
Gerry L DeNardo
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ABSTRACT: More than two decades of research and clinical trials have shown radioimmunotherapy to be a promising approach for treating various forms of cancer. Lym-1 antibody, which binds selectively to HLA-DR10 on malignant B-cell lymphocytes, has proved to be effective in delivering radionuclides to non-Hodgkin's lymphoma and leukemia. Using a new approach to create small synthetic molecules that mimic the targeting properties of the Lym-1 antibody, a prototype, selective high-affinity ligand (SHAL), has been developed to bind to a unique region located within the Lym-1 epitope on HLA-DR10.
Computer docking methods were used to predict two sets of small molecules that bind to neighboring cavities on the beta subunit of HLA-DR10 surrounding a critical amino acid in the epitope, and the ligands were confirmed to bind to the protein by nuclear magnetic resonance spectroscopy. Pairs of these molecules were then chemically linked together to produce a series of bidentate and bisbidentate SHALs.
These SHALs bind with nanomolar to picomolar K(d)'s only to cell lines expressing HLA-DR10. Analyses of biopsy sections obtained from patients also confirmed that SHAL bound to both small and large cell non-Hodgkin's lymphomas mimicking the selectivity of Lym-1.
These results show that synthetic molecules less than 1/50th the mass of an antibody can be designed to exhibit strong binding to subtle structural features on cell surface proteins similar to those recognized by antibodies. This approach offers great potential for developing small molecule therapeutics that target other types of cancer and disease.
Clinical Cancer Research 10/2007; 13(18 Pt 2):5621s-5628s. · 7.74 Impact Factor
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ABSTRACT: Selective high-affinity ligands (SHALs) were selected as substitutes for monoclonal antibodies (mAbs) to deliver radioisotopes to malignant tumors. Because a SHAL (5 KD) is considerably smaller in comparison to an antibody (150 KD), a significant therapeutic index (TI) enhancement for radioimmunotherapy (RIT) is anticipated. The antibody-antigen (Ab-Ag) model system chosen for the development of SHALs consists of Lym-1, a MAb with proven selectivity in non-Hodgkin's lymphoma (NHL) patients and its well-characterized Ag, the beta subunit of HLA DR10. Whereas Lym-1 is readily available, the subunit of HLA-DR10 is not. Native, heterodimeric (alpha and beta subunits) HLA-DR10 can be purified from Raji cells, which are known to overexpress this Ag. Inconsistent homogeneity between preparations of HLA-DR10 solubilized in the presence of detergents prompted us to express a recombinant form of the beta subunit of HLA-DR10 in Escherichia coli. Negligible production yields (<or=50 microg/L) were achieved by the expression of the full-length protein in a soluble form. By contrast, yields of 240 mg/L were obtained by expressing only the extracellular domain (ED) of the beta subunit of HLA-DR10 in an insoluble form (inclusion bodies). The recovery yield of refolded protein was 75%. Circular dichroism (CD) and Lym-1 binding studies indicated that the recombinant ED of the beta subunit of HLA-DR10 was properly folded. Therefore, this recombinant protein can be used as a surrogate for native heterodimeric HLA DR10 for the in vitro selection of SHALs and related targeting molecules.
Cancer Biotherapy and Radiopharmaceuticals 08/2007; 22(4):531-42. · 1.79 Impact Factor
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ABSTRACT: Laser tweezers Raman spectroscopy (LTRS) is a novel, nondestructive, and label-free method that can be used to quantitatively measure changes in cellular activity in single living cells. Here, we demonstrate its use to monitor changes in a population of E. coli cells that occur during overexpression of a protein, the extracellular domain of myelin oligodendrocyte glycoprotein [MOG(1-120)].
Raman spectra were acquired from individual E. coli cells suspended in solution and trapped by a single tightly focused laser beam. Overexpression of MOG(1-120) in transformed E. coli Rosetta-Gami (DE3)pLysS cells was induced by addition of isopropyl thiogalactoside (IPTG). Changes in the peak intensities of the Raman spectra from a population of cells were monitored and analyzed over a total duration of 3 h. Data were also collected for concentrated purified MOG(1-120) protein in solution, and the spectra compared with that obtained for the MOG(1-120) expressing cells.
Raman spectra of individual, living E. coli cells exhibit signatures due to DNA and protein molecular vibrations. Characteristic Raman markers associated with protein vibrations, such as 1,257, 1,340, 1,453, and 1,660 cm(-1), are shown to increase as a function of time following the addition of IPTG. Comparison of these spectra and the spectra of purified MOG protein indicates that the changes are predominantly due to the induction of MOG protein expression. Protein expression was found to occur mostly within the second hour, with a 470% increase relative to the protein expressed in the first hour. A 230% relative increase between the second and third hour indicates that protein expression begins to level off within the third hour.
It is demonstrated that LTRS has sufficient sensitivity for real-time, nondestructive, and quantitative monitoring of biological processes, such as protein expression, in single living cells. Such capabilities, which are not currently available in flow cytometry, open up new possibilities for analyzing cellular processes occurring in single microbial and eukaryotic cells.
Cytometry Part A 08/2007; 71(7):468-74. · 3.73 Impact Factor
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Rod Balhorn
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ABSTRACT: The protamines are a diverse family of small arginine-rich proteins that are synthesized in the late-stage spermatids of many animals and plants and bind to DNA, condensing the spermatid genome into a genetically inactive state. Vertebrates have from one to 15 protamine genes per haploid genome, which are clustered together on the same chromosome. Comparison of protamine gene and amino-acid sequences suggests that the family evolved from specialized histones through protamine-like proteins to the true protamines. Structural elements present in all true protamines are a series of arginine-rich DNA-anchoring domains (often containing a mixture of arginine and lysine residues in non-mammalian protamines) and multiple phosphorylation sites. The two protamines found in mammals, P1 and P2, are the most widely studied. P1 packages sperm DNA in all mammals, whereas protamine P2 is present only in the sperm of primates, many rodents and a subset of other placental mammals. P2, but not P1, is synthesized as a precursor that undergoes proteolytic processing after binding to DNA and also binds a zinc atom, the function of which is not known. P1 and P2 are phosphorylated soon after their synthesis, but after binding to DNA most of the phosphate groups are removed and cysteine residues are oxidized, forming disulfide bridges that link the protamines together. Both P1 and P2 have been shown to be required for normal sperm function in primates and many rodents.
Genome biology 02/2007; 8(9):227. · 6.63 Impact Factor
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ABSTRACT: The Clostridial neurotoxins, botulinum and tetanus, gain entry into motor neurons by binding to the sialic or N-acetylneuraminic acid (NeuAc) residues of gangliosides and specific protein receptors attached to the cell's surface. While the C-fragment of tetanus toxin (TetC) has been identified to be the ganglioside binding domain, remarkably little is known about how this domain discriminates between the structural features of different gangliosides. We have used electrospray ionization mass spectrometry (ESI-MS) to examine the formation of complexes between TetC and carbohydrates containing NeuAc groups to determine how NeuAc residues contribute to ganglioside binding. ESI-MS was used to obtain an estimate of the dissociation constants (KD values) for TetC binding to a number of related NeuAc-containing carbohydrates (sialyllactose and disialyllactose), as well as six (NeuAc)n oligomers (n = 1-6). KD values were found to range between approximately 10-35 microM. The strength of the interactions between the C fragment and (NeuAc)n are consistent with the topography of the targeting domain of tetanus toxin and the nature of its carbohydrate binding sites. These results suggest that the targeting domain of tetanus toxin contains two binding sites that can accommodate NeuAc (or a dimer) and that NeuAc may play an important role in ganglioside binding and molecular recognition, a process critical for normal cell function and one frequently exploited by toxins, bacteria, and viruses to facilitate their entrance into cells.
Journal of the American Society for Mass Spectrometry 08/2006; 17(7):967-76. · 4.00 Impact Factor
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ABSTRACT: Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for studying bimolecular interactions at the atomic scale. Our NMR laboratory is involved in the identification of small molecules, or ligands, that bind to target protein receptors such as tetanus neurotoxin (TeNT) and botulinum neurotoxin, anthrax proteins, and HLA-DR10 receptors on non-Hodgkin lymphoma cancer cells. Once low-affinity binders are identified, they can be linked together to produce multidentate synthetic high-affinity ligands (SHALs) that have very high specificity for their target protein receptors. An important nanotechnology application for SHALs is their use in the development of robust chemical sensors or biochips for the detection of pathogen proteins in environmental samples or body fluids. Here we describe a recently developed NMR competition assay based on transferred nuclear Overhauser effect spectroscopy that enables the identification of sets of ligands that bind to the same site, or a different site, on the surface of TeNT fragment C (TetC) than a known "marker" ligand, doxorubicin. Using this assay, one can identify the optimal pairs of ligands to be linked together for creating detection reagents, as well as estimate the relative binding constants for ligands competing for the same site.
Methods in molecular biology (Clifton, N.J.) 02/2005; 300:141-63.
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ABSTRACT: Clostridium botulinum neurotoxins (BoNTs), the most potent toxins known, disrupt neurotransmission through proteolysis of proteins involved in neuroexocytosis. The light chains of BoNTs are unique zinc proteases that have stringent substrate specificity and require exceptionally long substrates. We have determined the crystal structure of the protease domain from BoNT serotype A (BoNT/A). The structure reveals a homodimer in a product-bound state, with loop F242-V257 from each monomer deeply buried in its partner's catalytic site. The loop, which acts as a substrate, is oriented in reverse of the canonical direction for other zinc proteases. The Y249-Y250 peptide bond of the substrate loop is hydrolyzed, leaving the Y249 product carboxylate coordinated to the catalytic zinc. From the crystal structure of the BoNT/A protease, detailed models of noncanonical binding and proteolysis can be derived which we propose are also consistent with BoNT/A binding and proteolysis of natural substrate synaptosome-associated protein of 25 kDa (SNAP-25). The proposed BoNT/A substrate-binding mode and catalytic mechanism are markedly different from those previously proposed for the BoNT serotype B.
Proceedings of the National Academy of Sciences 06/2004; 101(18):6888-93. · 9.68 Impact Factor
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ABSTRACT: We used high-resolution atomic force microscopy to image the compaction of linear and circular DNA by the yeast mitochondrial protein Abf2p, which plays a major role in packaging mitochondrial DNA. Atomic force microscopy images show that protein binding induces drastic bends in the DNA backbone for both linear and circular DNA. At a high concentration of Abf2p DNA collapses into a tight nucleoprotein complex. We quantified the compaction of linear DNA by measuring the end-to-end distance of the DNA molecule at increasing concentrations of Abf2p. We also derived a polymer statistical mechanics model that provides a quantitative description of compaction observed in our experiments. This model shows that sharp bends in the DNA backbone are often sufficient to cause DNA compaction. Comparison of our model with the experimental data showed excellent quantitative correlation and allowed us to determine binding characteristics for Abf2p. These studies indicate that Abf2p compacts DNA through a simple mechanism that involves bending of the DNA backbone. We discuss the implications of such a mechanism for mitochondrial DNA maintenance and organization.
Biophysical Journal 04/2004; 86(3):1632-9. · 3.65 Impact Factor
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ABSTRACT: The conformation of the non-glycosylated recombinant form of the extracellar domain of rat MOG (rMOG(1-125)) dissolved in different solvent conditions was studied by CD spectroscopy. The results show that rMOG(1-125) exhibits a predominantly beta sheet conformation in aqueous buffer solution at pH 7.5 and that this 'beta-form' is stabilized by zwitterionic phospholipids, DPC and LPCP. The alpha helical content of the protein can increase from 9% to up to 20% when TFE or anionic detergent LPAP and SDS are added.
Protein and Peptide Letters 11/2003; 10(5):483-90. · 1.94 Impact Factor
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ABSTRACT: Protamine molecules bind to and condense DNA in the sperm of most vertebrates, packaging the sperm genome in an inactive state until it can be reactivated following fertilization. By using methods that enable the analysis of protamine binding to individual DNA molecules, we have monitored the kinetics of DNA condensation and decondensation by protamine 1 (P1) and synthetic peptides corresponding to specific segments of the bull P1 DNA binding domain. Our results show that the number of clustered arginine residues present in the DNA binding domain is the most important factor affecting the condensation and stability of the DNA-protamine complex prior to the formation of inter-protamine disulfide cross-links. The high affinity of P1 for DNA is achieved by the coordinated binding of three anchoring domains, which together in bull P1 contain 19 Arg residues. The single DNA molecule experiments show that sequences containing two or more anchoring domains have an off-rate that is at least 3 orders of magnitude slower than those containing a single domain. The use of Arg, rather than Lys residues, and the inclusion of Tyr or Phe residues in the hinge regions between anchoring domains provide additional stability to the complex.
Journal of Biological Chemistry 11/2003; 278(43):42403-8. · 4.77 Impact Factor
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ABSTRACT: Mitochondrial and nuclear DNA are packaged by proteins in a very different manner. Although protein-DNA complexes called "nucleoids" have been identified as the genetic units of mitochondrial inheritance in yeast and man, little is known about their physical structure. The yeast mitochondrial protein Abf2p was shown to be sufficient to compact linear dsDNA, without the benefit of supercoiling, using optical and atomic force microscopy single molecule techniques. The packaging of DNA by Abf2p was observed to be very weak as evidenced by a fast Abf2p off-rate (k(off) = 0.014 +/- 0.001 s(-1)) and the extremely small forces (<0.6 pN) stabilizing the condensed protein-DNA complex. Atomic force microscopy images of individual complexes showed the 190-nm structures are loosely packaged relative to nuclear chromatin. This organization may leave mtDNA accessible for transcription and replication, while making it more vulnerable to damage.
Biophysical Journal 11/2003; 85(4):2519-24. · 3.65 Impact Factor
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ABSTRACT: An experimental approach is described for determining protein-small molecule non-covalent ligand binding sites and protein conformational changes induced by ligand binding. The methodology utilizes time resolved limited proteolysis and the high throughput analysis capability of MALDI TOF MS to determine the binding site in a tetanus toxin C-fragment (51 kDa)-doxorubicin (543 Da) non-covalent complex. Comparing relative ion abundances of peptides released from the time resolved limited proteolysis of tetanus toxin C-fragment (TetC) and the TetC-doxorubicin complex every 10 min from 10 to 120 min of digestion revealed that the binding of doxorubicin induced a significant change in surface topology of TetC. Four of the twenty-nine peptides observed by MALDI MS, including amino acids 351-360, 299-304, 305-311 and 312-316, had a lower abundance in the TetC-doxorubicin complex relative to TetC from 10 to 100 min of digestion. A decrease in ion abundance suggests doxorubicin obstructs the access of the protease to one or both termini of these peptides, identifying doxorubicin binding site(s). Conversely, five peptide ions, including amino acids 335-350, 364-375, 364-376, 281-298, and 316-328, all had a greater abundance in the digest of the complex, indicating an increase in accessibility to these sites. These five peptides flank regions of decreased ion abundance, suggesting that doxorubicin not only binds to the surface, but also induces a conformational change in TetC.
Journal of the American Society for Mass Spectrometry 06/2003; 14(5):460-70. · 4.00 Impact Factor
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ABSTRACT: Two transition proteins, TP1 and TP2, participate in the repackaging of the spermatid genome early in mammalian spermiogenesis, coincident with the first detectable changes in chromatin condensation. Using an optical trap and a two-channel flow cell to move single DNA molecules into buffer containing protein, we have measured the rates of DNA condensation and decondensation induced by the binding of Syrian hamster transition proteins TP1 and TP2 and protamines P1 and P2. The results show that both transition proteins condense free DNA, with rates similar to those of protamine 1 and 2. DNA molecules condensed with TP1 were significantly less stable than DNA condensed by protamine or by TP2. Experiments conducted with a peptide corresponding to the C-terminal 25 residues of TP2 showed that this domain is responsible for condensing DNA. Experiments conducted with two fragments of TP1 containing arginine and lysine residues demonstrated that DNA binding by TP1 must involve more than these basic sequences. Zinc facilitated the condensation of DNA by P2 but not by TP2. The dissociation rates of TP2 and P2 from DNA were not affected by the addition of zinc.
Journal of Biological Chemistry 11/2002; 277(41):38895-900. · 4.77 Impact Factor
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ABSTRACT: A combination of computational methods, electrospray ionization mass spectroscopy (ESI-MS), and NMR spectroscopy has been used to identify novel small molecules that bind to two adjacent sites on the surface of the C fragment of tetanus toxin (TetC). One of these sites, Site-1, binds gangliosides present on the surface of motor neurons, while Site-2 is a highly conserved deep cleft in the structures of the tetanus (TeNT) and botulinum (BoNT) neurotoxins. ESI-MS was used to experimentally determine which of the top 11 computationally predicted Site-2 candidates bind to TetC. Each of the six molecules that tested positive was further screened, individually and as mixtures, for binding to TetC in aqueous solutions by NMR. A trNOESY competition assay was developed that used doxorubicin as a marker for Site-1 to provide insight into whether the predicted Site-2 ligands bound to a different site. Of the six predicted Site-2 ligands tested, only four were observed to bind. Naphthofluorescein-di-beta-galactopyranoside was insoluble under conditions compatible with TetC. Sarcosine-Arg-Gly-Asp-Ser-Pro did not appear to bind, but its binding affinity may have been outside the range detectable by the trNOESY experiment. Of the remaining four, three [3-(N-maleimidopropionyl)biocytin, lavendustin A, and Try-Glu-Try] bind in the same site, presumably the predicted Site-2. The fourth ligand, Ser-Gln-Asn-Tyr-Pro-Ile-Val, binds in a third site that differs from Site-1 or predicted Site-2. The results provide a rational, cost- and time-effective strategy for the selection of an optimal set of Site-1 binders and predicted Site-2 binders for use in synthesizing novel bidendate antidotes or detection reagents for clostridial neurotoxins, such as TeNT and BoNT.
Chemical Research in Toxicology 11/2002; 15(10):1218-28. · 3.78 Impact Factor
